Internat'ional S emtnar on Medtcal G enet'ics

August3L - September3, 1966

H.01 U 1967 19227 Hist.

Provided by the Maternal and Child Health Library, Georgetown University I I

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Reproducedwith permission by the U.S. DEPARTMENT OF HEALTH, EDUCATION, AND VELFARE, Social and Rehabilita- tion Service, Children's Bureau, from The AlobomoJournol of MedicolSciences, Vol. 3, No. 4, October 1966,(pp. 351-518,530)-

Reproducedby permissionfrom the ReynoldsHistorical Library' the University of Alabamaat Birmingham

provided by the Maternal and chitd Health Library, Georgetownuniversity ,i

li

INTERI\ATIOI\AL SEMII\AR Ii

on 123 MEDICAL GEI\ETICS

August 31 September3, 1966

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University of Alabama Medical Center

Birmingham, Alabama

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l Program Directors: f)r. Wayne H. Finley, Dr. Sara C. Finley 2 Sponsors:Division of Continuing Medical Education, Medical College of Alabafira; Post- sraduate Education, IJniversity of Alabama School of Dentistry; The Children's flospital, Birmingham, Alabama 3 Support: Children's Bureau, U. S. Department, of Health, Education, and Welfare, Project 413

university provided by the Maternal and child Health Library, Georgetown (ontents

352 > Welcome, S. R. Hill, Ir.

353 > Opening remarks, loseph F.Volker

354 > Welcome, Charles A. McCallum, lr.

355 > Genetics in expanding health programs for mothers ancl children. Alice D. Chenowetlt

359 > Biochemical genetics in medicine. Arno C. Motulsky

370 > Cleft lip and cleft palate in Iceland. Palmi itloller and loltn Dunbar

373 > Cenetics studies in human dental caries. Sidneg Finn

376 > Some old and new data on the genetics of human populations. L. L. Caoalli-Sforza

382 > Medical and dental findings in the Brandvrvine Isolate. CarI J. Witkop, Ir., Clnrles I. MacLean, PauI l. Schntid{, Joseph L. Henry

404 > Genetical investigations in mental retardation. /. A. Bii6k

408 > Clinical genetics at a population level. The ethnicitv of cliseasein the United States. ViAor A. McKusick

425 > Gene dosage effects in man. l6r6me Leieune

432 > Dermatoglyphics and chromosomes. lrene A. Uchida

435 > Reflections on 20 years experience of genetic counselling. J, A. Fraser Roberts

440 > Panel l: Gene function

447 > Panel 2: Diseases at the molecular level

456 > Panel 3: Diagnosis and management of metabolic abnoln-ralities

473 > Panel 4: Epidemiology of selected genetic diseases

483 > Panel 5: Clinical cytogenetics

496 > Panel 6: Indications for chromosome analyses

517 > Closing remarks, Joseph F. Volker

-:- University provided by the Maternal and Child Heatth Library, Georgetown Welcome

S. R. Hill, Jr., M.D.*

The faculty and the student body of the University of ,\labama l{eclical Center join n'ith the organizing committee to welcome all of you to this International Seminar on NIed- ical Genetics and to add to this welcome we would like to ask the Vice Presiclent for Health

,tffairs at the University of Alabama N{edical Center, Dr. .}oseph F. Volker, to speak to )'ou

GUESf }'ACULTY FACT]LTY

Jan A. Bddk, M.D., Ph.D. Joseph F. Volker, D.D.S., Ph.D. L. L. Cavalli-Sforza. M.D. S. Richardson Hill, Jr., M.D. Alice D. Chenoweth, M.D. CharlesA. McCallum,Jr., D.M.D., \,f.D. Valerie A. Cowie, M.D., D.P.M. Samuel B. Barker, Ph.D. Eric Engel, M.D. John B. Dunbar, D.M.D., Dr. P.H. Richard A. Finch, Nf.D. Margaret S. Klapper, N{.D. Frederick Hecht, M.D. Edwin M. Speed, D.N[.D. F. John Jackson, M.D. Charles A. Alford, Jr., N{.1). Cecil Jacobson, M.D. J. Claude Bennett, M.D. Robert S. Kroorh, M.D., Ph.D. Hcrschel P. Bentley, Jr., \I.I). Lejeune, Jerdme M.D., Ph.D. Charles E. Buttcnvorth, Jr., N{.1). Lillian Lockhart, M.D. CleralclL. Carlson, Ph.D. Arno G. Motulsky, M.D. Ceorge Cassady,N{.D. Ernest E. McCoy, M.D. Sarah F. Davis, trI.D. 'Ihomas Victor A. McKusick, M.D. W. Feary, Ph,D. Nfarshall W. Nirenberg, Ph.D. Sara C. Finley, M.D. William L. Nyhan, M.D., ph,D. Wayne H. Finley, Ph.D., \I.D. Donough O'Brien, M.D., F.R.C.p. Sidney B. Finn, D.II.D. Richmond S. Paine, M.D. Ralph Gilmore, D.M.D. J. A. Fraser Roberts, M.D., D.Sc.,F.R.C.p., II.R.S. Clarence E. Klapper, Ph.D. David W. Smith, M.D. Emmanuel Margolis, Nf.D. Robert L Summitt, N{.D. Russell S. McMillan, D.D.S.,Dr. P.H. Irene A. tlchida, Ph.D. Pcter B. Peacock,D.P.H., D.I.H., D.T.Nf.&H. Carl J. Witkop, Jr., D.D.S. Carlyn C. Tucker, I{.D. Hans Zellrveger,M.D. K. Lemone Yiclcling, \f .l).

+Dean, Medical College of Alabama.

_----a

University Provided bY the Maternal and Child Health Library, Georgetown Opening Remarks

losephF. Yolker,D.D.S., Ph.D.#

I would like to add my welcome to that of Dean Hill. \Vhenever I have a captive aucli- ence I behave like the fablecl scorpion who wished to cross a river but was aware of his limi- tations as a swimmer. While he was reflecting on his dilemma, along came a turtle who obviously was a talented mariner. The scorpion very promptly said to the turtle, "Will you carry me over the river?" The turtle looked at him and replied, "Do y,ou think I'm crazy? The first thing you'll clo if you get close is to bite and that will be the end of me." The scorpion pleaded, "No, I just want to get across the river. Please take me." The turtle heeded his request, and they started across. As they came to the middle of the river, the scorpion reachecl down ancl bit the turtle on the neck. The turtle, aware he was dyine, looked up at the scorpion ancl asked, "Why did you do it?" To rvhich the scorpion replied, "I can't help being the way I am." And as an administrator I can't resist the temptation to say a few words about the University and the Meclical Center. Obviously we are a very young medical center, and in terms of years we are a rela- tively young University. But like all universities, we have great aspirations. We recognize that as the university flourishes and gives leadership to the community, things so well. When this is not the case, the state and the nation suffer. Wherever a strong university system exists there is an unusual capacity to survive ad- versity. The rebuilding of Germany and .fapan after World War II give excellent support to this thesis. Conversely, the American people are very much aware that you can make all kinds of resources available to a country and it will come to naught if there is no intellec- tual focus to implement reclevelopment programs. We are hopeful, of course, that this University will be able to achieve competency in the traditional areas of teachin€i, research, ancl service. \4/e have macle, I believe, a modest beginning and have great hopes for the future. Your presence here is a stimulus to us and a reminder of what universities can do indiviclualll' ancl collectivelr'. Since it is an international symposium, I thought it might be appropriate to quote I'rom an essayby A. V. Hill, the physiologist, who said: It needs no historian to recall how learning, scholarship ancl art, on the one hanrl, and natural philosophy ancl technology, on the other, have from early claysbeen largely international in their stluggles, these were the only common interests of mankincl. It is pleasant to remember how philosophers and scholars coulcl, usually without hinclrance, even in time of war, continue uninterrupted their irrtercotrrse with other countries. A document now more than seven hundred years olcl recorcls the presence at Paclua o[ French, English, Norman, Provenqal, Spanish, and Catalan students. Later at Paclua twenty-two "nations" were represented, twelve from Italy itself, ten from beyond tl-re Alps. In the fifteenth century there were about one hundred French stuclents there, nearly as many English and Scottish, over three hundred German. In spite of all diffi- culties of transport and communication there was a very real international sensein the lrumane pursuit of learning. Hacl medicine, scholarship, and science had no other gifts

GeorgetownUniversity Provided bY the Maternal and Child Health Library, 354 VOLKER Ala. J. Med.Sci.

at all to offer to mankind, their habit of transcending language, nationality, and prej- udice would have made them, more perhaps than anything else, worthwhile. (A. V. Hill. The Ethical Dilemma of Scienceand Other Writings. New York, The Rocke- feller Institute Pressin associationwith Oxford University Press,1960. p 213). I hope these thoughts may serve as a keynote for this conference.Thank you.

Welcome

ChorlesA.

It is my distinct pleasure on behalf of tistry should have a vital interest in the area the faculty, administration, and studentsof of growth and development, and particu- the University of Alabama School of Den- larly the area of genetics. Even though the tistry to extend to you a cordial welcome Schoolof Dentistry in terms of years,might to the International Seminar on Medical be considered the embryo of the Medical Genetics. The University is proud to be Center, our interest in the field of genetics the host to the array of talent that has is long standing. We hope that through the gathered here for the purpose of sharing cooperative efforts of the scientific com- their scientific accomplishmentsin this most munity that important information from important area with their confreres. this field will permit the solution of many of the elusive problems of oral diseaseand It is appropriate that the School of Den- malformations.

tl)can of the lkhool of Dentistrv,

provided by the Maternal and Child Health Library, Georgetown University GeneticsIn ExpandingHealth Programsfor Mothersand Childrenl

On behalf of the Children's Bureau, I in genetics,i.e. as maternal and child health am pleasedto add my welcome to the dis- programs have expanded the opportunities tinguisheclparticipants in this Internation- for applying knowledge of medical genetics al Seminar on Medical Genetics.The Chil- in such programs has also increased. As dren's Bureau is proud of being able to identification of homozygotes,and especi- support the University of Alabama l\{edical ally of heterozygotes,increases, and as con- Center in its sponsorshipof this conference. trol of the manifestation of unfavorable I wish to take this opportunity also to genesbecomes a possibility, an understand- salute a talented husband-wife team, our ing of genetic principles is more and more hosts, for their contribution to medical useful not only for health personnel who genetics ancl for promoting an interest in serve families directly but for Program genetics throughout the State. All of us clirectors rvho chart the future course of thank the Finleys for the many man hours maternal and child health and crippled they have spent on our behalf arranging children's programs. this seminar. For many in the audience, the two health There is a great upslrrge of interest in grant-in-aid programs administered by Chil- geneticsin this country, and judging by a dren's Bureau, viz. maternal and child review of the medical literature, this inter- health and crippled children's are well est extends to other countries as well. known. The Children's Bureau has a vital inter- Nlaternaland Child Health services,since est in geneticsbecause of the nature of its they are a part of State Health Depart- pro€irams.Human and medical geneticsare ments, have developed along lines which necessarilyan integral part of programs to are traditional for public health in this promote the health and welfare of mothers countr)', i.e. they consistmainly of preven- ancl children, which is the Bureau's man- tive and health promotional services,with clate.Consequently, the beginnings of life, a minimal amount of what might be termed improving the quality of life, and prevent- medical care. There are exceptions; exam- ing or mitieating the effects of handicap- ples are: in Alabama under Dr. Harold ping conditions, whether mental or physi- Klingler, one of the participantsin this con- cal, are basic concernsof the Bureau. ference,the provision of maternity care in- Recent, rapid ddvancesin biochemistry cluding hospitalization for delivery; in a and cytogenetics have led to a hope that few States comprehensive care of prema- new knowledge of genetics will have prac- tures including hospitalization, transPorta- tical application in a solution of hereditary tion, medical and nursing care and follow- problems. In a general way the expansion up sen'ices;in l3 Stateshospitalization for of health programs for mothers and chil- r{'omen with complications of pregnancy. clren in this country has paralleled progress The earmarking of maternal and child

*"r, at International Seminar on Medical Genetics, health funds for services to mentally re- University of Alabama llfedical Center, Birmingham, Augu:t 3r, I966. tarded children beginning in 1957 and of 2Chief, Program Services Branch, Dilision of Health funds since 1963has had Services. Children's Bureau. crippled children's

Georgetown UniversitY Provided bY the Maternal and Child Health LibrarY' 356 CHENOWETH Ala. J. Med.Sci. the effect both of increasingthe amount of was cliscovered,advisors to the Children's meclicalcare and of stimulating an interest Bureau recommended that cytogeneticstud- in geneticsamong maternal and child health ies be made available to families with an staff. As evidenceof.the importance of gen. affected child. At that time the number of eticsto maternal and child health programs, laboratories performing chromosome analy- a few Stateshave employed full-time geneti- seswas relatively few and the main purpose cists;several utilize the part-time servicesof of the cytogeneticstudies which were done geneticists.Nearly every State has used its was to promote research;service to a family earmarkedfunds to develop multidiscipline was not the primary objective. clinics for the diagnosis,evaluation, treat- With new discoveriesin cytogeneticsand ment, and follow-up care of mentally re- biochemistryindications for such laboratory tarded children. Parent counseling is an examinations to be macle a part of the com- important component of the service,includ- plete evaluation of a child and his family ing when appropriate genetic counseling.A are increasing. Responding to a growing number of medical center programs have demand for service, the Children's Bureau extended their servicesto satellite clinics in has encouraged the development of cyto- neighboring cities or communities. By the genetic and biochemical laboratory services end of the fiscal year year 1966, the Chil- in medical centers associatedwith programs clren's Bureau was supporting, in whole or for mentally retarded or multiply handi- part, I34 clinics for mentally retarded chil- capped or congenitally cleformed children. rlren in the United States. At present l3 such laboratories are receivinp; The inauguration of a new program has support through special project srants from a characteristic pattern in the Children's the Children's Bureau. Bureau-a special consultant is employed; Another example of program expansion in-service and observation training pro- through a heightened interest in mental grams are arranged for staff; and an expert retardation is the new authority given the committee to advise the Bureau on pro- Children's Bureau to make grants to State gram planning and developmentis appoint- or local health departments for Maternity ed. The Children's Bureau advisory com- and Infant Care projects, under the 1963 mittee on services for mentally retarded Amendments to the Social Security Act. children has met regularly and many of Acting on a recommendation of President their recommendationshave helped chart Kennedy's Panel on Nlental Retardation, the course this program has taken. that prevention also be a part of the pro- When the prevenrion of the ill effects of gram, Congressappropriated funds for com- PKU appearedto be a possibility,screening prehensivemedical and hospital care servi- 'high of young infants was begun and an exten- ces to risk' maternity patients and to sive field trial of the Guthrie "inhibition 'high risk' infants in areasof concentration assay"test was carried out. With a network of low-income families. The objective of of clinics for the care of mentally retarded these projects is to reclucethe hazards asso- chilclren in operation, confirmation of diag- ciated with childbearing. It was hoped that nosis, treatment and follow-up care for af- the availability and accessibilityof mater- fected infants could be effectively imple- nity clinics to low-income patients would mented. Improved screening methods ind be increasedand that overcrowdinp;of pub- test for multiple inborn errors of metabo- lic hospitals would be relieved. There are lism are being sought. now 5l projects which have been approved Now, as recommendedby the Children's -most of them in laree cities. It is my Bureau Advisorv Committee, a long-term understanding that one is getting underway evaluation of children treated for PKU is here in tsirmingham. Nearly all projects in- in the planning stage. A protocol for the clude family planning services.Since the 'high submissionof data has been drafted. women served are risk,' a higher than When the translocation defect in addi- usual proportion of families may be in need tion to the trisomy in Down's Svndrome of genetic studies ancl counseling.

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provided by the Maternal and child Health Library, Georgetownuniversity Vol. 3, No. 4, 1956 GENETICSIN EXPANDINGHEALTH PROGRAMS 357

Another program development which has neurological deficits;^some are canclidates increasedthe interest of MCH and CC per- for cytogenetic study. sonnel in geneticsis the changing character The present Congressenacted legislation of the children who receive servicesunder which requires the extension of MCH ancl the auspicesof official crippled children's CC servicesto all parts of the Stateby 1975. agencies.The crippled children's program Such legislation was directed chiefly at the is a medical care program for handicapped problems of disadvantaged mothers and children or for children with conditions children in large urban areas,however low- which may lead to crippling. There is no income mothers and children in rural areas definition of crippling in the Federal stat- may also benefit from extensionof services. ute, but the trend is toward a broad inclu- Another new program with implications sive definition to encompass a wide spec- for geneticsis provided under the Maternal trum of conditions which may handicap a and Child Health amendmentsof 1965viz. child. The trend has been for State agencies the comprehensivehealth and medical care to expand their coverageof more and more projects for children and youth. These chronic conditions as funds become avail- grants are for projects which are designed able. A few Stateswill now accepr a child to provide continuing preventive and cura- with almost any chronic or long-term or tive services,both medical and dental, for complex diseaseor disability. At ttie present children who have not generally received time congenital malformations constitute consistent medical care and who live in an increasingly higher percenrage of the areas or neighborhoods where low-income total number of children servedby crippled families are concentrated. It is possible for children's agencies,roughly 30 percent. We projects to develop serviceswith a special are confident that many infants are being emphasis while providing comprehensive saved who formerly died. Also, the avail- care to all children of the area, for example, ability of servicesand more effective treat- a special program for children with a hered- ment has contributed to the increase in itary diseasecould be a part of the project. numbers in the crippled children's program. Twenty-three children and youth projects An example of this is the very grear rise in have been approved to date. N,Iostof these the number of children with congenital are in urban areas,some are in areaswhere heart defects under the program from 2,200 a maternity and infant care project is locat- in 1950to 25,000in 1963. ed, thus providing continuity of medical In recent years other conditions of inter- care of infants born under the maternity est to this conferencesuch as cystic fibrosis, project. The Federal share may be as much are being acceptedby an increasing number as 75 percent of the cost of both the mater- of States. Programs for hemophilia and nity and infant care and the children and sickle cell anemia are being developed by youth projects. States in the hope of identifying the child Shortage of trained manpower is a prob- early and preventing his disability. From lem which the 89th Congressrecognized in time to time the crippled children's pro- all the legislation it passed. The MCH grams are confronted with new problems, amendmentsof 1965included an authoriza- an example of one is the 1963-65rubella tion for training of professional personnel epidemic in this country which left in its for care of mentally retarded or multiply wake an estimated 20-30,000 babies with handicapped children. This is the first time congenital deformities. that funds to be administered by the Chil- Clinics for both mentally retarded and dren's Bureau have been designated for crippled children are seeing an increasing training. (I am told that the University of number of children who are severely and Alabama has been awarded a construction multiply handicapped. Children's Bureau is grant for a university-affiliated center. Con- now supporting 12 special centers which gratulations!) are providing services for such children. The Children's Bureau has authority to Many multiply handicapped children have use up to l?r/e percent of its maternal and

---- provided by the Maternal and child Health Library, Georgetownuniversity 358 CHENOWETH Ala. J. Med. Sci. child health and crippled children's appro- technological change.Congress has respond- priations for grants to State agenciesor in- ed to the ferment by passing a vast amount stitutions of higher learning for special of new legislation which is said to be com- projects of regional or'national significance parable only with the enactment of the which may contribute to the advancement original Social Security Act of 30 years ago. of MCH or CC services. N{any of these Tremendousopportunities for better health, training grants are for the purpose of education and welfare for all people are strengthening faculties and field training in now possible; to implement the new laws addition to the support of fellowships. Most creatively and wisely is a grave responsibil- of the cytogenetic and biochemical labora- ity ancl a great challenge for all of us. tory projects which the Children's Bureau supports have a training component. remarks In 1963, funds for research relating to DR. J. F. VOLKER: Dr. Chcnotvcth's has to maternal and child health and crippled bring into focus some of the planning that be done by this and othcr medical cent€rs. She has children's serviceswere Con- authorized by referrecl to the fact that by 1975 there rvill be lsgis- gress,the first funds given to the Children's lation that should provide comprehensive hcalth Bureau for health research.The purpose of service for people classified as mcdically indigent. thesegrants is to support studiesthat show We should appreciate that in the proposed plan by the promise of making a substantial contribu- the level of eligibility will be determined individual states. Morcover, it should be noted that tion to the advancementof health programs some states, i.e., New York, have set a level of indi- for mothers and children. Studiesrelated to gencv for a famil,v of four aftcr taxes and exemp- medical genetics are appropriate subjects tions at 56,000. I don't believc anyone has a good for Children's Bureau supported research. estimate of the nttmber of people that will benefit Nor do I think that Further evidence of the Children's Bu- under this t1.peof legislation. manv understand the scope of the program. It is reau interest in geneticswas the inviting of my recollection that bv 1975 the last of the D's, an ad hoc advisory committee on medical namelv drugs and dental carc, n'ill be included in genetics to Washington, D. C. in Nfarch these services.Dr. Chenorveth, clo vott know of an-v 1966to considerwith the Bureau what op- intelligent estimate as to thc cost of these programs and. the numbers of tlained peoplc that will be re' portunities exist for genetically oriented quired to operate them? approachesto program planning and servi- N{ore specificalh', health aclministrators are con- ces for mothers and children. At least two cerned u'ith numbers of mentallv retarded persons other conferenceson genetics besides this rvho rvill be eligible ttnder tten' Fctleral progralns there reli- one at the University of Alabama have also for cliagnosticancl treatmcnt scrriccs.Are able cstimatcsas to the nrttnbcr of investigatorsand been supportedby Children's Bureau funds. practitioners n'ho will bc ncctlctl to strpport these I have tried to give a brief summary of special programs? C)bviouslv thcsc are troublesotne the expanding programs for mothers and qucstions, but I think this group rvotrl

university Provided bY the Maternal and child Health Library, Georgetown BiochemicalGenetics In Medicinet'2

ArnoG. Motulsky,M.D.ft

Originsof HumonBiochemicol Genetics variability was likely. He predicted that idiosyncrasiesto drugs as well as various de- Many advancesin sciencehave occurred greesof natural resistanceagainst infections by applying concepts and techniques frorn might be explained by the "failure of mem- different disciplines to an unsolved prob- bers of a speciesto conform to an absolutely lem. Garrod, a British physician worked on rigid standard of metabolism." How huppy various rare metabolic diseasessuch as al- Garrod would have been to learn that an kaptonuria in the early part of this century. enzyme deficiency of the red cell (G6PD He was struck by the familial nature of this deficiency) predisposesto hemolytic reac- diseaseand notecl the increasedfrequency tions by drugs and at the sametime confers of first-cousin marriages among the nonaf- resistanceagainst falciparum malaria! Gar- fected parents of his patients.Talking mat- rod utilized conceptsfrom biochemistry and ters over with Bateson, the biologist who geneticsand applied them to human disease coined the term of genetics,these findings to found the field of biochemical genetics. seemedbest explained by the recently re- We can be proud that a physician-a true discoveredN{endelian lar,r's.Thus, each par- clinical investigator-made these discover- ent of an alkaptonuric patient u'asassumed ies which not only were of importance to to be a latent or recessivecarrier of the gene medicine but set the stage for the breath- for alkaptonuria, having derived this gene taking developmentsof molecular biology from a common ancestor. The double dose of recent years. of a specific factor produced the disease Garrod's work was far ahead of his time. rather than consanguinity by itself. Under His colleaguesrespected him, his students the influence of the physiologic chemist loved him, his peers electedhim ro be the Frederic Gowlancl-Hopkins and arguing successorof Osler'sprestigious chair at Ox- from alkaptonuria, pentosuria, cystinuria ford, but no one realized the general signif- and albinism, Garrod concluded that nor- icance of his work. Even geneticistswho mal metabolism was carried out by single should have known better ignored him and discretesteps, each probably under the con- the literature on physiologicgenetics of the trol of specific hereditary factors.Blocks at 1920's and 1930's fails to mention him. some particular point of intermediate me- Beadle'sand Tatum's work on neurospora tabolism clue ro cong;enitaldeficiency of an which firmly established the one gene-one enzymewould lead to excretion or accumu- enzymehypothesis was carried out in igno- lation of normal intermediateswhich ordi- rance of Garrod's contribution. narily could not be detected.Garrod named disordersof this sort "inborn errors of me- Investigotionson Sickle Hemoglobin tabolism" and ascribeda more general bio- It took another discoveryto establishbio- logic significanceto thesediseases (Garrod, chemical genetics as a discipline of great 1909).He maintained that just as srrucrure relevance to medicine. In 1945,Pauling, the varied between individuals, biochemical -Elr"n,"d famous chemist, and Castle, the renowned before the International Seminar on l\{edical hematologist, served on a committee deal- Genetics, August 31, 1966, University of Alabama Medical Center, Birmingham, Alabama. ing with matters of national scientific pol- 2 Fartiallv aided by Grant He 3091 from the National Institutes of Health, Bethesda, Maryland. icy. Castle told Pauling that red cells from fDepartments of Medicine and Cenetics, University of Washington, Seattle, Washington. patients with sickle cell anemia when de-

. -b+- Georgetownuniversity Provided bY the Maternal and child Health Library, 360 MOTULSKY Ala. J. Med. Sci. oxygenated became sickled and showed confirmed what appeared evident when birefringence in polarized light suggesting single amino acid alterationswere found in some type of unspecifiedmolecular realign- rnutty other abnormal hemoglobins' Amino ment. Pauling later concludedthat a molec- acid alterations of the protein chain had a ular abnormality of hemoglobin might best linear relationship to the mutational sites explain these phenomena and suggestedto within the gene specifying the information Harvey Itano, a graduate student with a for a siven protein chain. Analysis of these medical degree,to investigate this problem. chaini- coulcl provide detailed information After much hard work and many negative regarding the site of mutation in the DNA findings, it became clear in 1949 that the of the gene. Genes specifying the sequence electrophoretic mobility and therefore the of u*ito acids in protein chains were structure of hemoglobin of patients with termed structural genes and the most com- sicklecell anemia was indeed different from mon mutational alterationsof protein were normal hemoglobin. The somewhat puz- single amino acid substitutions' zling genetic findings that parents of pa- Study with microorganisms yielded full tients with sickle cell anemia were clinically undersianding of the genetic code (Niren- well but had sickledred cellswas explained berg, Ochoa). Triplets of nucleotide bases by finding both normal and abnormal hem- conlain information specifying which of the oglobin in their blood (Pauling et al., 1949). 20 amino acids is inserted into a Protein .|im Neel'sgenetic conclusions (Neel, 1949) chain ancl the composition of these DNA indicating that sickle cell anemia required triplets has become recently clarified' All the double dose of the sickling gene were knbwn amino acid substitutions in human confirmed by the biochemical data. Pauling hemoglobin can be explained by alterations realized the full general significance of of on"e of three nucleoticle triplets which thesediscoveries and pointed out that sickle comprisethe variouscoding units asworked cell anemia was the first example of a mo- out in microorganisms (Watson, 1965)' lecular disease. These finclings conform tc data on muta- The nature of the molecular abnormality genesis in lower forms of life and are of in sickle cell anemia was not clarified until in indicating [..ut biological importance 1957 (Ingram, 1957).Ingram, a ,voungbio- ihat the genetic code is rtniversal and ap,- chemist,worked in the intellectually stimu- plies to oieanisms as far apart as man and lating atmosphereof the Cambridge Labo- viruses. ratory of Molecular Biology. Here, Sanger Hemoglobin was founcl to be composed had establishedthe amino acid sequenceof of four polypeptide chains.Two of the four insulin. Crick and Watson had demonstrat- hemoglobin chains \t'ere different ancl were ed the double helix of DNA and Perurz clescribeclas the crancl 0 chains.Each chain ancl Kendrew were investigating the three- was founcl to be under control of a specific dimensional structure of hemoglobin and gene: the Hb crand Hb p genes-This find- myoglobin. By 1964, all these investigators irg has general significance fol protein had received Nobel prizes for their work. structure. Work with other proteins in re- By digesting hemoglobin and separating the cent years has shown that most proteins resultant peptides by various techniques, consist of several polypeptide chains and Ingram showed that the sickle hemoglobin therefore require more than one gene for molecule differed from normal hemoglobin their synthesis. The one gene-one protein by the substitution of only a single amino hypothesis has become the one gene one acid among almost 300 amino acids consti- polypeptide chain hypothesis. tuting the molecule. The stage was now set for a clear under- Abnormol Hemoglobins-Modelsof Molec- standing nature of the of mutational altera- ulor Diseose(Lehmonn ond Huntsmon,1966) tion of protein molecules. Later work with bacterial tryptoohane synthetaseby Yanof- Hemoglobin can be obtained freely from sky ancl his group (Yanofsky et al., 1964) blood and many investigators all over the

provided by the Maternal and child Health Library, Georgetownuniversity Vol. 3, No. 4, 1966 BIOCHEMICALGENETICS IN MEDICINE 361

\,vorldinitiated studiesancl slrrveyson hem- gotes hacl clinical symptoms. This hemo- oglobins. Within a few years, it became globin-Hb Chesapeake-was fotrnd to -+ clear that there was consiclerablebiochemi- have an arginine leucine substittrtion at cal variability of the hemoglobin.molecule. the 92nd amino acid resiclue of the o, Ilost hemoglobin variants were not associ- chain: 5 amino acid residuesaway from the ated with disease.If an amino acid substitu- heme-linked histidine of the o chain. Hb tion affects a portion of the molecule not Chesapeakeshowed a significally increased involvecl in its physiologic function (i... oxygen affinity in red blood cells as well as oxygen carriage), altered electrophoretic in the purifiecl state which presumably ex- mobility might result but if the stability of plained the polycythemia.Neither methem- the molecule is not otherwise impaired the oglobin nor increasedblood destructionwas altered hemoglobin may carry out its usual found in Hb Chesapeakepatients. Other function. However, a variety of hemato- casesof benign familial polycythemia may ogic diseaseswere found with amino acicl be causedby this type of abnormal hemo- substitutionsin the critical functional site globin. of the hemoglobin molecule (i.e. near the UnstableHemoslobin DiseasesAssociated insertion of the heme eroup). These include with Hemolysis (Huehns and Shooter, positions 5l-76 ancl 80-94 of the cr chain 1965):Occasionally an amino aciclsubstitu- and 57-76and 85-96ol the B chain. tion may not grosslyinterfere lvith function Hb M: (Geraldan

Georgetownuniversity Provided bY the Maternal and child Health Library, 362 MOTULSKY Ala. J. Med. Sci. boclieshave been detecteclparticularly after ing of sickle hemoglobin molecules (N,fara- splenectomy.The inclusion bodiesmay rep- yama, 1966). resent free o chains such as has been de- NIost other hemoglobin abnormalities tected in the hemolysatesof patients with have not produced any cliseasein the heter- unstable P chain hemoglobin anomalies ozygote state, presumably'because the mu- such as Hb Koln. Hb Seattle.and Hb St. tants neither interfered u'ith stability nor Mary's. In another abnormal hemoglobin function of the hemoglobin molecule. The of this sort-Hb Ube-l-the investigators reasons for blood destruction in patients believeclthat the abnormality consistedof homozygousfor Hb C, Hb E, ancl Hb D loss of reactivity of the sulfhyclrylgroup at diseaseis not yet apparent. position 93 of the B chain. The Thalassemias(\Iotulsky, 1964): In Hb St. Nlary's is :rssociateclrvith compen- this group of diseases,the senetic mutatioll sateclhemolytic cliseases.Although the spe- Ieadsto depressedsynthesis of a given hem- cific site of the abirormality has not been oglobin chain (i.e. a or B thalassemia)and identified yet, it is likely thar the substitu- causesreduction in the amount of hemo- tion affects the critical site of the hemo- globin in the red cells. Heterozygotesusu- globin molecule since the absorption spec- ally have only mild anemia but homozy- trum of Hb St. l\Iary,'sn'as abnormal, In gotesmay be severelyaffected. The genetic Hb Koln, the abnormality rvaslocalizecl be- mechanismscausing suppression of the hem- tween the 83rclancl 120th amino aciclof the oglobin genesare largely unknown. B chain (Pribilla et al., i965). However, one type of thalassemiacoulcl The more unstable the abnormal hemo- clearly be relateclto an unusual type of ab- globin, the more diffictrlt u ill it be to cletect normal hemoglobin-Hb Lepore. This l-rem- small quantitiesof the muranr hemoglobin oglobin is a fusion gene product consisting in the blood. Heat instability has been seen of both 6 and B hemoglobin chains. The E in most of the unstable hemoelobins and chain is a physioloeicgene product, differs may representa good screeningtest for strch by only l0 amino acid substitutionsfrom the disorclers.Unfortunately, tl-ristest u'ill not B chain, and forms the two non crchains of be infallible sincean innocuotrshemoglobin a minor normal hemoglobin: Hb Ar. The E abnormality-Hb ) Tacoma (pto arg gene is closely linkecl to the B gene. Non- serine) has also been associatedn'ith heat homologous crossing-overbetween the p lability (Baur and Nlorulsky,l965). and E gene producesa deletion of a portion Sickle Cell Disease;The reclcells of heter- of the 6 and B chains rvith formations of a ozygoteswirh sicklecell cliseasecan be made hybrid SB pol,vpeptide. Hemoglobin syn- to sickle in aitro but such carriersare quite thesisof the hybrid EBmolecule proceeclsat well under most circumstancessince more a reducedrate similar to that of the normal than 50% of their hemoglobin is of the 6 chain which onlv comprises 2% of the adult normal rype. With severe hypoxia, total hemoglobin and thalassemiaresults. such as at altitudesover 12,000ft.,|i uiuo Sincc hemoglobin can be easily obtained sickling may occur and splenic infarction and analyzed, the correlation of structure may be observed.The unusual circulation and pathologic function has progressedfur- of the kidney leads to hemoconcentration ther than for other geneticdiseases. Certain and relative hypoxia and may 'theproduce generalizationscan be made. Variability of renal sickling hemaruria in ab- moleculesis likely' to exist for other prorein ""a sence of structural renal abnormalities. and enz,vmes.Since mutations are rare mo- Patientswith homozygoussickle cell anemia lecular abnormaliti;s are likely to be rare. have no normal adult hemoglobin and will Occasionallya molecular abnormality may exhibit in uiao sickling with resultant orsan be of selectiveadvantap;e to its carriers and damageand chronic hemolytic disease.The its frequency u'ill becomesignificant. Since mechanism by which a substitution of a the sicklec:ll trait confersresistance against single amino acid produces severedeformi- falciparum malaria, the frequency of that ties of red cell shapeis believedto be srack- trait is relatively high in areasof the world

UniversitY Provided by the Maternal and Child Health Library, Georgetown Vol. 3, No. 4, 1966 BIOCHEMICALGENETICS IN MEDICINE 363 rvhere this type of malaria was endemic, ing G6PD cleficiencyexists among Nfediter- even though the homozygousstate was rela- ranean ancl African populations. The N,ted- tively lethal. The selective aclvantageof iterraneanmutation producesa more severe other genetic traits is less rvell understood type of enzymedeficiency with a wider spec- although malaria is likely to explain rhe trum of clrug sensitivity than seen among relatir..elyhigh frequency of the thalassemi- Africans. Recent studies in our laboratory as.When the activeor critical site of a mole- by Dr. Yoshida here helped to define the cule or its stability is affected,disorders o{ nature of the biochemical lesion in the Af- function may be expecteclsuch as various rican type of G6PD deficiency (A). When anemias, polycythemia and methemogtobi. red cells were fractionated by age, it coulcl nemia in the caseof the hemoglobin muta- be shown that very young cells from defi- tions. Physicianswill r"e -o.i of the dis. cient subjectshad almost identical enzyme ease-pro(lucingmutations. FIor,vever, if whole activity as comparedwith normals.Enzymo- populations are screened,benign and harm. logic properties were normal. Serologic ac- lessmutants will be more .o--o.r. tivity of mutant G6PD (A-) as assayeclby neutralization of antibody prepared against Glucose-6-PhosphoteDehydrogenose Defi- purifiecl normal G6PD also was normal. ciencyof the Red Cell-A Model for Inborn Column chromatographl', however, Errorsof Metobolism showed structural differenceslvhen the A- mutant Enzymesare proteins ancl the general les- was compared with normal and with the sons provided by hemoqlobin disorders electrophoretically identical rnutant with shoulcl apply to enzymatic abnormalities. normal enzyme activity (A+). These find- l\,Iutational effectsnright change the config- ings may be interpreted to indicate that a uration of an enzymeand distort the active structural mutation leads to more rapid site of the enzymeor interfere with the sta- degradation of the enzyme during red cell bility of the enzyme.The active site might agine. Since the normal enzyme is a hexa- be directly affected.Cofactors might not be mer, the processmay well relate to increased able to bind properly or subunit association subunit dissociation.In contrast,the Medi- or dissociation might be inrerfered with. terranean type of mutation was shown to Unfortunately, the amount of enzymewhich exhibit already diminished activity in young can be recoveredfor protein studiesis very red cells and exhibited g:osslyaltered sero- small so that basicknorvleclge regarding the logic activity. Its abnormal kinetic proper- nature of the biochemical abnormalities is ties as well as its chromatographic proper- lessadvanced than that of abnormal hemo- ties clearly establishthis mutant as another globins. However, enzyme deficiency has structural abnormality. been clemonstratedin many clisordersal- Other mutations are associated with ready. chronic hemolytic diseasein the absenceof Recl cell enzymesare more reaclily avail- any clrue or food adrninistration. Chemical able than most other material and more cle- stuclieshave shown that even in uitro a tailecl knowledge is available in this area. highly unstableenzyme is found in patients Abnormalities of G6PD are a sood exam- of this type. ple. GGPD rleficiency of the recl cell is a The relatively high frequency of G6PD heteroeeneousdisorcler. XIany different mu- enzymecleficiency in Africans and N{editer- tations may causealterations in G6PD ac- ranean populations appearsto be causedby tivity (Kirkman et al., 1961).Some mura- a protective advantage against falciparum tions changethe electrophoreticmobility of malaria (Motulsky, 1964).The discoveryof the enzyme and clo not causeenzyme defi- G6PD variations of the red cell illustrates ciency. They are clinically asympromatic. that abnormality affecting a single enzyme Others causeenzyme deficiencv but are clin- explained diverse types of hemolytic ane- ically harmlessunless certain ,lr.,g, or fava mias such as hemolytic diseaseof the new- beans are administered when l^emolytic born, clrup;induced hemolytic anemia, food anemia clevelops.A clifferentmutation caus- inclucecl hemolytic anemia, hemolytic

UniversitY Provided bY the Maternal and Child Health LibrarY' Georgetown 364 MOTULSKY Ala. J. Med. Sci. anemia by infection such as viral hepatitis chain production. In another type (Greek), ancl chronic nonspherocytichemolytic dis- there is only partial suppressionof B and E ease. Apart from G6PD deficiency, other chain production with smaller amounts of enzyme deficiencies affecting the red cell fetal (y) chain synthesis.The African type have been discovered to cause hemolytic could be explained by complete deletion of anemia. These include pyruvic kinase the adjacent B ancl 6 genes.This explana- deficiency, triose phosphate isomerasede- tion, however, is inaclequatefor the Greek ficiency and glutathione reductasedeficien- type of this syndrome ancl an operator rype cy. All of them are inheritecl as autosomal of mutation suppressingthe linkecl B and 6 recessivetraits. structure genes coulcl well explain both conditions. Control Mutotions in Mon? In orotic aciduria, two enzymes:orotidy- The discovery by Jacob and Monod lic pyrophosphorylaseand oroticlylic decar- (1962)of a classof mutations in microorgan- boxylase are depresseclto 20/o of their nor- isms causing interference with genetic reg- mal enzyme activity in the blood cells of ulatory elements rather than with structu- heterozygotes.Since the two enzymes are ral enzymeprotein has elicited much inter- closelylinkecl in E. coli they might also be est ancl was recently rewarded with a Nobel genetically linked in man ancl both be de- prize. If such mutations existed in man, pressed by a control type of mtrrarion one rvould expect severeenzyme or protein (Smith, 1966). deficiency.Since regulatory mutations affect In acuteintermittent porphyria, increased the function of linked structural genes,sev- E-amino levulinic synthetase activity has eral related enzyme or proteins might be been demonstratecl.A control type of muta- affected. The structure of the affected pro- tion has been postulated (Granick, 1965), tein or enzymewould be normal. If there is particularly since this enzyme can be in- enzyme deficiency,serologic testing should duced experimentally by the same drugs not show any cross reactive enzymatically which precipitate the human disease in inactive material. Increased,rather than de- genetically predisposedcarriers. Structural ficient enzyme activity also might be ex- studies on the human enzyme, however, plained by controller mutations although have not yet been carried out, ancl a struc- structural mutations at allostericsites of an tural mutation is not ruled out. enzymemaking it lessresponsive to inhibi- tion by small moleculeswould be an alter- Phormocogenetics-EnzymoticVoriobility os q Model for Diseose native explanation. Many inborn errors in Susceptibility man have been ascribed to regulatory muta- Several examples of genetic disorders tions but none have been proven yet. In which predispose to clrug reactions have several instances,where a regulatory muta- already been mentioned. Thus, Hb Zurich tion was postulated, more detailed study and G6PD deficiency of the red cell both revealed a structural mutation such as in predisposeto drug-induced hemolytic ane- the African type of G6PD deficiency cited mia. above. Increased pseudocholinesteraseac- It has become clear that some dnrg reac- tivity (Neitlich, 1966)in a family wasshown tions and a good amount of variability in to be associatedwith an electrophorerically responseto drugs may be causedby eeneti- demonstrable structural mutation rather cally determined biochemical differences than with a control mutation. between inclividuals (Kalow, 1962). Pro- However, three diseasesremain where a longed apnea following administration of goocl casecan be made for the existenceof succinylcholine is often caused by eenetic regulatory mutations. In one type of heredi- alterations of pseuclocholinesterase,the en- tary persistenceof fetal hemoglobin (Afri- zyme rvhich normally inactivates succinyl- can type), there is complete suppression of choline. Severalclifferent mutants affecting the aclult type ,B and I hemoglobin chains pseudocholinesterasehave been recognized. which is fully compensaredby fetal (y) The most common t1,peis found in 3-4lo of

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the population who are heterozygous for genetic differences can be expected. this trait and representsa strllctural muta- Liver alcohol dehydrogenase is mainly tion of the pseudocholinesterasemolecule. concerned with breakdown of ethyl alcohol. About l/3000 individuals are homozygotes. The finding of an atypical alcohol dehydro- A rare mutation Presentswith no enzyme genasewith higher in aitro enzymaticaffin- activity and is known as the silent allele. ity for alcohol in some human liver speci- Heterogeneity again has been founcl in sev- mens is of some interest (Wartburg et al., eral casespresenting with no enzyme activ- 1965).Carriers of this mutation should be ity. In some cases,cross reactive material more tolerant of alcohol than the rest of the could be found. Sera with cross reactive population. Many Japaneseindividuals re- material might represent examples of con- act with marked vasodilation to the admin- trol mutations but other explanationscoulcl istration of alcohol. The mechanismis un- alsoexplain thesefindings. Individuals who known and raises the question whether are homozygous for the different pseudo- vasoactivepeptides are releasetlin these in- cholinesterasemutations as well as mixed dividuals. heterozygotesfor two different mutations Drug resistance may be genetically con- (such as those carrying the enzyme for the trolled. Increased pesudocholinesteraseac- atypical common mutation as well as for the tivitv is a rare mutation and causessuc- silent mutation) were found to be drug sen- cinyiocholine resistance just as defective sitive. A simple screeningtest basedon dif- pseudocholinestereseactivity causes succi- ferential inhibition of the enzyme by en- nylcholine sensitivity (Neitlich, 1966). zyme inhibitors has been developedfor use Marked resistanceto the action of anticoag- in agar gels (Harris, 1963)and has been ex- ulant drugs such as warfarin and dicumarol tended for test tube use in our laboratory. has been observedas a rare mutation in one Both heterozygotesand homozygotescan be family (O'Reilly and Aggeler, 1965). detected. The examples of genetically determined Differencesin biotransformation of ison- drug reactions may serve as models for the iazid (INH) are causedby genetic differen- interaction of genetically determined bio- cesin acetylationbetween individuals (Mo- chemical variability with exogenous agents tulsky, 1964). "Slow inactivators" of the of "disease".The enzymeabnormality alone drug have high blood levels, lack an acety- is harmless as is the conventional dose of lating enzyme in their livers, and are homo- the drug. However, when the usual dose of zygotes.Individuals who are heterozygotes the drug is given to an individual with en- have intermediate blood levels of the drug zymedeficiency, a drug reaction or "disease" while homozygotes for the acetylating en- occurs.Neither the genetic abnormality nor zyme have low blood levels. Other drugs the drug alone cause difficulties. The ad- such as some sulfa drugs, Hydralazine and ministration of the drug to the genetically Nardil appear to be inactivateclby a similar susceptible individual however causesdis- mechanism. The frequency of peripheral ease. By analogy, other diseasesmay be neuropathy induced by INH is very much caused by such interaction of environmen- higher among slow inactivatorsthan among tal agents and genetic variability. In the rapid inactivators.The distribution of the more common diseases,however, more than various genetic classesis trimodal rather a single enzyme abnormality may be ex- than unimodal when appropriate tests are pected so that the constitutional basis of being used. There is a higher frequency of such diseasesmust be sought in the cooPer- individuals with the acetylating enzyme in ation of several genetic factors rather than Oriental populations. These findings are of in a single specific enzyme deficiency. general significance.Whenever a difference of drug responseis seenbetween genetically BiochemicolScreening for Inborn Errors of (Menkesond Migeon, 1966; rliffering populations and a cliscontinuous Metobolism Perry et ol, l%6) response is obtained when a sufficiently large number of inclividtrals are studiecl, The brain is highly sensitive to various

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metabolic clisturbancesin the growing in- synrp urine clisease.The principle of these fant. Many clifferent inborn errors of me- tests consists in suppression of growth of tabolism are associatedwith mental retarda- certain bacterial strains in the presence of tion. These include phenylketonuria, galac- some of the abnormal metabolites present tosemia,maple syrup urine disease,histidi- in the blood of affectecl patients. Simple nemia, goitrous cretinism, homocystinnria, urinary screening tests are available to cle- Hurler's syndrome,methemoglobinemia due tect a variety of these clisorclers (Perry et al., to cliaphorasedeficiency, hyperuricemia as- 1966). These tests can be clone in any clini- sociated with choreathetosisand self-de- cal laboratory and do not require special- structive behavior and severalother amino izecl equipment (Table l). Amino acid acidicurias.llfental retardation is also more screening can be clone chromatographically common in some other hereditary clisorders or with amino acicl analysers in certain lab- such as myotonic dystrophy, pseudohyper- oratories. Refore potentialll' harmftrl treat- trophic muscular dystrophy and neurofibro- ment is started, consultation rvith a special- matosis.Although theseconditions are not izecl laboratory is always inclicatecl. classifiecl as inborn errors of metabolism, Several years ago, we clevelopecl a simple the existenceof mental retardation sugsesrs screening test for G6PD cleficiencl' baseclon a more generalized systemic involvement reduction of the dye brillianf cresyl blue affecting the brain. (N{otulsky ancl Campbell-Kraut, l96l). The In some diseasessuch as homocystinuria test has been widely used for the cletection ancl methemoglobinemiadue to cliaphorase of G6PD cleficiency but alons n'ith other deficiency,mental retardation is only seen screening tests for this condition must be in some patients with the disease.The rea- used with caution during hemolytic epi- sons for the presenceor absenceof mental socleswhen the older red cell populations is retarclationare not clear yet but may be re- destroyecl ancl only )'oung red cells remain latecl to the interacrion of the metabolic in the bloocl. A normal screening test result error with other genespredisposins to men- may then be obtained in the presence of tal cleficiency(Fialkou' et al lg6b)- prophy- G6PD deficiencl'. A clye test basecl on the lactic treatment involving clietary manage- same principle has been clevisedby'Reutler ment is highly promising in phenylkeronu- for galactosemia and may prove helpful in ria and galactosemiaand theoreticallyhelp- mass screening. Beutler recentl,v also has ful in maple syrup urine diseaseancl homb- developecl simple fluorescence screening cystinuria.Early diagnosisis thereforeman- tests for the cletection of recl cell enzyme clatory so that treatment can be started as abnormalities which clepencl on conversion soonafter birth asis possible.Bacterial inhi- of the recluceclpvricline ntrcleotides DPNH bition assaysare availablefor phenylkeronu- or TPNH to DPN or TPN. ria, salactosemia,histiclinemia ancl maple It rvoulcl be unn'ise at this time to pass

TABLEI URIN-{RY SCREENING TESTS IN SO\{E INRORN ERRORS OF \IET.\ROLIS\I ASSOCI,{-TED \I'ITH \TENTAL RETARDATION

trtaple Phenylke- Homocysti- G alacto- Tyrosi- SyrttP Hurler's Urine Test tonuria Histidinemia nurla semta ne nlla Disease Syndronte Ferric Chloride ++ Test Cyanide Nitro- + prusside Test Benedict's Tcst + -+ 'IestN itrosonapthol + Dinitro Phenyl- -t + + -r hydrazine Test Cetyl Trimethyl + Ammonium Bromide Test

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laws regarding screening of all newborn Carrier tletection of autosomal recessive babies for a variety of biochemical genetic traits is genetically of great interest but diseases.Extensive studies need to be per- practically of less importance than detec- formed on an investigational basis to rule tion of heterozygotes for X-linked traits. out some of the difficulties which may be The clinically normal sibs of patienrs with expectedin this work. autosomal recessivedisease have a zft chance of being carriers. The chance that their Corrier Detection mates are carriers equals the population Population studies for gene frequencies frequencyof the heterozygoussrate (i.e.Zfs and the requirements of genetic counseling for phenylketonuria). Recurrence risk of make it desirableto have testsavailable for the diseasefrom such a mating is very low (% ll50 x r/a = l/300 for phenylketo- the detection of heterozygotes.If the en- " zyme or protein abnormality in a given nuria). Sistersof patients with X-linked dis- disorder is known, specific assayscan be easeshave a r/2 chanceof being carriers. If devised. In general, the enzyme level of they are carriers, Vz of their sons will be heterozygotesis 50fo that of normals (acata- affected-a much higher risk. Carrier detec- lasemia, methemoglobinemia due to dia- tion of clinically important X-linked dis- phorasedeficiency, galactosemia, pseudocho- easessuch as hemophilia and pseudohyper- linesterasedeficiency, pyruvate kinase de- tropic muscular dystrophy has made some ficiency, histidinemia) (Harris, 1963).This progress in recent years. Antihemophilic finding by itself is gooclevidence that regu- globulin levels and creatine phosphokinase lation of enzymeactivity occursat the gen- levels (as eviclenceof some muscle disease etic level rather than by feedback inhibi- in clinically normal heterozygotesfor mus- tion. Overlap of heterozygotevalues with cular dystrophy)can be assayed.Apart from the normal range is observed in most dis- the potential problems posed by isoalleles, orders where enzymeactivitv was measured. the effect of random inactivation of the X When a structural clifferenceof the enzyme chromosome (Lyon hypothesis) adds a fur- can be demonstratedas an essentialpart of ther complication to X-linked carrier detec- the analysis (i.e. electrophoreticdifferences tion (Stamatoya?nopouloset al., in press). or the differential inhibition exhibited by According to this concept, approximately pseudocholinesterasevariants) such overlap Vz of the maternal and Vz of the paternal X disappears.The explanation for overlap for chromosomesin a given female are geneti- normal and heterozygoteson enzyme activ- cally inactive. Depending upon the time of ity assayspresumably relates to the exist- inactivation cluring embryologic clevelop- ence in the normal population of several ment and depending upon possibleselective genetically differins enzymeswith variable mechanismsacting on thesecell populations enzymeactivity in the normal range. There in Jater life, the ratio of active maternal to is excellent direct proof for the existenceof paternal chromosomesmay not be 50/50 such isoalleles in the case of red cell acicl but nray range from ll99 to 99/1. Conse- phosphataseand highly suggesriveeviclence quently, if in a given casemost of the nor- from correlation studies in relatives for glu- mal genesare active and the mutant genes cose-6-phosphatedehydrogenase and pseudo- are inactive, test results of such a heterozy- cholinesterase.Other more indirect evi- gote will fall in the normal range. F{owever, dence suggests that the phenomenon of careful assayof a large number of known isoallelesmay be quite general. When a normals and obligatory heterozygotesshould heterozygote inherits a high capacity nor- help to solve some of these problems and mal allele from one parent and amutant allow probability staremenrs whether a allele with negligible acriviry from the given person with a value in the zone of other parent, the level of enzyme activity overlap belongs to the normal or heterozy- in the heterozygote may be similar to that gote population. Centralized specialized of normal individuals with rwo doses of laboratories will be required for this im- low capacity isoalleles. Portant work.

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Summory eote traits is possible. Ol'erlap of heterozy- sotes ancl norrnals may be causeclby the ex- l. The history of human biochemical istence of isoalleles in the normal popula- senetics is reviewed with special references tion. Screenins of heterozygotes of X-linkecl to Garrorl's ancl Pauling's contributions. cliseases (muscular rlystrophy and hemo- 2. Recent knowledge on the correlation lrhilia) is practically more important than stntcture is of gene structure ancl protein screening Ior heterozvgotes of autosomal reviewed. cliseases. 3. The infltrence of various nltltat.ions on REFERENCES Hct- the hemoglobin molecule is cliscussecl. Ilarrr, L,. \\'. and A. G. ]Iotulsky. 1965. Hemoglobin Taco- erozygote structural mutations affecting the ma-A 8-Chain Variant Associated u'ith Increased Hb .4.,, Humanger-retik, l: tj2l -ti34. (larachc, critical active site of the hemoslobin mole- S., D. J. \\'catherall and .f. B. Clegg. I966. Polv- c'r'themia Associatcd rvith a Hcrnoglobinopath,v. J. Clin. cule may lead to hemolytic anemia, clrtrs Invcst.'15:813-822. I:ialkorv, I'..1.,.1 . A. Ilrorr'der, R. S. Sparkcs and A. G. sensitivity, polycythemia ancl methemoglo- \lotulskr'. l{)tiir. \Icntal Retartlation in llethcmoglobine- harm- nria I)rrc to Diaphorase L)eficiencl. N. E. J. llcd.273: binemia. Other mutants are trsually tt-10-u45. Iess unless they interfere rvith the ph1'sio- ()arrorl, ,\rt hibald F.., Inhorn Errors of trIetabolism, Oxford [.-nircrsitv I'rcss (l.on

provided by the Maternal and Child Health Library, Georgetown University Vol. 3, No. 4, 1956 BIOCHEMICALGENETICS IN MEDICINE 369

-f -fh. I't'rrv, . L., S. Hansen ancl L. llar:l)ougall. 1966. tlrinarv Stamatoyannopottlos, G., I'apalantroporrlott, Chr. Rako- -fcsts Slrcenine in thc Prcvention of \lcntal f)eficiencv. poulos and A. G. llottrlsky. Detection of Gltrcose-6-Phos- Clan. lIcd. Asso. I. !,)ir: 89-9ir. phatc Dehydrogcnase Dcficicnt Hctcrozygotes, lllood, ilt I'ribilla, W., P. Klessc, K. Betkc, H. Lehmann and D. J)rcss. Wartbtrrg, J. von I'apenberg and H. Aebi. 1965. Att Ilcalc. 1965. Hacmoglobin-Koln-Krankhcit: Farnilare hv- J.P., Atlpical Human Alcohol Dchydrogcnase. Can..T. Rio- po<.hromc haemolytischc Anamic mit Hamoglobinano- chcm. 43: 889-898. ntalie, Klinischc Wochcnschrift. 43: 1049-l0ir3. \\Iatson, James I). 196-o. trIolecular Biology of llte Cene. \\!. Smith, t.. H.Jr., O. trf. Hugulo, Jr. and J. A. Bain. 1966. A. Ilenjamin, Inc. (Ncrv York). Hereditary Orotic Aciduria in Tlte i\Ietabolic IJasis ol Yanofsky,-C., B. C. Carlton, J. R. Guest, D. R. Helinski aIr

r----- provided by the Maternal and chitd Health Library, Georgetownuniversity Cleft Lip and Cleft Palateln lceland*

Polmi Moller,D.M.D.r ond JohnDunbar, D.M.D., Dr. P.H.2

Although I rvill report specificallyon tl-re for most part. Out of approximately three occurrenceof cleft lip and/or cleft ualate hunclred specimens, only six showed evi- in Icelariders,I would like first to introciirce clence of caries. Of these only one showecl some background concerning the Univer- modern enamel type caries. Periodontal sity of Alabama lleclical Center's associa- cliseaseof severe form rvas quite lvide spread tion r,l'ithresearch in that country. as eviclencect by bone resorption arouncl In l96l the National Institute of Dental the teeth. Heavy calculus cleposits were Research awardecl the University of Ala- common. Ftrrther evidence of the solid Ice- bama School of Dentistry a grant to sup- lanclic teeth rvas provided by an Irishman port a survey of selectedoral conditions in visiting there in 1862.* Iceland. Icelanclrvas considered to be ideal for studies with possible long-term follow- Dried fish is the most extensivelvusecl article of food; and ling and cod are the most desired up implications. The country is small in sorts.It is impossibleto eat it until it hasbeen area and its 185,000population is fairly rvellpummelled on a stoneanvil, with a sort of accessibleand highly literate. The exisrence sledgehammer,formed by a round stonewith a of a population registry, in which every hole drilled through it for the har-rdleto pass tlirough; but even aftcr this severeorcleal, it Icelander is listed by name, birthdate ancl reqtriresIcelandic teeth and skill to eat it. address,was most encouraging. Not only could direct sampling procedures be insti- \\'ith this backgrouncl, Dr. Joseph F. Vol- tuted, but the registry would be exceedingly ker (the principal investigator), Dr. Palmi useful in locating follow-up casesin later Iloller ancl I visitecl Iceland in lg6l. Full years, in locating persons rvith knolvn cooperation from the Icelandic schools of cliseases(such as cleft lip or palate) and in medicine and dentistry and frorn public locating family members for studying health off icials \\'as assured. Dr. Moller familial frequency of an anomaly (such as cornpletecl a study of dental diseasesin pre- rnissingteeth or torus formation). school children in the summer of 1961.The The study of caries and periodontal str-rdy permitted valuable contacts to be cliseasewas felt to be especiallyinreresting rnacle with physicians and dentists arouncl in the light of severaladditional facts.The the country. In the period February skull collectionsof early Icelanders,both at through December, 1962, the survey of oral Peabodymuseum and the University of Ice- cliseasesof Icelanders five years old ancl land, include good specimensof teeth and older r\,'ascompleted. Of a total sample size jaws dating from before the year 1000A.D. of 3,916 (taken from the population registry (settlementtimes) to the middle of the lgth using a randorn system),3,451(88.2 percent) century. I examined the University of Ice- persons were located and examined. land collection and observed sound teeth In the summer of 1963 Dr. Palmi Moller undertook a preliminary study of the inci- *O.. given by Dr. John Dunbar for Dr. Palmi ilIoller who was continuing his work in Iceland at the time of the clence of cleft lip and palate in Icelancl. International Seminar on Medical Genetics. Universitv of Medical Center, Birmingham, Ala., September I, The objectives of the summer study were to fJAS:-" r Associate Professor of Dentistry. Universitv of Alabama cletermine the completeness of reporting of School of Dentistry. 2 Assistant Dean, University of Alabama Medical College and School of Dentistry, Coordinator of Research Grants, *.On..d, C. W., The Northwest Peninsula of Iccland, Profersor of Dentistry. Longmans, Green and Co., London, 1867.

Georgetownuniversity Provided bY the Maternal and child Health Library, Vol. 3, No. 4, 1966 CLEFTLIP AND CLEFTPALATE IN ICELAND 371

theseanomalies and if possible,to estimate The average weight at birth, holr,'ever, \,vas their ratio to live births. The feasibility of a little lower for the cleft group. gathering pedigrees in selected caseswas Looking at the distribution of sexes by also to be determined. This study and sub- rnorphologic type of cleft, the Icelandic sequent cleft investigationshave been sup- data is in agreement with that of most other ported by the National Institute of Child places. Males have clefts of the lip more Health and Human Development. than twice as frequently as females. Females The results of the preliminary u'ork en- experience isolated clefts of the palate couraged us to try to establish pedigreesfor more often than males. The overall inci- every living Icelander. This aspect of the dence of one affected birth in every 485 project was begun in the summer of 1965. live births is the highest rate reported for Dr. Moller has just returned to Iceland and a Caucasian nation. will spend a year there to complete the col- In the current phase of study, begun in lection. the summer of 1965, emphasis is placed on The data to be presented in this paper compilation of pecligrees. A family history are the result of theseinitial stucliesby Dr. questionnaire was designed and used in Moller. He has published one paper in the interviews with the parents of the patients. Arch. of Oral Biology (1965)and presented They were asked to identify by name, age another at the 1966annual meeting of the and address all close relatives. Close rela- International Association for Dental Re- tives refers to several generations of the search.Please bear in mind, then, that I am patient's family, including grandparents, presentingthis r,vorkat Dr. N,Ioller'srequest parents, children and brothers and sisters. becausehe is out of the country. It was possible in the summer of 1965 to Icelandic law requires the attending phy- establish pedigrees for 80 of the then knor,r'n sician or midwife to report the presenceof 108 affected cases. birth anomalieson birth certificates.These Of the B0 cases so far completed, 19 or recordswere the first to be scrutinized dur- almost one-fourth had close relatives u'ith ing the preliminary phasein the summer of similar anomalies. A total of 3,025 close I963. It soon became apparent that there relatives \\,'ereidentified. Among them were was incomplete reporting of oral clefts on 28 affectecl persons, a ratio of I affected case birth records, particularlv where the cleft for each 108 close relatives. Parenthetically, dicl not involve the lip. To test the possible Dr. \Ioller \vas in contact lvith more than reliability of birth plus maternity hospital 2,400 of these relatives, the remainder of recordsas a sourceof cornpleteinformation, lr,hom were not available for intervierv. seventy knorvn cleft cases were checked In the limited material collectecl in the againstthis dual source.Then surgical hos- summer of 1965 there are striking cliffer- pital recorclswere added. The combined ences in the frequency of oral clefts among sourcesresultecl in essentiallycomplete re- the close relatives on the paternal side, on porting, the maternal side and in chilclren of af- To check the incidence of oral clefts fectecl cases.Approximately one out of each during the period, .1956-1962,all records 36 children of affected cases were them- were coverecl.A control group r,r'asselectecl selves affectecl. One out of 86 close relatives by taking the child born next in the hos- on the maternal side n'ere affectecl in con- pital after the affectedcase. Family histories trast to one out of 327 on the paternal sicle. were taken for 65 of 68 affected children A high degree of similarity of cleft types born in the periocl 1956-1962and for 50 betr.veen patients and their affected close control children. Familial associationwas relatives rvas observed. For patients with t found in 46 percent of affected patients cleft lip, whether or not there was palatal and in 7 percent of the controls. involvement, over 90 percent of their af- No striking associationwas observecl with fected relatives had the same type of cleft. maternal age,birth order or seasonof birth. Only 42 percent of the affectecl relatives of

university Provided bY the Maternal and child Health Library, Georgetown 372 MOLLERAND DUNBAR Ala. J. Med. Sci. isolaterl cleft palate cases hacl the sarne DR. DUNB.{R: I trttst that Dr. Moller and l)r. cleft type. [dwards already hal'c tnet'in Iccland and discttsscd this point and othcr common interests. In closing may I add that the results DR.J. A. FRASER ROBERTS: I have been verv given are preliminary. Dr. X,Ioller'swork interested indeed in this paPer. Wc have had some cluring the year ending in the fall of 1967 comparable studies going at the Hospital for Sick the first qucs- shoulcl complete the cornpilation of pedi- Children in London. If I understood tion right, our findings are not quite the sanrc. gl-eesfor virtually all affectecl persons in Where there are two or morc rclatives af fectecl the cotrntry.Arrangements are being made other than the sibs,we find that in about a quartcr to keep the collection trpclaterl in ftrtrrre of the cases, these affected relatives are on both yezrrs. siries of the family. That's one finding and tltat idea that harelip, with ------*- seemsto negate the original or rvithout cleft palate, could be dtre to a recessivc gene of low penetrance. l)R. DAVID SNttTH: As part of anothcr study on DR. DUNBAR: \\'e tnttst itrclttde 1'ott in thc tlrc associationof malformation u'ith cleft lip and/or correspondence, Dr. Roberts. cleft palate, I had an opportunitv to inten'ierv fiftr DR. HANS ZELL\\'IEGER: Horv frequently in families in rvhich there rvas cleft lip arrd/or clcft vour casesdid ,vou find association with other tnal- palate, and in 48 percent of these therc was a posi- formations? I think just to talk about cleft lip antl tive family history of other individuals with the cleft palate is overly simplifying. We know that anomaly. In those families having rnore than onc some of the chromosomal aberrations show cleft lip family member affected, it was alwavs on one sidc and cleft palate. \{re have thcn the definite auto- of the family-the paternal or maternal. I n'as curious somal dominant condition rvith the labial pit, and in this study if there u'as more than one affectcd then the associationwith other malformations which individual beyond the propositus. Were the affecte

provided by the Maternal and Child Health Library, Georgetown University GeneticStudiesln HumanDental Cariesr

SidneyFinn, D.M.D.2

The teeth ancl associatecl:rrches provide this estimation is foo high ancl that hcrcclitv cxcellent material for genetic study, primar- plays only a minor role, if any. ilv because they are visible to the investiea- The evidence that hereclity plays a role tor, are morphologically different, and are in clental caries initiation ancl/or progres- rigicl enough that they can be measured sion has been derived mainly from the fol- accurately in all climensions. The excellent lowing three types of evidence: l.) animal morphologic studies of Lunclstrom, (1948) stuclies, 2.) human family studies, and 3.) Dahlberg, (1962) Osborne ancl co-r,r'orkers, human twin studies. Each of these sources (1958) ancl Garn, (1957) among others, ;,1, of eviclence will be cliscusserlbrieilv. test to the thoroughness with which liris Animol Studies: rnaterial is being utilized. The hererlitarv By selective breeding of rats through aspectsof human clental caries,unfortunatc- many generations, caries resistant ancl caries ly, are much more complex because clental susceptible strains of rodents have been cle- clecay is a multifactoral cliseasedepenclent velopecl. That these strains breecl geno- upon l.) a susceptible tooth surface to typically true has been determinecl in the caries attack; and 2.) a conclucive environ- following manner: a.) By placing caries ment depenclent upon both extrinsic ancl resistant or caries susceptible strains of rats intrinsic variables. Borh the teeth them- with genotypicaliy opposite mothers rrntil selves and the environment of the teeth weaning, they have founcl that the caries can be influenced bv hereditv. This is well scores remained the same as the genotypical illustrated by the total lack of caries cler.el- strain from whence they were born; b.) By opment in thc clefective teeth of inclivicluals depressing the oral flora of carics resistant with amelogenesis imperfecta or the raml)- ancl caries susceptible strains of youns rats ancy of caries associated rvith congenital with penicillin, then inoculating these ani- agenesis of the salivary glancls. The first mals with feces from both strains o[ rats, cliseaseis clue to a defect in tooth structure; the subsequent caries scores were typical of the latter is clue to a moclificarion in the their own genotypic strains; c.) By cross- oral environment. Both are genetic:rllv in- fluenced. mating genotypically opposite mothers ancl fathers, the subsequent caries scores of the Since dental caries is multifactorai anci offspring fell between those of both certainly polygenic, it becomes clifficulr l)arent pure-brecl strains. therefore to assessthe relative irnportance For further ancl more complete informa- of heredity in the disease etiolog,v. Dahl- tion, the reader is referred to the works berg and Dahlberg (1942) arremprerl ro of Hunt ancl Rosen, (1961 and 1962) Shaw, assess this relative importance from tr,vin (1960ancl l96l) and Konig (1965)anrl their studies and concluded from statsticai cteduc- co-workers, among others. tion that heredity ar mosr was equally as important as the environment ancl \,vas at Humon Fomily Studies: the least half as imporrant. ()thers feel rhat These studies have offered reliable evitl-

t,O=n ence that there is a genetic component. at I ntcrnational Senrinar on lledical Cenetics, Uni'r'ersity of Alabama N{edical Center, Rirmingham, Sep- Among the investigators in this fieicl rnust tcmber I, 1966. 2Professor of Dentistrv and Chairman, Department of be mentioned Klein (1946 and 1917) ancl Pcdodontics, University of Alabama I,Iedical Center School of Dcntistrr'. co-workers, (1938 and 1940) ancl lli;i)k &

b .-.- university provided by the Maternal and child Health Library, Georgetown 374 F|NN Ala.J. Med.Sci.

Grahnen.(1953) been undertaken to determine whether Klein in 1940 dividecl 4,416 unselected caries resistance is genetically inf luencecl. school children into groups that had had Only a few studies, unfortunately, have no cariesexperience and those that had six applied statistical analyses to their observa- or more decayed,missing, or filled teeth at tions, ancl onll' a ferv have determined ten years of ?ge, allowing an additional zygosity serologically'. Although a large carious tooth for every increasein year of number of studies have been conducted, the age. The siblings of the 148 immune chil- discussion in this paper is limited to those dren had lower average caries scoresthan which, it is felt, offer reliable statistical the siblings of the susceptiblechildren. In evaluations. a later study among 5,400 personsof Japa- Bachrach and Young, 1927, founcl slight nese ancestry, he found that the siblings differences in correlation coefficients of definitely reflected the cariesscores of their caries prevalence in teeth of 130 pairs of parents.He alsofound in a natural fluoride monozygotic twins and l7I pairs of like area that the children of parents of either and unlike sexed dizl'gotic twins. These high or low caries susceptibility reflected differences, lvhich favorecl the identical the cariesexperience of tireir parents.Since twins over the like-sexed fraternal twins, both groups had the benefit of increased were not statistically significant. They con- fluoride and thus more resistant teeth, yet cluded therefore that there were no dif- showed marked differences in degrees of ferences benveen the nvo groups and that susceptibility, it rvould appear that the therefore genetics pla;'ecl onll' a very minor fluoride content of the tooth surface may' role in this disease. not be the phenotypic expressionfor caries Dahlberg and Dahlberg in 1942 found resistance. in their study of 37 pairs of monozygotic In the exacting study by Book & Grah- twins and 99 pairs of like and unlike dizy- nen (1953), they selectedforty completely gotic twins that 24 per cent of the identical caries-freemale conscriptsfor militarl' sen'- twins and 28 per cent of the like-sexed ice and examined 162 related indivicluals. fraternal twins were discorclant, but these These propositi were compareclto a group differences clid not attain significant pro- of 92 relatives of. 23 individuals selected portions. Thel' felt, holvever, that there from the same military conscripts. Thel' was a genetic influence. concluded from a thorough analysisof the Osborne, Horou'itz, ancl DeGeorge, 1951, data that there was a genetic factor or fac- utilizing the tooth surface as the unit of tors which apparently determined the re- measurement in contrast to the entire tooth sistanceor susceptibility to dental caries, unit useclin the two previous studies, found since parents and siblings of the caries-free a significant difference in caries ratios be- aclultshad a significantly lower cariesindex tween the 30 monozygotic twin pairs and 19 than clid the control group. It must be clizygotic tH'in pairs. Thel' concluded that noted that related families may have the identical twin pairs \r'ere significantly more same diet and foocl clislikeswhich might alike in caries experience than dizygotic account for some of the familial caries twin pairs. scores. Mansbridge, 1959, usinp; fingerprints and These studiesoffer information that there physical characteristics to identify the type is a genetic component but offer little of tn'ins, found that the 96 pairs of mono- evidence as to the factor or factors con- zygotic nvins shorvecl greater similaritf in troiling this expression. caries experience than the 128 pairs of like- sexecl clizygotic twins ancl that either twin Humon Twin Studies: type had FTreatersimilaritv than unrelatecl The twin study method first suggested paired children. by FrancisGalton in 1876offers a fruitful Goodman and co-n'orkers, 1959, used techniquefor evaluatinggenetic influences serological testins to establish zygosity in and a fairly large number of stuclieshave their study, as did Finn and Caldwell, 1963.

university Provided bY the Maternal and child Health Library, Georgetown Vol. 3, No. 4, 1966 GENETICSTUDIES IN HUMANCARIES 375

TABLE 1 '[OO]-H 'I\VINS ABSOLUTE DI\IF AND SURFACE VARIANCE BETWEEN NIO,r-OZYGOTIC AND DIZYGOTIC TWINS AND A COMPARISON WITH U.\RELA'I'ED CHII,DREN

DAIF incisors DMF lirst molars (permanent) DtrlF incisors and first rnolars Ase ) 8 years Age ) 7 Years Age ) 8 years Teeth Surfaces Teeth Surf aces Teeth Surfaces Monozygotic (Vr) 0.56 0.82 0.60 4.48 I .16 5.30 Dizygotic (V1) t19 4.98 1.00 9.58 3.22 14.56 F Value 3.96 6.07 1.66 2.t3 9i7 9J4 Confidence level 0.000r 0.0001 0.10 0.025 0.005 0.005 Monozygotic 0.56 0.82 0.60 4.48 l.16 5.30 Unrelated 3.r2 5.96 1.56 20.58 4.68 25.61 F Value 5,5 I 7.26 2.60 4.59 4.03 4.83 Confidence level 0.0005 0.0005 0.01 0.0005 0.0005 0.0005

In the Goodman srudy using 19 monozy- been macle, but a great cleal of work re- gotic and 19 dizygotic twin pairs there was mains to be done, ancl encouragementin a significant differencein intrapair variance this field is certainlv desirable. with the identical twins showing less dif- REFERENCES

ference.The study thar Finn and Caldwell Bachrach, F. H. and NI. Young. 1927. A comparison of reported using 35 of the degree of resemblance in dental characters shown in sets monozygoric and pairs of twins of identical and fraternal types. Brit. Dental 3l setsof like-sexeddizygoric twins, having J. XLVIIT, 1293-13M. Book, J. A. and H. Grahnen. 1953. Clinical and genetical a mean age of l0 years, the calculated co- studies of dental caries. Il. I'arents and sibs of adutt highlv resistant (caries-free) propositi. Odont. Revv. 4:l-53, No. l. efficients of variance indicated statistically Dahlberg, A. 1962. Genetic aspects of evolution of the human dentition. Genetics and Dental tlealth. Neu' York: significant differences between the mono- NfcGraw Hill Book Co., Inc. Chaptcr 10, pp ll3-121. zygotic and dizygotic rwin Dahlberg, G. and B. Dahlberg. 1912. Uber Karies und pairs and an andere Zahnveranderungen bei Zrvillingcn. Uppsala even greater variance between the monozy- Lakerf. Forh.47:395. Finn, S. B. and R. C. Caldrvell. 1963. Dental caries in twins €iotic twin pairs and pairs of unrelared -1. A comparison of the caries erperience of rnonoz\'- gotic twins, dizvgotic twins and unrelated children. Arch. children. There is a statisticallysignificant Oral Biologv. 8:571-585. intrapair difference in the Galton, Francis. "The History of Trvins As A Criterion of caries scoresin the Relative Powers of Nature and Nurture," Journal of the incisor tooth surfacesbut not in the Royal Anthropological Institute, 5:391-406, 1876. those Garn, S. and A. Lewis. 1957. Relationship benteen the of the first permanent molars. When molars sequence of calcification and the sequence of eruption of the mandibular molar and premolar teeth. J. Dent. Re- and incisors are combined, the differences search.36:992-995. Goodman, H. O., J. E. Luke, Samuel Rosen and Emanuel are significant. The variance between rhe Hacket. | 959. Heritabilin of dental caries and some monozygotic related salivary components. J. Dent. Research. 38:662. twin pairs and unrelated chil- Klein, H. and C. E. Palmer. 1938. Studies on dental caries: clren is highly significant. familial resemblance in caries experience of siblings. Public Health Report (\fashington), 53:1353-1364. Klein, H. and C. E. Palmer. 1940. Dental caries in brothers It must be remembered that although and sisters of immune and strsceptible children. N{ilbank like-sexed twin pairs reside in the l\Iemorial Fund Quarterlr,, I8:67. same Klein, H. 1946. Dental caries (DI{F) experience in relocated homes and have the same environments. children exposed to $ater containing fluorine. II .J.A.D.A. 33:1136-ll4l. unrelated children not only have a differ- Klein, H. 1947. Famih and dental disease, caries experience among parents and offspring erposed to drinking water ent inheritance but also have a different containing fluoride. Public Health Rcport (Washington) environment, 62:t247-t253. which probably accountsfor Konig, K. G. 1965. Caries resistance in erperimental animals. a large per cent of the intrapair differences Caries-Resistant Teeth (A CIBA Foundation Svmposium). London: J&A Churchill, Ltd. in this group. Lundstrom, A. 1918. Tooth Size and Occltrsion in Tu'ins. New York: S. Karger. There therefore appears to be a genetic Nfansbridge, J. N. 1959. Heredin' and dcntal caries. J Dent. Research 38:337-347, influence in the suscepribilityor resisrance Osborne, R. H., S. L. Horowitz and F. \'. DsGeorge. 1958. Hereditary factors in tooth dimensions, A studv of the to dental caries.Whether these influences anterior teeth of twins. Angle Orthodont. 28:87-93. Rosen, S., H. R. Hunt and C. A. Hoppert. lg6l. Importance act tirrough salivaand the oral environment of the genot-vpe on susceptibilin' to dental caries in the or through the morphology or location rat. J. Dent. Research 40:352-354. of Rosen, S., G. T. Coleman, A. C. Sawant, H. R. Htrnt and the teeth in the oral cavity or rhrough their C. A. Hoppert. 1962. Effect on caries of cross-breeding caries-resistant and caries-susceptible rats. J. Dent. Re- chemical or physical structure or both has search 4l:1033-1036. Shaw, J. H. and D. Griffiths. 1960. Partial substirution of not been determined as yet with certainty. hexitols for sucrose and dextrin in caries-proCucing diets. Attempts are being J. Dent. Research 39:377-384. made to ascertainwhat Shaw, J. H. and D. Griffiths. 1961. Relation of protein, the genetic pathways are. A beginning has carbohydrate and fat intake to the periodontal syndrome, .f. Dent. Research 40:614-621.

-r-- provided by the Maternal and child Heatth Library, Georgetownuniversity SomeOld and New Data on the Geneticsof HumanPopulations1

L. L. Covolli-Sforzo,M.D;)

goocl llr. Chail'n]:rn, Laclies autl Getttletnetr, I out, keel)irrg lttltl exllitttclirle the tltrcs n,oukl like to start by thanking mv hosts rtntl strl-,irressinsor limitins the clifftrsion of lor givine me the opl)ortunity to visit this the bail ones. \\'e knorv that isolation gives very' interesting city antl Universitl', antl I chances I'or incleltetrcleuttleveloprnent, ancl hope vou rvill not mincl if I hr sit s. Pavia Scction, Istitttto di Gcnctica, Utlivcrsin of I)lvirt, gories o[ genetic tntits: the t'are ones :tncl I'ar,ia. Itah. -flris t':tt'cones arc trstrally ACKNO\VI-EDGL,IIENT: uork h:rs bcetr sttpportc

university provided by the Maternal and child Health Library, Georgetown Vol.3, No.4, 1966 GENETICSOF HUMANPOPULATIONS 377

the earth if we look at large areas but as trrre at a given time, a cross-sectionin time soon as we look at small areas we find that of an evolutionary process. Second, other they are heterogeneously distributed. This polymorphisms are not changine in time; fact is essentially, one may say, a statistical they are seemingly stable, or as we also call artifact, as it is a consequence of drift. The them, "balanced," and therefore there must reason is very simple. When a mutant arises, be some factors that keep them still. it may leave no clescendants at all, it may It is useful to put some of the best-known leave one, or more, at random. These again polymorphisms on a suitable scale to show may have, by chance, zeto, one, few, many hou' much these polymorphisms change clescendants. In any case the final fate is from place to place. When one tries to rep- usually extinction. But before extinctior:t resent polymorphisms, one usually gives a there may be a rise, especially for mtrtants n orlcl map rvith isophenes (ot curves of that are not very aclversely selectecl. If the equal frequencies of phenotypes). I find it mutants are at an aclvantage they will al- very difficult to get a really good measure most certainly increase, but this lvill hallllen of the variation from such a world map. seldom. The frequency of a trait is the ratio For a s1'nthetic measure of variation, one between the number of bearers of the car-- shoulcl preferably use variance. But it is rier ancl the total number of individuals in then convenient to stanclardize it so that it the population. Such chance fluctr-ratious can vary betlveen zero (for complete homo- rnay sometimes increase the nttmber of geneity) ancl oue, as the nraximum that the bearers, but what is likely to make a trait variation can attain, irrespective of the frequency especially high is when the cle- mean frequencies. One thus obtains a sort r-totirittutor is small and the numerator is of scale on which one can comPare the not zero, as it happens in many small iso- variation of different polymorphisms. latecl populations. So one may say that a Here one fincls that the ABO blood .otr..r"tttition of rare traits in some small sroups are among the least variable, in communities is almost a statistical artifact; ro-e contradiction to what has been said it is often, in any case, the consequence of a in the literature so far. N{NS bloocl groups also are not very variable. Amons Rh blood ^purell' statistical process. Polymorphisrns-But if rve look at thc sroups there is one allele (Rh,) which is frequent traits, ancl by frequent \'ve mean hiehi)' r'ariable and others which are less not less than l% (formine the so-callecl voi'iolrl", and there are other variable Poly- polymorphisrns), \t'e ma)' fincl verl' consider- rnorphisms-Gm haptoglobins, Diego, PTC iUt" aifferences between populations. Polr'- (phenr'1thiocarbamicle tasting) Lervis, Duffy. morphisms have been investigatecl a great Unfortunately', in spite of much work by rleal but we still knolv little about thenr. verv man,v people, the knowletlge of the \\re do kno\\,, horvever, that there are very causes behincl these polyrnorphisms is very many polymorphisms. In fact, some recent lirnitecl ancl there have been alnrost as many inveitigations ln enz)'mes strssest that if clon nfalls of explar-rations as llew theories' you take zlr1rsttr'trte and s[tldv it ver,v care- The onlv thins lve knorv is th:rt ABO and fully, you fincl that it is almost alwal's poll'; Rh sive rise to incompatibilitl' phenomena, morpl'tic, or at least in a high proportion of as is lleil knolvn to the clinician' Ancl we cases. Therefore we mttst think there is a also knol' that this is not a strfficient expla- very great cleal of genetic cliversit,v,some of nation. In fact it cannot sive rise to a stable rvhich we carl see. \Ve ma)' then ask, rt'hy polvnrorphism; on the contrary it shourlcl are inclir,'itluals clifferent frorn one another ,lettrof ii. I must atlcl that thc ABO Poly- in this \l'a\', an(l wh1' arc these clifferences rnorphisrn seems stable in the sense that it clillerent {rom one place in tl-re 'lvorlcl to is not /oo variable, but it is variable, of another? There are essentially t\\'o reasons corlrse, from one PoPulation to another. It why this happens. First, evel'y species is is interestins, however, that the stability of evoh'ing ancl so are \\,'e,ancl therefore some some of these polymorphisms shown here of these polymorphisms are simply a pic- :rt worlclwicle levels for the httnlan species

- ^ UniversitY ProvidedbytheMaternalandChildHealth Library, Georgetown Med. Sci. 378 L. L. CAVALLI-SFORZA Ala. J.

one frorn is alscl valicl at a wider level. In fact, in the populations comltletely seParate(l Prirnates r.t,e find some sort of PT'C and the other, of size N, the amount of variation is .\BO polymorphisms. One cannot say to- rvhich can accumulate because of drift I of cl:ry, htlvever, if the alleles recognizecl in a fraction of the time available, ancl how much Primates are itlentical to those that can bc population size. We clon't know founcl in man. Anyhow, it suggeststhat the time has been available for accumulating poly,morphisms that are more stable have the observecl clifferences between ethnic iurvivecl for tens of millions of years and so groups in man, but some anthroPologists lnust have a stabilizing factor. Others are woukl accept a million )rears even. There- of tl-re perhaps more transient :rncl they may be fore rve can see that at least some thote- that are variable over the r','hole variation ma,Y have been rleterrninetl bi' rvorlcl. Unfortunately, toclal', r've can only clrift, even if the ltoptrlation size is as high nrake these vague statements, but theoreti- as ten thousancl. Unfortrtnately, holvevet, ltse calh'lr,e knorv that for a stable equilibrium u'e clon't kncxv rvhat fienre lt'e shoulcl lreterozy'eotesmust be, at least r-irioit(ro-g(, for N, especiallv since \ve knolv so little at all aclvantage over hotlrozl'eotes. As lt abor,rt the more ltrimitive populatiotls ancl ol happens, holvever, not all the finclings on abotrt rrhat liallpenecl irl the past historl' heterozrgote advantage nith resl)ect to anv l]1alt. /li's- of tl-rese polymorphisrns have beeu con- Rcconstrttctiort of tltt: cuoltttionary b:en firnrerl. The only case in rrhich we are still tory,of ?tlatl.One of ottr itltercsts has reasonably sure that there is heterozvgotc that of exploiting, in as ratiollill a tn:tuttr-'t- tlif- :rclvantage in given conclitions is that oI as possible, the kno'lvleclse on existine tht sickle cell anernia, ot't u,hich mttch has beeu ferinces in httmau grotlPS so as to trace histor'1' of rnall. This has le- said to )'ou in this s1'mltosittm by Dr. evolutiouan' Ilotulsky. rluirecl the creation of sevet'alnew methocls; Drif t-IJnfortunately lve clon't knorv for instance, one is clesienetl to lreastll'e mnch about the reasous lr,ht' polymorphisms svnthetically' the senetic clistauce, being are there ancl we can only make some baseclon knorvu genetic traits betrt'een Poll- guesses.\Ve may try, however, to cliscover ulations. Our svstem uses sllherical trigo- rvhy clifferences have been establishecl in nometr)' in orcler to rePresent the cliffer- clifferent populations. \Ve tnav thus try i-o ence betr.l'een t\\'o populations for three explore the reason why, for instance, there alleles as in the ABO svstem. \Ve put the the are no .\ or R bloocl grouPs among Ameri- square root of the gene frequencies on can Inclians (or, more exactly, no B bloocl tliree coorclinates, incl then every popula- groups, as there are some A in a few north- tion lvill be a point on the sr-rrfaceof a ern ribes) while there are A ancl B blootl sphere. If we have tlvo points correspond- groups in other populations. Theoretically ing to two PoPulations, the way to measure zrt least, we can say that the reason coulcl be thi clistance is to take the angle betrveen either a difference in selection, that is, the raclii benveen the centre of the sphere American Inclians have been exposecl to a ancl the two points. To those of 1'ou rn'ho different kind of selection from the rest o[ are statistically minclecl it u'ill be clear that the worlcl, or it may be clrift, tha[ is, ratr- this is only the extension to many dimen- dom fluctuations. Some specific investiga- sions of the angular transformation, which tions throw some light on this point but lve has the advantage of having variance con- r,r'ill limit our interest to exploring theoret- stant throuehout most of the range of vari- ically if clrift can be responsible for the vari- Irtions. Ancl a stanclard I'ariance is what you ation of ABO groups observed in the whole want for a variation cltte to clrift-for in- world. stance, to make it incleltenclent of the gene Drift can be responsible for these var- frequency. \'\Ie have I'ery largely used this iations. (Naturally even if drift could be, transformatiotl to meastlre genetic distances this doesn't mean it is responsible.) What ancl I think it fills an important gap in this the graph says is that if vou take several fielcl. This proceclure was sup;eested to mc

university Provided bY the Maternal and chitd Health Library, Georgetown Vol. 3, No. 4, 1966 GENETICSOF HUMANPOPULATIONS 379

by Sir Ronald Fisher. The resuits for clif lation structure, to see holv much varia- ferent geneticailr,-inclepenclent loci are cun]- tion is possibly clue to drift ancl, as a cort- ulatecl b,v adrling the chorcls (squared) cor- sequence, of the \{'ay in ll'hich they ex- respondine to the angles, ancl taking the change genes, the size of the groul)s, etc. square root of the sum, in such a ll'ay' that Norv there are still a few "primitive" popit- the full substitution of an allele by another lations in existence in the lt'orlcl ancl they at a locus is the unit in the cumulative shoulcl be stucliecl before they clisappear. genetic distance thus calculated. We have, trIaybe I can give you here some clata to thanks especially to the collaboration of Dr. shorv that interesting conclusions can be Anthony Eclu,ards, cleveloped several meth- obtained if one tries to s,vnthesize the in- ods to stucly the way in rvhich present-da)' formation from several genetic svstems. In populations may have evolved from .an fact I am surprisecl that this has not been originally uniclue population. We have clone before. I aclmit thotrgh that our re- made a few analyses on existing material sults are on the verge of sisnificance. Some in r,r'hich we have taken l5 poptrlations. of the conclusions shoulcl not be trustc(l three for each continent, ancl sr-rbjectcrl entirely because the methocls for testins them to anulrsis. The "lthvlopenetic tree" sienificance are still unclerclevelopccl and shown is arnons the mo:t likely ones sus- the anal,vsis is incomplete. Havins adclecl gesteclbr tlic an;rlvsis anrl looks surltrisinelv this n,orcl of caution, I hoPe )'ou \,\'otl't "acceptltblc." mincl if I tell you the couclttsions. There is rntrch more inf'ormation a.r'ail- In Africa live manv interestine pctlttrla- able, brrt the rc are statistical clifficulties tions and among those livinc in the most clue to thc fact that most ltopulations alc prirnitive conditions are the Pyernies. They' investis:rtecl only for a fraction of charac- live in separate groups in thc ecltratorial ters, ancl the characters investigated change forest, the most important ones beine the for even' population. -\n analy'sisrvas maclc \Vestern or Babinga Pygmies :rncl the ltast- basecl on sevel'itl ethnic grolll)s, usins thc ern or N{buti P1'gmies. Lust .}anuary' I average of seven poprrlations: Africans, joinecl friencls of Pavia tlniversity on a Europeans, extl'a European Clatrcasoicls(foi journey' to the Central African Republic. this purpose ..\nlericans al'e sl-oupecl u'ith I founcl that most of the Pvgrnies there clo Europeans), Ilonscllians, ,\rnerican In- live in rathcr primitivc contlitions, both dians, L,skirnos,,\trstr-aliansor Jlelanesians. from an economic anci a sauitary' point of Genetic clistances\\.ere calc'ulateclfor about view. "Prirnitive" shoulcl not be taken :rs l3 loci ancl a total of 33 alleles. Here asain rlerogatory. These people :tre extremely we get alt earlv split, sel)aratine ,\siatic charmins, kincl, ancl I thirrk intelligent, from Afro-Eurol)eilns, ;rnrl sonrelr'hat later anrl in fact I was srlrprisecl :rt the nttmber separation betlieen .-\lricans ancl Euro- of pharmaceutical proclucts thel' knor,v end peans. Thcse splits :rre still a matter ol' the rliscoveries thel' have tnitcle. Ancl in conjecture as I arn still not entirclr, hapltv fact my colleagues lvere there to stu

university Provided bY the Maternal and child Health Library, Georgetown 380 L. L. CAVALLI-SFORZA Ala.J. Med.Sci.

analyze(l thanks to the collaboration ol i[ I'}ygmies look r]ore like one anotheu several laboratories (Dr. Van Loghem's in th:rn they look like other Negroes, and Amsterdam, Dr. M. Siniscalco's in Leir-len, obviously look more like Negroes than Dr. lVlodiano's in Rome and Dr. De Carli's Caucasoicls, a ftrrther cotnparison of in- in the Genetics Department at Pavia). The terest is that lvith one group living in South blood sroups ancl almost all possible other .\frica, the Rushrnen, rvhich is considerecl markers of these people were investigatecl. lry some anthropolosists to be the oldest Dr. Laura Zonta helpecl n'ith the collrplrter .\frican grolrp. Ditl the Prgmies ancl Busli- analysis. rnen stern from an originallv unique grouP It is interestins to carry ollt :r conrl)arl- rr,hich then sPlit :tnrl aclaPterl to clifferent son between Caucasoids as a 1-roint of ref- livins conrlitions, the Iltrshmen living in erence, Negroes ancl Western Pvgmies. it .sotrth A[rican cleserts antl the P1'ernies in happens that Caucasoicls are a soocl lloint trclpical forests? The ans\ver at the mo- of reference because they tencl to occtlpv nrent is lirniterl bv the fact that the com- a fairly central position in the llorlcl usins. lrarison can be ur:tcle for otrlv the ferv lor.i sene frequencies as coorclinates. In fact l'or rvhich \\'e ltave information cornmon Caucasoids almost corresponcl to the tvorltl to all the sroups. I anr hoping that this averase. Comprtrting the total genetic clis- inlornration rvill soon be itrcreasetl. Nat- tance for l4 loci (each rvith several alleles), rrrallr', the [er,r'er ]oci )'otr ltave, the less one obtains a distance of l 65 betrvecn valicl is tlte inlormation. .-\lso I have not Pygmies anrl Caucasoids, 1.45 benveen Nc- lulrpenclecl stanclarcl errors because rt'e al'e sroes and Caucasoicls, ancl 0.15 benveett still a little uncertain about the computa- I'}ygmies antl Neeroes. I rvotrlcl remincl loLr tion of stanclarcl errors in these systems. that the unity in this scale is one sene sttb- \\restern Py'gmies are a little nearer to the stitution. With a total clistanceof l.-15out Negroes that strrrouncl them than to the of 14 loci examinecl. it means tl'rat at each Bushmen, rvhile the Eastern P1'emies are locus there is on average l0 percent sene nearer to the Bushmen than to the Ne- substitution. This may be a biasecl sar-nple groes, ancl the tentative conclusion that we of loci because we have selectecl polvmor- rlran' here is that it is possible ancl perhaps phic loci, ancl thus the proportion of lOou even likelv that Pvgmies ancl Btrshtnen gene substitution may not be r alicl for an- formecl originallv a single sroup. They may ot.her set of loci. The (listances betu'een have forrnecl the original African group- Caucasoirl and Negroes ancl benveen Cau- I r,vill shon' \'ou some eviclence in that di- casoid antl Pyemies are sirnilar-Prgtnics l'ection-ancl thel' have perhaps hybridized are different, they are more extreme. Ne- to a clifferent extent lvith surrouncling pop- groes and Pyemies are similar betr,veen rrlations. It is possible that our Pygmies themselves but they still shorv e fair cli[- har.e hrbriclizecl u,ith Negroes more than ference. l)r. \Iottrlskr''s P1'ernies. Another reason It is interesting to conlpare Negrocs, that makes Dr. ]Iotulskv's Pvgmies per- Western Pygmies ancl Eastern Pvsrnies. haps a little more Pygmies than mine is Thanks to the courtesy of Dr. ]Iottrlskr'. I that his Pvgmies are perhaps a little smaller have been able to obtain unptrblishecl llata than mine. on Eastern Pygmies, of u'hich he anah zetl Can one cotrsicler these Pygmies as a blood samples some )'ears ago, at a timc 1>roto-.-\frican population? The attsr.t'eris when that region was more peaceful than verr' probablr' "r'es", o[ the basis (coming it is now. Unfortunately the comparison out verv clearlv from the bloocl eroup data can be carried out onlv for a smaller num- obtainecl b1' Dr. Nijenhuis in Van Loghen's ber of loci than before. As far as one can laboratorr') of the cltaractersthat have been say at present, Western Pl,emies ancl East- I'ormerlv consiclerecl specifically African. ern Pygmies are sholvn to be nearer to each Rho is believecl to be a mostlv,.\friczrn allele other than Pygmies (especiallv the Eastern l)resent onlv in a ferv Etrropeans but in 60 ones) resemble Bantus. to 70 percent o[ Rantus. If vou take Pyg-

) University Provided bY the Maternal and Child Health Library, Georgetown Vol. 3, No. 4, 1966 GENETICSOF HUMANPOPULATIONS 381 rrlies and Bushmen, 90 to 95 Percent of your gene variance estimates,your genetic them are Rho. Even more tyltically African distances,and the general concltrsionsof is Fy (a-b-), which is l)resent in Euro- your studies? l)eans :rt a very low freqtlency, but in 80 DR. CAVALLI-SFORZA: I rlon't think it l)el'cent of Africans anrl in 100 percent of is likely to be true that we have that many the Pygmies. This looks like good evidence, alleles. Theoretically, it is unlikely that with all the limitations characteristic c:f there are very many allelesin a polttrlation. historical eviclence, that Py'gmies are proto- In any case,the type of variatiot-tyotl sug- African. sest may affect perhaps onlv the scale of () uestions: distances. DR. VICTOR NIcKLISICK: Professor DR. RALPH PLATOLI: I rvonclerif you Cavalli-Sforza, I wanted to ask what huP- would like to speculatea little further about pens in Pygmy-non-Pygmy crosses. Is there why the Pygmies and Rushmen are more any eviclence of single major gene resPon- African that Africans in resartl to these sibility for the Pyg*y state? allelesyou have stucliecl. DR. CAVALLI-SFORZA: This is one of DR. CAVALLI-SFORZA: I think there the problems we would like to investigate are essentiallytwo possibilities.One is that but we still don't have any material. There the Pygmies are a group that has been al- certainly are some hybrid grouPs and all most fully isolateclfor a long perioclof time, I can say is that they are grossly interme- while the present Banttts may have split rliate in stature. We haven't measurecl from the proto-African stock and mixed to them, but this is one of the problems we some extent with other grouPs. The other will be intereste(l in, if we are able to carry possibility is that Bantus and Pyemiesmav the research further. have been exposed to the same tyPe 'rf DR. N{cKUSICK: Would you exPand on selectionof different intensity; the Pygmies your differentiation between isolation and in a more intense rt'ay ancl the Bantus itr clrift, as in your list of four major factors a lessintense way. in evolution. DR. ARNO N'IOTULSKY: Whenever DR. CAVALLI-SFORZA: Yes. I have al- sickle trait and G-6-PD deficiencyare meas- ways been at clifficulty to distinguish fully ured in different populations in a very large between isolation, migration and drift be- number, they have tended to run rather cause these three concePts seem to have parallel. In other worcls,if sickling is high, only two clegrees of freedom, using loosely G-6-PD cleficiencyis high. The only excep- a statistical term. Sometimes people say tion we noted were these Pygmies referred isolation and migration are separate fac- to. Now we wonder-because there is some tors. I don't like to give them as separate evidence that they might have been dis- factors because I consider migration as a easedancl Dr. Nance from Nashville who reciprocal of isolation. What I usually do later on investigatedthem was also there- is to use the worcl "population stntcture" we woncler if our tests might have been to cover everything-isolation, migratiotr, falsely negative; in other \t'ords,if the low rlrift in acldition to assortative mating. Al- frequencies of G-6-PD deficiency might though I have been interestect in this prob- have been due to the fact that they hacl lem for a long time, I have never been able young red cells. Did you do G-6-PD fre- to find a very goocl definition for this ternr, quencies and sickling in your population, r,r'hich I like to use as including all the and how did they run? factors that are not mutation or selection. DR. CAVALLI-SFORZA: They are both DR. WALTER NANCE: If what you call low. but G-6-PDis almost absent,while sick- an allele actually turns out to be a set of ling is presentat a frequency lower than in hunclrecls or perhaps thousands of different the Bantus.We have no epiclemiologicalin- base sequences which cocle for the same formation yet to tell if malaria is low there. amino acid seqtrence, holr' \,1'oul

UniversitY ProvidedbytheMaternalandChildHealth Library, Georgetown Medicaland DentalFindings in the Brandywinelsolatel

Corl J. Witkop,Jr., D.D.S.,M.5.,* Charles J. MocLeon,M.S.** PoulJ. Schmidt,M.D.,*** JosepfiL. Henry,D.D.S., Ph.D.****

Introduction the eeographic extent of the isolate, the availabilitl' of church baptismal From 1955 to 1963 a joint sttrclyof the ancl mar- riage recorcls, historic, genetic,meclical, dental ancl socio- ancl the u'illinenessof the iso- late members to logic, aspectsof a triracial isolate resicline participate in the stu(ly, protocols \\'ere clesiened rnostlyin Charlesand Prince GeorgesCoun- to investigatesev- eral aspectsof isolate ties,Nfarylancl was instittrted by the Human the in clepth. Source material the Genetics Rranch, NIDR, in' cooperation ancl number of entriesby indi- vicluals for each of these protocols are r,vith Dr. .|oseph Flenry, Dean, Sihool of shorvnin Table Dentistrv, Howarcl University; and Fr. l. Thomas Harte, Chairman, Department of Historyond Descriptionof the lsolote Sociology,Catholic University of America This population has been designatedthe (Witkop et al., I95O).This isolatewas first Branclvrvineisolate after a small villase in b.rought our attention when three pa- ,to southern Prince GeorgesCounty, N,Iarylanrl tients, related as cousins,rvere seen at Ctrit- as it is the location of the ereatestconcen-

provided by the Maternal and child Health Library, Georgetown university Vol. 3, No. 4, 1966 FINDINGSIN THE BRANDYWINEISOLATE 383

thy among the Guineas of West Virginia. unusual frequenciesamong isolates.For er- Since 1955studies by the Human Genetics ample, a house by house census,field anrl Rranch have found several cliseasesr,vith clinical examinations amons the Halin'a

TABLE 1 PROTOCOLS USED IN THE RRANDYWINE STUDY BY SOURCE OF }IATERIAL AND NU}IBER OF ENTRIE,S

Number ol Protocol Source tr[aterial PersonsEntered History of Isolate Archives of \Ian'land \Iarriagcs & Baptisms(Harte, 1958) Parish Recordsof l9 Churchcs from 1793to 1956 9,255 Baptisms 4,266 Ilarriages Sociologic Family Interviervs 150 Households Kindred Individual Interviews 1r,7 5r Sibship Schedule Individual Interviervs Including Deccascd Sibs 6,840 of Living Family Grorrp Sclrctlulc Family Interviews 6,834 \Iedical & Dental Histories Individual Intervien's Living Drrring 5,r28 Study Period trIedical Examination Patients 2,639 Dental Examination Patients 2,819 Blood T1'ping Blood Samples 2,108 Laboratory Examination Blood, Saliva, Urine 2,648 Special studies Sclected Patients Eye Examinations 99J Admitted to Clinical Center, NIH 153 Admitted to Dcntal Clinic, Horvard Univ. 534

TABLE 2 REPORTED CAUCASIAN-NEGRO-A}IERINDIAN RACIAL ISOLATES IN RUR,{L AND S}IALL URBAN CO\I}IUNI- TIES OF EASTERN UNITED STATES: 1950 OR 1960 BY GROUP AND STATE (}IODIFIED F'RO}T BEALE' I957)

PoPulationin Isolate State I 950 1960 Cajans Alabama 2,790 Creoles Alabama, trIississippi 910 \lelungeons (Ramps) Alabama, Kentuckl', Tennessee,\'irginia 15,510 N anticoke -l\{oors DeIarr'are 530 Dominickers Florida 60 Lumbee Indians (Croatans) North Carolina, South Carolina, Georgia, \{anland 27,381 Pea Ridge Group Kcntucky 100 Sabines or Hauma Indians Louisiana 2,29r Red Bones Louisiana 5,r70 Natchitoches il'fulattoes Louisiana 200 St. Landry Mulattoes Louisiana 240 Rapides Indians Loursiana 90 Guineas \l'est Virginia, Ohio, \Iarlland 3,100 \Vesorts (Brandywine) \Iary land 3,050 5,128 Goulds New Jersey t,420 Jackson Whites Nerv Jersey, Nerv York l,280 Bushrvhakers Nerv York 100 Person County Indians (Cubans) North Carolina 406 q I{aliwa Indians North Carolina 3,689 1t() Portuguese North Carolina 320 Rockingham Strrry Group North Carolina, \'irginia 2,930 Carmel Indians Ohio 450 Darke County Group Ohio r30 Vinton County Group Ohio 190 Keating N{ountain Group Pennsllvania 100 Pools Pennsylvania 460 Brass Ankles South Carolina 2,320 Turks South Carolina 280 Adamstown Indians ( l\Iattaponi) Virginia 130 Rrown People Virginia 710 Chickahoininy Indians Virginia 820 Issues Virginia 300 Potomac Indians Virginia 230 Rappanhannock Indians Virginia 430

Georgetown University Provided bY the Maternal and Child Health Library, 384 WITKOP,et al Ala. J. Med. Sci.

rl t i t '-"i' ..t)

1

f ',:* 1

\-^' f,*,,,i* o*[ \ -"---' ', Brrr rrro!1ffi--pRrs[NrlolrcqR AS n qonxuNrri pR uMSStMtLATtDSTE*iN * -A ,,---4 1- V,- \ I ,-

. '"{ .. i!i.€'t ,.s

Inclians of North Carolina founcl 70 .ur"ltt"t" f'he Ilranclyrvine population of rodav of hereclitarl'benign intraepithelial dysker- l'epresents a breecling isolate in u'hich most atosis (\\ritkop et al., 1960), 24 casesof rnarriage l)artltcrs aIe selectecl among per- Sjogren-Larsson Syndrome (\Vitkop and sons having one of fiftecn sul'narnes.Not all Ilenrl', 1963)ancl 50 casesof cleft palare in family lines are historicallv inteerated into the isolateu'hich totals 3,742persons. These the present poptrlation in the same manner frequencies (l :19 HBID, 1:160 Sjcigren- (Harte, 1959). From baptismal ancl marriage Larsson clisease,and l:75 cleft palate) are records and from kindrecls clerivecl from in- the highest for these cliseasesreportecl for terviews, it is evident that seven family lines any pol)ulation. constitute the "core" of the isolate; the re-

)

provided by the Maternal and Child Health Library, Georgetown University Vol. 3, No. 4, 1956 FINDINGSIN THE BRANDYWINEISOLATE 385

TABLE 3 TABLE 4 -fRIINDS tN N{ATE SELECTION I'ATTERNS OF COR[' DISTRIBUTION OF SIBSHIPS FROI'{ I82O TO 1963 BY N,IAT,T-SAND FEJ\IAI,F,S E NDOGOT\{Y CLASSIFICATION

I 820-1909 r910-1%9 1940-1956 Endogomy Number ot Group Sibshibs Percent Marriage Tyhes No. No. %No.% Core I 638 64.6 Males Marginal 249 9.8 totals zt9 100.0 331 100.0 308 100.0 Outside 650 25.6 cndogamous 229 92.3 286 86.4 238 77.3 cxogamous l9 7.7 45 13.6 70 22.7 Total 2537 r00.0%

Females totals 224 100.0 266 100.0 226 100.0 County, N{innesota for the same period was endogamous 212 94.6 9C9 87.2 156 69.0 68.2 percent; approximately the same as 34 12.8 70 31.0 exogamous 12 5.4 Brandyu'ine women for the same period. H".*, T. J. (1959) This is especially interesting in lieht of the frequency of overt hereditary cliseasefountl rnainins eight have entere(l the grouP at in the Rranclyr,vineisolate. some time later than 1850.Prior to 1850 The exact origins of the core of the iso- there is no recor(l of an intermarriage be- late are unknolvn. However, r'ariotts famih tween a "core family" and what is desig- traclitions find some substantiation in r-ire nated as a "marginal family" i.e., one that historical records of the Archives of N'Iary'- has been absorbecl into the sroup after land and in tracing lineages throueh bap- r850. tismal and marriage recorcls of the l9 Cath- While the mating pattern still reflects olic Church Parishes which encompass Lhe considerable endogamy there has been .r Brandywine area. Evidence for one strch steacly increase in the proportion of exoga- line is presented here, for by history it is mous marriages from lB20 to 1956 (Table the family that introduced hereclitary clenli- 3). An endogamous narriage is defined as nogenesis imperfecta (to be cliscusserll:rter) either core-core or core-marginal, while the into the isolate. cxogomous matings are core-outsicle or rnitr- The Baxtor family most likelv hacl its einal-outsicle. The proportion of sibships rlrisins in a marriage performecl sometirncr resulting from various tvpes of matine's rluring l678 or 1679 on the Estate ol' \Iaj,r' from 1820 to 1963 are shorvn in Table 1. William Boarman lvhich \vas locatecl in Two families, Praxtor and lJaxtor* accoultt an area now knor,r'n as Br1'anto\,vlt. Ch:rrles 1"r). for over half of the core sibships (Tablc Calvert, I-ord Baltimore, harl brought r,r'ith While the isolate still demonstr:rtes a colt- hirn from Englancl an Irish nrairlsen':rnt it is in thc siderable degree of encloganly, named Illeanor (Nell) Butler anil also ir l)rocess of breakins ul). This has accomlt:t- Negro servant narnecl Charlcs rvith lr,horrr nied a graclual srowth of suburban housing he resicleclat the lJoarman ltlant:rtion (Ncrv- rlevelopments peripheral to the Washins- rrlan, 1956). After an al[erc':rtion u'itl'r Loltl ton metropolitan into rural ure-t the small llaltimore, in rvhich shc u'as n'rrlncrl th:rt clusters of Branclywine homes. While the endogamous mating pattern of the Brancly- rvine population is consiclerable even for I)rsrRrBUTroNoF ffi.1l;-rr,s rjR()\rr820'r() l1)ri3I]\' t".\\ilL\' 5t RN.\\ill the period 1940 to 1956, it is still less than that reported for other populations. Ken- Nttntber of Sibsltilts I'er<'crtl necly (1943) repor:s that for the years 1931 7l(; 1.)./ to l94l in the poptrlation of Ner.n'Haven, 22i 13.9 Connecticut, Neeroes \^'ere 94.4 percent en- tt,1) i 0.3 closamous; 90.1 percent ancl Italians I +r) 8.{) .fews 131 8.2 85.5 percent. Nelson (1943) reported the 126 enclogamy index for rural women in Wright r2r 7.3

I otal i {i:l tt I r)0t) *Pr",,,l,rrr1ms are uscd for family narnt's.

Georgetownuniversity Provided bY the Maternal and child Health Library, 386 WITKOP,et al Ala. J. Med.Sci. she and her children would become slaves. children, 10,085slaves, and 404 "other free Miss Butler and Charleswere married in the people" (Klapthor and Brown). One hun- estatechapel by Father William Hubert. In rlred ninety or 39 percent of the latter had I763 two grandchildren of this union sued nine of the sixteen surnamesfound in the Richard Boarman for holdine them in group toclay. Four of these were classified slavery.Their lawyer, John Hall, basedhis in the Census also as mulattos; five were petition to the court on the point that the found among both free white and mulattos. Iaw stated that any offspring from a slave In adclition,individuals having three of the \,vomanand a free man was to be considered current Brandywine surnames were classi- slave,but was unclear when the mother was fied only as white. Also of interest is the the free parent. In 1770 the case was ad.- fact that individuals having thirteen differ- judicated in favor of the plaintiffs ancl ent non-Brandywine names rvere classified William and N{ary Butler obtained their as free Negro. freedom.2 This court decision established Thus, a distinction had been made be- the point of Iaw known as the "right of free tween mulattos, approximately half of issue" in that offspring of free women ancl whom had Brandywine surnames,and free slave men would be considered free, bur Negroes,of whom only one person had a offspring of free men and slave women Brandywine surname. Further recognition would be slave.The Western Shore Judge- of this group as a distinct ethnic element in mentss record cases in which thiity_five the population is that at the earliestcenter members of the Baxtor family obtained of the Branclywine population, Bel Alton, freedom and compensationin lancl and to- the church of St. Ignatius separatedwhites, bacco.In thesecases relationship to Charles Brandl'wines and Negroes in their seating and Nell were stared so that f kindred of arrangement (Dodson and Wooley, 1943), about 100 persons could. be constructed. and evidence from the separation of the Unfortunately, this kindred extends for_ burial plots into white, Negro and Brandy- ward only to 1790while the kindred based wine areaswith headstonesdating back to on church records dates back only ro l gZ0, 1793indicate that thegroup was established so th-atpositive identification of the lineage as a recognizedethnic and social block at of the present Baxtor family cannot t,e least by 1790and possiblymuch earlier. attributed to this source. The testimony in the Butler-Boarman The Praxtor family is also known to have trial,2,sthe Colonial Censusof 1776-1778,8 lived on the Boarman Estate from 1700 to and the U. S. Censusof 1790eindicate thar 1778 as evidencedby wills of the Boarman the isolatewas well establishedby 1790and familya and by the will of an Elizabeth roughly numbered from 150 to 350 persons. Praxtor5'6 and by a draft notice of her son, Since 1790 most of the descendantsof Charles,who died in servicein liig.i NIar- these families have resided in the Brandy- riage and baptismal records substantiatea wine area.Some migration did occur in the family tradition that the Lynn family re_ 1830'sand in the 1890's.Components of at sulted from an illegitimate relationship be- least two large families migrated to Point tween a mixed-blood woman and a de_ of Rocks, Maryland about 1832 where a scendant of John Ffanson, the president subclone of the isolate resides today. At under the Articles of Confederarion. least ten Helray and Praxtor families mi- Other sourcesof evidence of the origins grated to Pennsylvania between 1890 and of the core families as a resulr of legilly 1905. Other migration from this group is proscribed matings (McKilty, 177g)u-ong local i.e., into Baltimore or Washington or all three racesin the early daysof the coloni involves individuals who marry outside the are given by Harte liOolj and Gilberi group and leave the community. In all but ( I e45). a few instanceswe rvere able to obtain cen- In the first national censusof 1790sthe sus and kindred information concerning population of Charles County was listed as the offspring of thesemigrants. 10,124free white men, white women and At the time of the present study there Il I ) UniversitY Provided by the Maternal and Child Health Library, Georgetown Vol. 3, No. 4, 1956 FINDINGSIN THE BRANDYWINEISOLATE 387

TABLE 6 colrp.,\RrsoN oF FREQUENCTES OF GENES FOR VARIOUS BLOOD GROUP ANTIGENS, PHF]NYI,THIOCARBA- rrrDlt TASTE (NoN-TASTER) AND HAPTOGLOBTNS IN AFRICAN NEGROES, ENGLISH, AIIERICAN INDIAN, NIARYLAND NEGROES, AND BRANDY1VINE

African .4merican trIaryland N egro English Indian BrandywineJ Negro No. dr No.Ct No.& No. & No. & (Ref.) Freq . (Ref .) Freq. (Ref.) Freq. (Ref.) Freq. (Ref.) Freq. A .139 124 (46) .279 3459(47) .006 78 (48) .107 2468 (72) .212 26t .{r .120 .209 0 .078 .l54 A: .019 .070 .006 .030 .057 B .148 .061 .013 .l33 .136 o .7t4 .660 .981 .760 .654 ( r (cde) .181 1345(49) .390 1798(50) .039 78 (48) .176 2497 (72) .243 266 7q\ Ro (cDe) .630 .026 .403 .445 *. r' (Cde) .019 .012 100 .026 .009 r" (cdE) .005 .0l0 :. .002 R1 (CDc) .088 .414 .t)15 .307 .U/J Re (cDE) .077 .145 9RO ".008 .l4l R' (CDE) .003 .058 .005 .061 *.426 (19\ NI .455 112 (46) .532 l1r9 (50) .801 i8 (48) 2ti07"* .514 158 s .r24 qoa .449 .154 .169 I{S .072 941 .369 .094 2+7 (72) .l 18 * l\.fs .382 9Rq 4\' '261 .408 NS .051 .080 .079 .060 '041 *.586 Ns .493 .390 .l l9 .1Ja (53) Fyt .082 108(5t1 .413 r 166(52) .J* I 78 (48) .233 2'D83(72) .140 200 *.004 K .009 r14 (54) .037 566 (55) .006 78 (48) 2553(72) .018 158(72) *.420 (58) .lk" .782 105(56) .5r7 2or (57) .469 78 (48) r32o (72) .13r 305 --49 * P .838 I 14(56) .490 500 (53) ,/o (48) .211 1853(72) .871 3oo(60) He .0r4 1428(62) o 387 (63) .007 78 (48) .0016 929 (72) .019 4500(7t; * Se (se) .062 I 14 (54) .4t'7 lll8 (61) 0 40 (59) .523 873 (72) .380 (53) *.039 PTC (r) .164 74 (65) .561 440 (64) .141 489(66) 2327 (72) .34r 120 (72) t'7 9\ IlPt .670 r92 (67) .390 218 (68) .480 II8 (70) .4t2 514 (69) .540 101

flncludes large number of related persons. *Frequencies incompatible rvith any amount of admixture of parcntal populations. **Based upon 2607 samplcs rvith Nf and N antisera onl)'. (46) Chalmers, Ikin, and NIourant, 1953a. (47) Ikin, Prior, Race, and Taylor, 1939. (48) Pollitzer, et al., 1962. (49) Livingstone, Gershowitz, Neel, Zuelzer, and Solomon, (50) Race and Sanger, 1950. I 960. (52) Ikin, Kopec, Mourant, Parkin, and Walby, 1952. (51) Zoutendlke, Kopec, and }fourant, 1953. (54) Barnicott and Larvler, 1953. (53) N{ourant, 1954. (56) Ikin and Nlourant, 1952. (55) Dunsford, 1919. (58) Rosenfield, Vogel, Gibble, Ol.rno, and Haber, 1953 (57) Race, Sanger,Allen, Diamond, and Niedziela, l9irl. (60) Miller, Tannor, and Hsu, 1950. (59) Chou'n and Lewis, 1953. (62) Chalmers, Ikin, and Nfourant, 1953b. (61) trIcConnell, 1962. (64) Harris and Kalmus, 1949. i63) Landsteiner, Stratton, and Chase, 1934. (66) tr{atson, 1940. (65) Barnicott, 1950. (68) Allison, Blumbcrg, and ap Rees,1958. (67) Neel, Robinson, Zuelzer, Livingstone, and Sutton' l96l' (70) (69) Nferritt, \\ritkop, Robinette, and Schmidt' 1961. 'l'hisIlory, I963. t72) studv. (71) Greenn'alt, l96l .

From Rucknagcl, l9t were approximately 5,128-+- 40 living incli- groes and some whites. Prior to Worlcl War vicluals who could reasonablybe included II, the members of the group either clicl not in the Brandywine population distributecl attend school or attendecl small local over the District of Columbia, Prince schools, that were comprised mostly of Georges and Charles County, Maryland. Brandywine students. With consolidation They are still predominantly farmers,semi- of the school system, they lvere forced to at- skilled laborers and domesticsresiding in tend predominantly Negro schools, a factor small groups or villages within both pre- now contributing to the accelerating break- dominantly white or predominantly Negro down of the isolate. Since World War II, neighborhoods and constitute an acknowl- over 30 have attended colleges most obtain- edged social block. Becauseof their consid- ing degrees in teaching, nursing and agri- erable land holdings, they enjoy an eco- culture. nomic advantageover the majority of Ne- The physical appearance of the groul)'s

University provided by the Maternal and Child Health Library, Georgetown 388 wrrKoP,et al Ala.J. Med.Sci. members vary from frank Negro at one ex- that the exact biological parents of each treme to typical Caucasianat rhe other. The child be stated and that this information majority have straight black or brown hair; woulcl not be revealed to any other member some,especially the older ones,have Indian of the isolate. facial features; and blue eyes are not un- The Sibship Schecltrle arranged offsprine common. An admixture o[ various physical by trnique numbers based upon the family types can be found in one sibship. There is surname, the order of the sibship inter- often a considerable dissociation of other viewed and the birth orcler of the indivicl- physiognomonic featnres,so rhar light col- ual. An attempt was made to recorcl the re- ored individuals may have flared nasal alae, sults of all pregnancies. Items pertainine to thick lips, etc. The averageBranclywine per- the birth ancl childhoorl cliseasesof an in- son has light colored skin which becomes

l

provided by the Maternal and Child Health Library, Georgetown University Vol. 3, No. 4, 1965 FINDINGSIN THE BRANDYWINEISOLATE 389 mate year of birth, death, spousesoffspring, taken of every patient with a positive sick- albinism, etc. These latter data were used as ling test, dentinogenesisimperfecta, or sus- kindred information only. pected congenitally missing teeth. Paraffin Items for which we felt there might be stimulated saliva was collected for secretor some hesitancy in revealing, such as legit- factor and lactobacillus counts which were imacy status,diseases, etc. were crosschecked performed by the method of Hadley (1933). by asking each member of a sibship about Environmental data were obtainecl for these items for every other member of the fluoride content of drinking warer ancl sibship. Examinations were held either in background radiation. Repeated samples mobile trailers or in a field clinic estab- from ten home wells distributed through- Iished in Clinton, Maryland. Examinations out the area varied from 0.01 to 0.17 ppm were conducted during the school vacation of fluoride. Scintillation countsof warer and period, June to September,during the years soil samples indicated no area of unustral 1956 through 1960. In a few instances ex- radiation exposure.A monazite beach was aminations were given in the homes. Be- discovered in an adjacent counry (Calvert) cause examinations could be done more with radiation levels from two to three rapidly than interviews, approximately half times background, but none of the Brandy- of the people were examined before they wine population have ever lived in the area. were interviewed. Medical examinations were done by med- Kindreds for the entire population and ical students under supervision utilizing a for plotting the findings on examination modified P.H.S. history and medical form. and from Sibship Scheduleswere construct- From each individual examined a venous ed from the kindreds obtained from indi- blood sample was drawn using vacuum vidual interviews and from baptismal and tubes containing E.D.T.A. On each sample marriage records. a capillary hematocrit (McGovern, lg55) Ascertainment for any particular condi- and sickle-cell preparation using two per- tion depended upon the nature of the con- cent sodium metabisulfite were performed. dition. Sickle-cell hemoglobin was ascer- A Wright stained peripheral blood film was tained only by laboratory examination. made from a finger puncture. The plasma Dentinogenesis imperfecta was ascertained was separated for haptoglobin electropho- by interview of the individual and his rela- resis(Smithies, 1958). The cellswere washed tives, and also ascertained at the time of and a portion hemolyzed (Drabkin, 1946) dental history and examination and again for paper electrophoresis (Smith and Con- on medical history and examination. The ley, 1953).Fetal hemoglobin determinations number of persons entered in the various (Singeret al., l95l) and quantitative starch protocols are shown in Table I. block electrophoresis(Kunkel et al., lgbi) Clinical and Laboratory Criteria of hemolyzateswere performed on bloods in which the hematocrit was below 36 percent The dental examination was done by or the film significantly abnormal, dental students under supervision. Dental or on bloods in which the caries, (DMF-def index, excluding dentino- hemoglobin electro- phoretic genesis imperfecta), congenitally missing pattern appeared abnormal. Agar teeth, dentinogenesis imperfecta, fluorosis gel electrophoresisat pH 6.5 (Robinson et (Dean, 1942), enamel hypoplasia, enamel al., 1957)was performed on selectedbloods opacities (hypomaturation), rongue lesions, to exclude the presenceof hemoglobin D. Fordyce spots, clefts, tori, arch shape and Red blood cell blood typing was per- occlusion were routinely recorded utilizing formed using standard techniques and uti- standard or previously established criteria lizing the following antisera: Anti-A, Ar, (Witkop and Barros, 1963).The peridontal A2 (Ulex), He, B, C, Cw, c, D, Du, E, e, M, index (PI) of Russell (1956) was used to N, S, s, Fy., K, k, Jku, Jko, P, Di", Le". score periodontal disease.During the first Phenylthiocarbamide (PTC) rasretests were two years, full mouth dental x-rays were performed using squares of filter paper

provided by the Maternal and child Heatth Library, Georgetownuniversity 390 WITKOP,et al Ala.J. Med.Sci, rvhich hacl been soaked in a saturated solu- ly reaclily and yet would reflect the clesree tion of PTC ancl clried. Saliva \vas exam- of endogamy of the parents. ined for secretor status. The laboratory clata An endogamy index \'vas constructecl in were pr-rncheclon one IBNI carcl per person, the following lvay. If all four sran

-t University Provided bY the Maternal and Child Health Library, Georgetown Vol. 3, No. 4, 1966 FINDINGSIN THE BRANDYWINEISOLATE 39r

TABLE 8 ined would reflect a true frequency sample ALBINIS}1 of the isolate, particularly when dealing Numbers and Percent of Persons Affected in the Historical, Living and [xamined Portions of the Isolate with a condition such as sickle-celltrait that coulcl be cletectedby laboratory means only. Nttmber of Number of Percent Two parametersillustrating our efforts in Pol'ulation Persons in Persons of Persons Base Population Base Affected Affected this direction are that there was no signifi- Dental Exams cant difference in the proportion of people r 950-l 960 2,819 36 1.3 Sibship Schcdulcs with sickle-celltrait in the yearly subsam- Alive 1956-l9tiO 5,128 60 | .2 ples (Rucknagel, 1964),and that there was Historical I820-l960 r l ,751 69 0.6 no statisticallysignificant clifferencein the proportion of personswith clentinogenesis imperfecta examineclclentally ancl those as- name only her offspring lvould be exclud- certained in the Sibship Scheclulein the en- ed. From a population clynamicsvier,vpoint tire livine poptrlation (Table 7), or albin- we clo not knor,v in the casesof siblings ism /Table 8). two ancl three rvhether the isolate has lost the Rrandywine genes of siblings rwo ancl Descriptionqnd Frequencyof Normol Troits three or gaineclthe Y genesof their spouses qnd Diseoses until their oflspring marry. If their off- spring marrv back into rhe group then the The phenotypic frequencl, of the blood isolate has sained the Y senes.If the off- groups,blood tyl)es,PTC tasteability, secre- spring marry other Y's then the isolate has tor factor and haptoglobins are siven in lost genes from siblings rwo and three. Table 6. In light of the complex biological Therefore membership in the isolare is ex- interrelationships ancl inclications that so- tended for our purposesto anyonewho hacl cial factors influence certain traits (see at leastone half of his seneticmaterial from dentinogenesisimperfecta later), variances the isolate.From kindiecl chartings ancl in- are not given and Harcl,v-Weinberg equi- terview data, there \,vere5,128 -+ 40 such librium was not tested. We feel. however. individuals alive during the years of data that the mean frequenciessi\.'en should be collection 1956-1960.Forty personsinclude valicl. those who coulcl not be located or known The frequencyrof the sene for sickle-cell illegitimate persons for whom the exacr parentagewas unknown. hemoglobin (HbB) among the 2578 persons examined hematologically was 0.1045,ancl At the beginning of the study it was real- ized that all persons in the isolate would for hemoglobin C (HbB) was 0.00174.AII of. not submit to examination; most frequent the carriers of the latter were the offspring among thesewere males during the working of Brandywine outgroup marriages, further period, ages20 to 45 years.An attempt was climinishing the magnitude of Hb; fre- made to selectpatients for examination on quency within the isolate.This is the high- the basisof isolatemembership rather than diseasestate hoping that the sample exam- est frequency of the HbB €ieneyet reported

TABLE 9 'I'HE DISTRIBUTION OF ALBINIS}I BY ENDOGAMY INDEX AND SEX OF EXA]\IINED SAN{PLE

F ernales trl ales

Endogam.y Number Number Percent Number Number Percent Index Examined A ft'ected Af f ected Examined .4f f ected Af t'ected Total 0, r 320 0 0 907 00 0 2 316 o 0.6 9,\7 2 0.8 0.7 3 890 l6 1.8 759 16 2.1 1.9 Total 1526 I8 t.2 r293 18 r.4 1.3

SexX2-0.258 P=.50 Endogamy Index X2 - 14.866 P < 0.0r

provided by the Maternal and chitd Health Library, Georgetownuniversity 392 WITKOP,et al Ala.J. Med.Sci.

TABLE 10 Albinisrn present in the sroup is of thc I)ISTRIBU'TION OF DENTINOGENE,SIS IITPERFECTA SIBSHIPS BY SURNA}TE AND ENDOGA}'IY GROUP generalizecl complete type classifie

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il Vol. 3, No. 4, 1956 FINDINGSIN THE BRANDYWINEISOLATE 393

perfecta without bone cliseaseoccurs in ated with bone clisease,blue sclera, or any about one in 8,000 North American white other stigmata of osteogenesis imperfecta. school children (Witkop, 1965). Certain There was no increased frequency of bone family lines have introduced the condition fractures among Persons affected with den- into local areasso that it is unequally dis- tinogenesis imperfecta comPare(l to unaf' tributed in the population. Nlany affected fected persons from the isolate. Blue sclera families in New England for example trace did occur in the isolate but was of the in- their ancestry to a passengeron the Moy- fantile type ancl not the slate blue tyPe flower (Finn, l96l). In the central Atlantic seen in persons with osteogenesis imller- statesmany affecteclfamilies trace their ori- fecta. The frequency of infantile blue sclera gin to a German family which settled in was not significantly clifferent among Per- Pennsylvania in the 1700's. Amons the sons affectecl with clentinogenesis imper- twelve propositi founcl in a survey of 96,471 fecta compare(l to persons without the tooth school children in Nlichigan (Witkop, 1965) defect. No case of osteogenesis imperfecta three of the families traced the origin of the was founcl in the isolate. clefectto an ancestorby the name Baxtor. Among the 164 persons examinecl, 147 Family tradition among the Brandywine were also examinecl racliographically. Nine- isolateplaces the sourceof the defectamong teen persons affected with the cliseasewho the Baxtors. It is interesting to note that were not inclucled in the complete clental among all the Rrandywine sibshipsin 1956- examination were subsequently seen either 1960that the frequency of the trait is high- at the Clinical Center, N. I. H', or at the est among the Baxtors so that 27 percent of Dental Clinic, Howarcl University, ancl re- all affecteclsibships are Raxtors who consti- ceived full-mouth roentsenograms. Of these tute only 6 percent of the total sibships 166 persons, eight children had atypical (Table l0). The rate of rlentinogenesisim- dental finclings in their primary teeth perfecta by enclogamy index (Table I l) which coulcl be characterized as "shell demonstratesthat it is definitely in the core teeth." These cases, illustrated elsewhere of the is,olate. (Witkop, 1965), occttrred only among chil- Dentinogenesis imperfecta was unassoci- dren who hacl a parent affectecl with clenti'

TABLE t2 SEGREGATION OF DENTINOGENESIS IMPERFEC'IA BY GENERATION

Number ol Condi!.ion Sibships IJnhnown (Sex ol Condition and Sex Alfected Parent) Aflected Unaf f ected fJnknown Unhnown Total Generation 0 22 l0 0 3 g male ll l0 0 female II 00 0 I unknown 00 00 0 0 Generation I 213 I'r 0 0 l8 male l6 20 0 8 female t7 30 0 l0 unknown 00 00 0 0 Generation II ll 37 38 19 t0 104 male 520 19 16 0 JJ female 617 193 0 cq unknown 00 00 l0 l0 Gerreration III 32 83 69 t7 6 t75 male t4 4i, 32 Il 0 88 female t7 38 376 0 8l unknown t0 00 6 6 Generation IV 62 150 l2r 26 32 329 male 29 77 56 13 0 146 female 33 73 65 13 0 t5l unknown 00 00 32 32 Generation V 35 50 39 ll 3 I03 male 14 26 194 0 4g fcmale 2t 24 207 0 5l unknown 00 o0 3 3 Total t44 335 273 73 5l 732 male 64 r75 r29 44 0 348 female 79 160 r44 29 0 J J.) unknown l0 00 5l 5I

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il 394 WITKOP,et al Ala. J. Med. Sci.

nogenesis imperfecta. The histological changes in the dentin of these "shell teeth" were identical to those seen in teeth show- ing typical lack of pulp chambers ancl root canals. Ftrrther, four cases of shell teeth were observecl over a periocl of seven years, and the l)ernlanent teeth in each instance clemonstratecl radiographic and histologic Fig. 2. finclings oI typical clentinogenesis imper- The great frequency of this cliseasein the fecta. It u'as concludecl that shell teeth are Brandywine population is clearly an exam- an expression variant of the dentinoeenesis ple of a founder effect resardless of selec- imperfecta gene and not a separate clisease tion. Trvo big families in lB52 to 1872 es- entity. Another variant r,l'asobserved four tablished it firmly' in the isolate. times; three times in the primary clentition All the selection effects cliscusseclbelor'r' of chiklren lrncl once in the molar teeth of are very subtle by comparison. The periocl, an arlult. Rarliosraphs of these cases clem- 150 years, is too short for effects of such onstrate(l normal-sized pulp chambers and magnitucle to occur by selection onlv. root canals. Hou'ever, the clinical appear- Dentinogenesis imperfecta is classified as ance, color, rapicl attrition, shape, ancl his- a clominant genetic clisease in lt,hich the tological linclinss were those of dentino- changes are so striking that practically no genesis imperfecta. The children were from difference has been founcl betrveen histon' three separate sibships, each with an affect- and examination as to rvhether a person is ed parent. The aclult woman with normal affectecl or normal.* Perhaps it shotrlcl be pulp ancl root canals had an affectecl father termecl co-clominant, even though there has ancl affecterl children. f'hese tr!'o variatiois been no reportecl observation o[ a homozy'- in expression of the gene are adclitional sote. Only one sibsl'rip of rvhich both par- examples of those clescribed in a previous ents were affecterl has been sttrcliecl (Figure publication (Hurser er al., 1956). 2). This sibship sugseststhe possibilitl, that From kinclred clata, two lines of clentino- the homozvgous state is lethal. There n'as genesis imperfecta were found. The two no cliscernible cause for the repeatecl abor- progenitors of these lines were born in 1852 tions, each occtrrring at t\r'o ancl one half and 1836, ancl it is not known how they were months gestation. Unfortunatelr', the abort- related to one another. Family history states ecl fetuses were not save(l for histoloeical that it n'as introduced into the isolate by a examination. Causes other than homozy- sea captain from Liverpool, England r,r'ho gosity for the clentinogenesis imperfecta could for had chilclren by two differenr mixed blood gene be postulatecl the abortions in Figure 2. Although the parents L 2.06 women in Charles County. We have been ancl B 59.01 are not relatecl closer than half unable to iclentify this source in baptismal thircl cousins, a recessive genetic cause for or marriage records however. It appears the abortions or inbreecling effect in the that the only source dentinogenesis imper- mother could be listecl amons other possi- fecta in the population of southern Mary- ble mechanisms. The trait is apparentlv land is the Brandywine population. We always expresseclin any person who receir.'es have done numerous examinations of Prince the gene which should follorv the elemen- Georges County school children for other tary laws of inheritance: l) that sibships reasons and have found dentinogenesis im- with one affected parent should segregate perfecta only in persons of Brandywine one to one with respect to the disease; 2) ancestry or from a few recently arrived families from Pennsylvania or New Etg- -I" u separate study in \Iichigan, 42 propositi gave 650 histories about the fact of having or not having dentino- land. No case from old line southern Mary- genesis imperfecta in their families; history was- founcl to be in error once, in that a person said to be normal lr,as land white or Negro families have come to found to be affected on examination. No dif fere nce be - orlr attention. tween history and examination was cncountercd in thc Brandywine study.

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TABLE 13 I)T-N'IINOGF,NESIS IN'IPERFECTA BY S[,X

Percent Standard A ff ected I'nat'| ected Total A ffected I)euiation

NIale I /t) 129 304 l) / .1) 2.\t'o Female t60 144 304 l-r2.6 2.gth TOTAI, r).)l) 27\ 608 'f hc rl ffcrcncc of proportions affected in the two sexes is significant at 109/o levcl.

that Lrnder the assumption of panmictic ness of the affectecls with respect to their matins and the absence of selection the fre- o\vn unaffected siblings is the observecl quency of the sene for the cliseasein the ratio. The gross clifference nithout regard population shoulcl not chanse from senera- to the parameters such as seneration ancl tion to seneration. It h:rs been h1'pothesizecl name \,\rereas follorvs: of 732 inclir.'iclualsin that the fitness of people affectecl with sen- sibships with affected parents, 335 \,\rere etic clisease shoulcl be reclucecl. Hor,rever, themselves affectecl, 273 \'vere not affected many sttrcliesappear to contain an excessof ancl 124 clied before the conclition cotrlcl be affectecl lteople not entirell' accountecl for juclgecl.* Of the former trvo classes,55.lfo by the classical corrections for rnethocl of \{,'ere affectecl anrl 44.9J2,,rvere unaffectecl. ascertainment. Further, a quasi l)opular The observeclcleviation from 50fi, of 5.1% view hoirls that genetic cliseasesseem to in- \\rasequ:rl to 2.5 stanclarcl cleviations; abont crease in frequency in isolatecl l)ol)ulations. the 1c[ level of significance . I)entinosenesis imPerfecta in the Rranclr'- Table l2 is a finer breakclon'n of the ses- rvine poptrlation seemed to be strch an cx- resation of affectecl families. f-he latio rr'as :rmple; so an analvsis of selection factors apl)arently constant over the last three sen- rvhich may I'rave influencerl this sittration erations (Table l4), btrt cloesshorv a slieht rvas macle. sex clifference. There is no knon'tt explana- The nrethocl of sttrcll,userl is baseclon the tion for this slight sex clifference shon'n in theoretical seeregation ratio of I : 1, auto- Table I3. matically sivins equal test ancl control pop- The clifference 55.1:44.9 r'nislit be ac- ulations for observations. The metho(l is countecl for by an ascertainment bi:rs lather known to have pitfalls particularlv nncler than a true clifference in nature. If for example, the ascertainment of the less than l)erfect ascertainment of the con- clitions sttrcliecl (Krooth, 1955). All of the clisease\vas very incomplete then the prob- inclivirluals knor'r,n to have been affectecl bv ability of a sibship being founrl :rnrl inclucl- the disease throuehout historv of the popri- ecl in the stucly rvoulcl be iouehlr, propor- lation toeether u'ith all of their siblings tional to the number of affec.terlr:hilclren in rvhether aflectecl or not were sturliecl lr,ith it, but inclepenclent of unaffectetl chiklren respect to their seneral fitness, tl:eir fertil- in it. A bias in favor of affecte

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TABLE 14 PERCEN'I OF PERSONS AFFECTED AMONG DENTINOGENESIS I}IPERFECTA SIBSHIPS BY GENERATIONS

Percent Standard Alf ected Unallccted Total Alf ected Deuiation

I t3 I l8 72 rr.r%

II ct 38 IJ 49 5.7%

III 83 69 r52 55 4.0% IV 150 r2r 27r JJ 3.r% V 50 39 89 56 5.4% JJJ 272 605 No differences are significant. affected siblings would not be counted in er people could be ascertainmentbias. Some the study. They should be counted as un- percent of the older people had no teeth at affected siblings of an affected individual, all, and the dentist in spite of the fact that even though their affected sib does not ex- he gave a dental examination, was forced ist. This will introduce an artificial dearth to rely on historical account for ascertain- of normal cases and bias the segregation ment. The theoretical adjustment for the ratio in favor of affecteds. case in which no parents were ascertained There is Iittle evidence in this case to is given by Fisher (1934).The adjustment show that the older generations had some- applied to these data reduces the segrega- what less perfect ascertainment than the tion ratio to a lessthan statisticallysignifi- younger generation. Among affected sib- cant one. If such a bias did occur it would ships which were observed by dental exam- be expressedin the sibships with few mem- ination in the NIH study from 1956 to bers; the fewer the numbers the greater the 1960,55.6 percent were affected, practically probability of none of them being affected. the same as the percentage obtained from Table l5 shows the evidence for such a bias interview (55.1 percent). in these data. Also, among the people who were given Of the twenty-five one child sibships rep- complete dental examinations there is a resentedin the sample, l6 were affected and slight decreasein incidence with age of the only 9 were unaffected. Of the 17 two child examinees.The causeof this dearth in old- sibships, 22 of. the members were affected

I AFFECTEDMALE o UNAFFECTEOFEMALE DTo LEGITIMATE t}p ILLEGITIHATE

------al -F -t-r -T-l', ----r--1 ftT | i vi t- i L-J r L-J I I I I I I I I INCORRECTLYA A rnconnEcrLY EXCLUDED-V I vrttcLuoEo til sruDy FROMSTUDY o- STUOYPOPULATION Fig. 3.

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TABLE 15 remains practically the same as before the SEGREGATION OF DENTINOGENESIS TMPERFECTA correction as follows: BY SIBSHIP SIZE Suppose r people of the parents' g€Der?.: Sibshib Number of tion are misclassified.Suppose further that Unalf ected Dif Size SibshiPs Af f ected, lerence the ascertainment of the childrens'genera- 7 I 25 16 I any sibship which I r7 22 r2 l0 tion is perfect and that 3 l0 16 14 2 had affected children led to an investiga- ^ a b 13ll: tion that disclosedthe affectedparent. Then Total l-4 67 46 2r all the r misclassified parents had complete- Greater than 4 268 22t y ly unaffected sibships. The distribution of Total s35 273 48 family sizes of these r people would there- fore be about three and only 12 were unaffected. The ten (A) I Ps child sibships were split almost equally, l6 affected and 14 unaffected. In the six four child sibshipsl3 members were affected and (a)t' ,r, I I were unaffected. The probability of all 2 sl=l the members being unaffected in sibships of fam- of more than 4 children is so small as to be where P" is the expected proportion beyond the bounds of the accuracy of the ilies to have size s in the population from Then statistics being applied here. Further, there which the r parents were drawn. was no birth order effect, so that the ratio t of affected to unaffected among first born s (a)'Ps from 3 was 55:45, not significantly different fr the 55.1:44.9 obtained in data from the en' sample. z (L)' qr, tire sl-l However, the usual adjustments for such number of unaffected individ- a bias cannot be properly applied to the is expected be included but who were Brandywine Study. The proposed adjust- uals who should they had no affect- ment is predicated upon the proposition lost to the study because usual Poisson distri- that only children were ascertainedand not ed sibs. Assuming the value is equal to parents, and therefore small sibships bution of family sizes,this their I having only normal children who have an - r where I is the average family size. affected parent are missed.That is entirely 2 the population of unaffected untrue of the Brandywine Study. If such Therefore, I unaffected children were left out of the sibs is short - r from its true value due to study, their parent must either have been 9 However, the r par- entered erroneously in his own generation these missed sibships. so that the unaffect- as an unaffected sib, or else he must have ents were misclassified are under- been an affected child of some affected in' ed sibs are over and the affected The dividual in the generation before who had estimated by . due to them. proper r : Uf r no other affected children and who escaped estimatesshould then be Af ascertainment himself, and so on. In the 2 (; - t) where A and U were the origi- Brandywine study the probability of these compound evasions is negligibly small. It nal estimates. TABLE 16 can fairly be assumedthat nearly all of the ILLEGITIMATE CHILDREN OF PARENTS AFFECTED sibships which were incorrectly left out of WITH DENTINOGENESIS IMPERFECTA the study'as unaffected siblings had an af- fected parent who was misclassified as un- Af f ected Unalf ected Total I 6 These misclassified parents must I\fale Parent affected. Female l0 23 now be reclassified as affected with the re- I t5 29 sult that the ratio of affected to normal Total t4

G-

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TABLE T? I'r-RI-ILI'[Y OF I'I-RSONSAI'FECTED WITH I)1.-\I'l\O(;E\l:SIS I\IPF-.RFEC'f'\ ROTH SE,XE,S

Alfected I'naflerted

Degrees I)egree.s of Auerase 0f .tt,errtqe Freedom Nunber F recdotrt .\'l rnl;c r' No. of of .\tandard. -\'o. of ot' \lattrlard Generatiort Parents Offspring I)et'iation Parert t s Off.tltrirtt I)t't'itttiort I)iflerence .\i,!ttilicattce 0+r+rr 52 r. (i .:i -t.l 11) .t .i 0.0i III 83 1.0 .2 (i(r 2l .'l r.6 0.00t IV laO ().f.r .07 l2l .i .0s 0.1 N.s Total 285 2.7 .l 2.rI :: .i .i 0.0I

An estimate can be macle for r in this There is one other major area likely to populatiol] Llsing the restrlts observecl for a cause irn ascertainrnent bias: illegitimate small sibship by fittine the follou'ins srsrem: offsprine. Dtrrins the sturlv all illegitimate r exP( -xtz\ ( x/2) - i offsprine \\'ere obtainecl rvhenever possible. I exP ( -x/2) ( x/2)2 _ l0 In this poptrlation, nearl\. all illegitimatc chilclren are raiseclrvith their mothers. The -r42) o I .*P ( ( x/z)' fac't of illesitirnacv is often not kno'wn. An 2 illesitimate chikl is sirnply raisecl witl-r his 'lr'ho r exP ( -x/2) ( )"/2)1 _ 2 half sibs, in mAnv casesare themselves {i lesitimate. Geneticallv there are t\{o sepa- an([ othel' tern]s of consequence. This calctr- rate Problems: lation clepenclson size assurneclfor the hy'- l) Fan-riliesin rvhich the mother is affect- pothetical 1ieo1tle. Of course, the averase eil. Assurning her spouse is unaffectecl the family size of these hypothetical r people sesresation of her leeitirnate children will cannot be observecl, but since all Branclr'- be l:l Since onlv a few percent of the pop- wine people, b1' er.er1,sesregation attelnpt- ulation at large n'ith u'hom she misht be e(1, h:n'e averase family size of betrveen involverl carr\, the rlisease,her iliegitimatc three ancl six chilclren, it is reasonable that offsprine n'ill segresate1:l also. these are soo(l bouncls. This rielcls from 2) \\tith affectecl fathers horvever, therc r - 15 to r - I+. is :r chance his illegitimate chilclren mielir For this ('aseat hancl then, :rssilrlrinsall be rvith \\'omen n,ho are outsicle of the the clil'lerence in the srnall sibships ro be stLlrh'. \\'hatever af fectecl chilclren com(l cltre to ilscertainrnent bias, the 2l cases from such unions n'ill have a ntuch greatet' shorvn al'e (lrre to about lJ or l5 rnisclassifi- probabilitr. of beins cliscovererl ancl as- cations. f'ltc tnre l-esults shoulcl then be signecl to the tr-tre bioloeical father, than approxirrl:ltel\ : n'ill be trtre of normal chiklren (Fieure it). a{fcctctl 3b0 5J.Z(,;, T'o tletermine tliis sorlrceof bias, we must rrn:rllectecl 29-1 Ib.7o:, cor]ll)ill'e the observecl ntrrnber of illegiti- virtu:rllt, tlrc sunrc as befer-c. nlrrtc rtl'fecterl irnrl trnal'[ecte rl chilclren ol'

TABLE 13

IiI.,RTII,II'\' I'E,RSO\.S ,\IIFI.,C]-I'ED \\'ITH I)}-\ I I\O(;I,\ T-SIS I \I I'r-RIrI-C'I",\ IrF. \ t.{ l_ [_.s

lfftrtrd

D egre e.s of .ltteraqr .lr,(raqp Freedonr .\'tnr D"r .\'l rrrbr r No. ctf of .\tuttdarrl ol Generation I'arertl.s Off.sltrirr!!, I ) et,iat iort ()ff.sbrin.s I)ifferentr \itrrilitunce 0+r+II 25 i.8 .-+ r.5 .3 N.S. III 38 3.0 .3 -.3 N.S. IV 7:l 1.0 ,I 'fotal 0.tt 0.2 0.t0 136 2.5 2:i 0.2 N.S.

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TABLE T9 FER-TILITYPE,RSONS AFFECTED WITH DENTINOGENESISINfPERFECTA N{ales

4llecled Unaff ected Degrees Degrees of Auerage ol Aaerage Freedom Number Freedont Number No. ol of Standard No. of of Standard C eneraiiort Parents Ot'fsPring Deaiation Parent.t Of f spring Deaiation I)it'lerence \i grtilicance 0+r+rr 2 t- 5.3 0.4 23 4.3 0.4 r.0 0.05 III 15 1.7 0.3 32 r.6 0.2 3.r 0.001 IV i7 O.ti 0.r 56 0.5 0.1 0.1 N.S. Total r19 2.8 0.2 l il 2.2 0.2 0.(; 0.05

affected fathers. The clifference between affected children. As a first approximation these two categories is the estimator of the one might guessthat these illegitimates in- amount of the bias introducecl by this kind troduced into his sibship incorrectly will of error (Table l6). just about balance out the unaffecteclille- The loss of males' chilclren from the gitimate children of his own rhar are lost ro study has clearly occurred. But there are the study. It is of courseimpossible to make only four illegitimate affected children of any more specific estimatesof thesevalues. affected fathers, in the data, clearly no An examination of the fertility of the in- source of g;reat bias. There were two such clividuals with D. I. and their unaffected unaffectecl chilclren. So both affected ancl siblings shows that throughout rhe hisrory unaffectecl illegitimate offspring were lost, of the diseasein this population there has irrespective of clentinogenesis imperfecta. been a consistentdifference between them. One woulcl suppose from this that by seg- The calculations in Table l7 are fertility regating the population as to the sex of the figures but they are not accorcling to the affected parents and considering the ratio standard practice for calculating fertility. from affectecl mothers, against affected fa- Roth men and women are useclas the popu- thers that this would also cliscloseany dearth lation basefrom one generation to the next. of unaffectecl children on the part of the Sincemost of the membersof the later gen- father. However, there is one further com- erationshave not yet completecltheir repro- pensating factor involvecl and that is illegit- ductive lives, the figures for those genera- imacy of children due to the unaffected tions are lower than the true fertility. For wives of affected men. For the same reasons each individual including both sexes the as above, the unaffected women who have total number of offspring, regardlessof the relations outsicle the family will have them number of matings, are counteclfor his en- with unaffectecl men in the population at tire life. Those individuals who had no larse, ancl therefore her chilclren will be un- affectecl. If these illegitimate children are -r-HE (;s raisecl in the sibship in DrsrR r* r"ll:"ru ritouro, o r, sr BL r N the family of the \,VITH'' AND"t WITHOUT DE,NTINOCE,NE,STS affected male then his segregation ratio will "iftilu?Ifi.i"l il+%kL .)'f '" o be balancecl too highly in favor of the un- Family Name TABLE 20 or Endogamy Percent ol Percent of Group ol Affected Unaf lected Av E R A G E ri"tiftti r orA L Spouse Siblings Siblings l)it'f erence *Y ?t i.qtl^T If" A 22.8 26.5 -3.7 B 12.8 | 5.9 -.). t Means Standard Detiation Other Core 3l .4 25.7 ft)./ 1950-60+ t 1940-50 r9i0-60 1940-50 Total Core 67.0 68.I -t.t Praxtor 3.034 4.44 .18 .23 I\farginal 4.6 2.3 Baxtor 2.989 5.44 .44 .60 Y 28.4 29.0 -t.2 Corc 3.030 5.26 .13 .16 Total Out 33.0 3r.9 +t.t Y 2.5t9 3.78 .15 .20 Degrees of Freedom 150.0 *Not fertility. 132.0 **Decade of birth No differences are significant.

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TABLE 22 SCHOOL GRADE CO\TPI-ETEI).I'HL,IROF PERSONS AFTECTED WI-[H DE,NTINOGENESIS I^\IPLRFECTA Arr-l) UNAFFEC]'I-DSIBLINGS BY SEX

trf ales F emale.s Lln- Dif - Un- Dif- Name Aff ected alfected f erent sie. Affected aff ected f erent sie. * A 6.3 8.3 2.0 .05 8.0 8.2 fi9 None B 5.8 8.0 92 .05 /.o /.u 0.2 None Other Core 6.8 8.0 t9 .05 7.9 7.2 -4.7 None Total Core 6.4 8.0 1.6 .01 7.8 -0.1 None ()9 Outside r0.0 -0.8 -. l0 8.4 10.0 1.6 .10 Total 7.8 8.3 0.5 .05 1.6 7.9 0.1 None Cases 32 29 39 49 None progeny are includecl in the calculations. differ in their mating patterns. The expla- The degreesof freedom column in the fer- nation for this differential male fertility is tility tablesbelow represenrthis population not obvious and not at all amenable to base.The first and most obvious phenome- proof by the kind of evidencethat has been non to be found in the fertility tables is the gathered in this study. Only the grossest constant and considerable difference be- sort of surmise can be offerecl as to the tween the affected and their unaffected sib- calrseof differential. It is not at all clear lings. The differential consrirutes abour rvhether the clifference is psychological, 15% of the total number of offspring. social or biological. Horvever, some hint Tables l8 and 19 break down the fertilitl' that this high affecteclmale fertility mav be data by sex. They clearly demonstrare the clue to psycho-socialfactors is obtained, if same phenomenon as found in the segrega- graclecompleted in school is a measure of tion analysis above. The affected uncl un- these factors. Table 22 indicates thar af- affected females do not shorv a significant fected core males complete on the average difference. It is enrirely within rhe male 1.6 grades less than tht:ir unaffected sib- population rhar the difference resides. lings. There is a known differential fertilitl, Two other findings of clental interest between ingroup matings an(l outgroup shotrlcl be mentioned. A correlation was matings (Table 20). If a higher proporrion founcl between PTC raste ability ancl clen- of unaffected siblings marriecl outsicle the tal caries such that tasters had approxi- group than affected siblings, this coul(l rnatell' 28 percent loH'erclef rates than non- account for this clifferential.Table 2l is an tasters (Chung et al., 196+) (Table 23). analysis of that hypothesis.Ir shorvs that OPaque n'hite sl)ots on enamel of teeth the affectedancl trnaffecteclsiblines clo not (Ttrrner's teeth), not associateclwith hieh

TABLE 23 CARIT-S EXPERIENCE IN THE PRI\IARY DE\TITION OF -I'ASTETHE CHILDRE\ IN THE I]RANDY\\'INE POPT'LATION, RY ARILITI' TO PTC

N on-tasters T ast ers IlIean Number of del Teetlt Age Number ill ean l{ttn ber f,l earr Pe rcent in Years Examined def Examined del Dif f erence r-2 93 .3 5l.l 3-1 5l 2.6 r44 1.8 30.8 5-6 49 3.9 172 J.l 20.ir 7-8 9q c.t r77 J,.) 10.8 Percent Caries Free Perccnt Percent Number Caries l{umber Caries Examined Free Examined Free o-4 83 55.4 256 66.0 r6.l 5-9 98 25.5 461 32.5 21.5

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TABLE 24 WHITL, OPAQUE ENAMEL SPOTS (Turner's Teeth) BY SEX AND ENDOGAIIY INDEX IN f'HOSE RL,CEIVING DENTAL EXAMINATIONS

Females Males

En' No. No. No. No. Total % dogamy Exam- Posi- Exam- Posi- Exam- Posi- Index ined tite 10 ined tiue % ined tiae ,e 92 0, 1 320 23 297 t.t 617 l.u 99 I 315 JI 10.8 236 9.3 551 10.2

J 890 u9 13.4 759 90 I1.9 I 649 12.7 Total r525 176 l l.5 r292 135 10.4 2817 ll.l x, - 13.048P < 0.01.

ingestion of fluoride, showecla statistically fected siblings contributed significantly to significant associationwith endogamy in- the apparent increasein the frequency of dex at lessthan the one percent level (Table this trait by generation in addition to the 24). The endogamy index cloesshow a cor- initial contribution made by the founder relation with the coefficient of inbreeding, effect. It is suggestedthat psycho-socialfac- but caution shoulcl be taken in ascribing a tors may be involved in this clifferential direct associationof white spots with an male fertility. inbreeclingeffect. The phenotypic frequenciesfor clentino- genesisimperfecta (5.6 percent) ancl tyrosi- Conclusions nasepositive albinism (1.2 percent) are the The Brandywine isolate had its origins highest reported for any population, and in matings between Caucasians,Negroes, the gene frequency for sickle-cellhemoglo- and possibly Amerindians from 1660 to fin (0.1045)is the highest reported for a 1800.Several family lines contributed to the U. S. population. isolate which was a recognizedethnic seg- Chilclren who were tastersfor PTC had ment of the population of southern Nlarv- 28 percent lower clef scores than non- land by 1790.At this time, the isolate num- tasters. bered from 150 to 350 persons. Genetic \Vhite opaque enamel spots showed a markers inclicate that the present genetic statisticallysignificant increasein frequency pool cannot be a simple aclmixture of rep- with an increasing enclogamyindex of af- resentativeCaucasian, Negro, and Amerin- fected persons. dian gene frequencies. The phenotypic ancl gene frequenciesfor It appears that the Branclyr,r'ineisolate normal ancl the more frequently observed owes its peculiar gene distribution in great abnormal traits are given. measure to the founcler effect. In addition to the founder effect, other factors have Acknowledgments contribllterl over a period of generationsto The authors are indebted to N'Irs.Hazel the establishment of a gene pool that is Ryon Dyson and NIrs.Shirley SkeneRLrtters unique for the Brandywine isolate. A sig- for collection of the interview data. N{r. nificant segregationbias in favor of persons blood Webster C. Leysholl for -[oaneroupings, affecteclwith dentinogenesisimperfecta was N{r. Ronald Robinette and N'Irs. Gaiser founcl in various comparisonsof the data Eckert for hemoglobin electrophoresisancl which coulcl not be accounted for by the haptoglobin determinations,ancl Dr. Robert usual methods of ascertainmentcorrection O. Wolf for secretor cleterminations.Dr. or by illegitimacy. This bias was of greater Donald L. Rucknagel ancl Dr. Laurence magnitude in favor of affected males in Schneidermandirected the medical exami- each generation (Table 12) than for af.- nations and the collection of the hemo- fected females.Analysis of the fertility of globin data' persons affected with dentinogenesis im, REFDREN.ES perfecta shows that differential fertility of Allison, A. C., B. S. Blumberg and ap Rees. 1958. Hapto- globin types in British, Spanish, Basque and Nigcrian affected males compared with their unaf- African populations. Nature (London) l8I:825.

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il 402 WITKOP,et al Ala. J. Med.Sci.

Ilarnicott, ^\. A. and S. D. Larvlcr. 1g53. A study of the Ikin, \V. \{'. and Lewis, Kell and Lutheran, p A. E. N{ourant. 1912. The frequency of and blood group st.stems the Kidd blood-group and the ABH secretion in anrigen in Africans. NIan b2:24. West African negroes. Amer. Ingram, V. NI. J. I'h1s. Anthrop. n.s. ll:83. I958. Abnormal human haemoslobins. I. llarnicott, N. A. I'he comparison of normal hurnan and sickle-cEll haemo- 1950. Taste deficiency for phenylthiourea "fingerprinting". in African Nggloes and Chinese. g!.oPilr by Biochem. Biophys. Acta. _ Ann. E,ugen. it:Z+A. 28:539. Beale, C. L. 1957. American triracial isolates,their status Kcllv, C. H.- an.d pcrtinS!!q to genctic research.Eugenics .1. W Lawlah. 1946. Albcrs-Schiinberg .,,and euart. 4:1g7. discrse-a familv survev. lleguez, A. I9{3. Ncutropenia Cronica }{aligira }'amiliar 'prernarltr['.esiOeniialRadiologv 47:b07. Kennecll',. R I943. propinquity con Granulaciones Atipicas de los Lcucocitos. Bul. Scc. J. \. Ctrbana Ped. l5:900. _,,and_ ethnic cndogamv. Anr. .]. Sociol. 48:580.^ Klapthor, \[. l]., anrl I'. D. l]rilr.n. Chalmers,J. N. Nf., E. W. Ikin ancl A. E. lfourant. l9:r3a. Tlte Hi.story of Oharles Countl', Iluryland. Laplata, The A-BO, IINS and Rh blood groups of the Nigerian.s. \larlland. Charlej County Ann. [,ugcn. l7:168. Tercentenarv, Inc. pp. 204. Krooth, R. Chalmers, N. NI., E. W. Ikin S. 1955. Use of the fcrtilitics of affected individ- J. and A. E. Nlourant. lg53b. uals and A-s.tudy of two unusual blood group thcir unaffectcd sibs in estimation of fitness. antigens in \\'est Am. Huntan Africans. Ilrit. Nfed. 2:178. J. Genetics n.o. 4, 7:325. J. Kugelman. T. I). and E. \'an Clrcrnoff..{. I. and J. C. Liu. 1961.The amino acid corn- J. Siott. lg(il. Tr.rosinase position of hcmoglobin. activit! in melanocltcs of hrrman albinos. J. Invest. Der- II. Analvtical Techniqr.res.Blood mar.37:73. I /:t)-i. Xqlkg!, H. c., R. Cepcllini, Clrorln, B. and N{. Lewis. 1953. The ABO, N{NSs, p, i?lt. t.. \Iulter-Eberhard and J. \\'olf. I957. Obserrations on rhe nrinor Ltttheran, Kell Leuis, Dulf1, and.f, jdd blood g.oupi ar.1 basic hemoelobil"n componcnt in the blood of normal indiriduals pa- t-Le secretor sratus of the Blackfoot Indians 6f Albe.t", an? phys. ticnts rvith tlralassemia. J. (llin. Inrcsr. 36:1615. -..(lanada.{mc_r. J. Anthrop. n.s. l l:369. [.andsteiner, (,htrng,.C. S.._C. IVitkop, K., W. R. Strlltton and II. \\'. Chase. 193.1. J. -caiiesJr. and J. L. Henn,. l96{. A An gcnetic srudy of dcnrat rvith" (o _agglutination reaction rtith some human bloods, spccial referenre

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il Vol. 3, No. 4, 1956 FINDINGSIN THE BRANDYWINEISOLATE 403

yrhorcsisof human hemoglobins rrith special reference to hacl a verY lalge roll. This partictllar trait scentctl the incidence and clinical significanceof hemoglobin C. to be inherite

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il GeneticalInvestigations In Mental Refardationi

Mental retardation is a phenotypical trait qualitatively different origin. Here mental or symptom, usually defined in terms of re- retardation, usually severe, has resulted sponse to an intelligence test. The border- from major lesions inflicted on the brain lines between normals and retarded are during development in an individual who conventional and related to the distribution was predestined for an IQ anywhere within of IQ scoresin the population from which the limits of the normal variation. the tested individuals have been drawn. Such lesions may be caused by a variety By any definition, mental retardation has of genetical as *eil environmental fac- a complex origin in which a variety "r of en- tors. In this context, however, the genetical vironmental as well as genetical factors are determinants are mostly specific major gene instrumental. A critical evaluation of the mutations or chromosome mutations which extensive literature indicates, firstly, that provide the basis for the additional two genic factors constitute significant causesof types of genetical mental retardation. This variation of tested intelligence and, second- description is no more than a useful ap- ly, that these factors are of three different proximation. A considerable overlapping is kinds. to be expected between the curves as genet- The first type of genetical differences ical and non-genetical brain lesions occa- causingmental retardation is of a quantita- sionally may lead to slight intellectual im- tive nature. The accumulated familv and pairment, only. population data on the variation of iested The possibility of a disadvantageousef- intelligence strongly support the explana- fect on intellectual developmenr of a single tion that the genetical component of the dose of the recessivegene for microcephaly variance is polygenic, i.e., representing the which was suggestedin one of our earlier combined effect of several pairs of genes. investigations (Bddk et. al., 1953)has been In consequence, one group of the men- studied in more detail by Kloepfer et. al., tally retarded should represent the minus (1964)in three communities in Louisiana. variates below a conventional level of the The observations, distribution of testedintelligence. including psychometric tests of patients, relatives and a control Mental retardates of this type are quan- group, clearly indicate that the heterozy- titative variates only. From a biological gotes averagedbetween ten and twenty-five point of view they belong ro the samepopu- IQ points less than the controls. lation as those scoring higher IQ values-. Consider- ing the incidence of genetic microcephaly, Since the early investigations of the dis- estimated at one in twenty-five thousand tribution of IQ scores in random popula- births for every microcephaiic there would tions some fifty years zgo, it has been be about three hundred heterozygotes. known that this distribuiion is, in fact, Even if the lowering of the slightly but significantly skew. More vari- IQ test response of the heterozygoteis ates than expected fall below an Ie value relatively moderate, the total of 50. The explanation of this skewnessis loss is more significant on the popula- an accumulation of minus variates of a tion level than the loss through the micro- cephalic lTlr..rt.d patients who will remain very few before the Inrernarional Seminar on Medical Genetics,_Septem!'erl, I966. Universiry of elabama M;Ai;;i in numbers. Uenter, Birmingham, Alabama. 2 Institute for Medical Genetics, Uppsala, Sweden. The results of those investigations need

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il Vol. 3, No. 4, 1966 GENETICALINVEST.IGATIONS 405

further confirmation in other populations. such determinants are associated with easily However, they are of sufficient interest to recognizecl clinical synclromes or with rather sug€iestsimilar stucliesof heterozygotesfor uncharacteristic phenotypes. Investigations other types of well clefineclrecessive condi- of isolates, not selected because of previous tions associateclwith mental retardation. knowledge or hints about the occurrence of In the followine, I shall limit my cliscus- many patients with a peculiar disease, are sion to one type of genetical investigation likely to provicle adequate material for using an epiclemiologicalapproach for the genetical analysis of mental defect. study of rare recessiveconditions. Investigations of morbiclity and mortality One of the important objects of the in- in geographically relatively isolatecl com- vestigations on mental retarclation is the munities is just one of the approaches in differentiation of the patients in diagnostic genetical epidemiology. In such isolates the or preferably etiological entities. genealogical analysis often shorn'sthat the For the detection of clinical ancl geneti- major part of the present population origi- cal entities a specialtype of family data is natecl from a small number of ancestral particularly useful. The rationality of this pairs perhaps 5-7 generations earlier. If so, approach is that specificmajor gene nlirtil- it indicates a relative biological homogene- tions, in particular rare recessiveones, by ity as compared to surrounclins popula- chance may accumtrlate in geographically tions. isolated subpopr.rlationshaving a relatively Sweclen is one of the relatively few coun- small gene flow. tries in which opportunities for this type of Epidemiological investigationshave often epiclemiological investigation still are ex- revealed accumulations of otherwise rare cellent. Reliable parish registers are avail- diseasesor defectsin isolated communities able starting from the micklle of the lTth (Symposium, 1964). If analysis of family century and they include special registra- data from such an i:olate showsthat a par- tion of indivicluals rvho are blincl, cleaf- ticular condition is inherited, it is also very mute, epileptic, mentally retarclecl or men- likely that all affected individuals carry tally ill. identical copies of the genetical factors re- An example of this kincl of work is an in- sponsible for the conclition. This means vestigation undertaken in three North- that we are dealing with a clinical and Swedish communities locatecl about 60 miles genetical entity. north of the Arctic Circle (Book, 1953). The On the other hand, inclex cases(propo- total population was about 9,000. The reg- siti) selectedfrom hospitals or institutions istration of the mentally retarded was lim- serving large population groups, who ap- ited to those who had a severe defect (upper pear to belong to the same clinical eniiry, IQ value of 60-70). One of the main objects may neverthelessconstitute a sample of of the investigation was to look for clinical- mixed etio-geneticalas well as non-genetical ly distinct types which were more likely to entities. be founcl among the low-grade defectives. Our knowledge of rare recessivedisorclers As a result of the fielcl work, a total of 99 comesto a large exteni from isolate stirdies. caseswere registered as living on the census Most of these conditions are characterized da,v. The prevalence figures were for males by outstanding, if not always,unique, clin- l 2 percent ancl for females 1.0 percent. ical signs. Epidemiologically, and geneti- As expected the majoriry of these patients cally it is quite clear that the isolate studies did not show any characteristic clinical are heavily biasecl,but this bias can be uti- signs (i.e.75 percent of them). Ten percent lized for special purposes.The fact that iso- had Down's syndrome. f-he remaining l5 lates give accessto etio-genetical determi- percent, however, were different. All had a nants of relatively homogeneityhas perhaps congenital and stationary condition charac- not been sufficiently appreciated. More- terized by symmetrical spastic motor r-lefect.s, over, it makes no difference whether or not predominantly of the legs and severe mcn-

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il 406 BOOK Ala.J. Med.Sci. tal clefect. No eviclence of environtnental tions were unclertaken by the same investi- causation was founcl. gator, Dr. T. Sjiigren (1966)' As the population of the three parishes When the pecligrees of these Patients increased to 18,500in 1960, the follow- \,\''eremarle up, it lvas founcl that all except hacl was limitecl to two of them rt'itll a one belonsecl to the same peclieree complex. up study population of 13,300in 1960. The eenetical-statistical analvsis eave suffi- cient evirlence for the concltrsion that this The new registration raisetl the total \,\''asa conclition causecl b1' a sinele recessive number of proltositi and secontlary casesto gene clifference. We felt jr-rstified to regard 318. Of these 66 were exclucleclbecause of this "senetic spastic oligophrenia," as it oc- milcler forms of mental retarrlation (IQ) currecl in this population, as a clinical and 75), not beine mentalll' retarclecl at all, or genetical entity. The investigation hacl alscr because they belonsecl to specific cliaenostic shor,l'n that there are conclitions of specific catesories. Some further 50 1l:rtients \vel'e genetical orisin u'ithin the heterosenotts excluded for the onh' reason tl-Iat the sen- sroup of cerebral spastics, altl-roueh thel' ealosical anall ses sholvecl that they were might occur exceptionall)' onll', in other ttnrelatecl u'ith the orieinal lletlieree colll- populations. plex of 1930. Another exatnple is the following investi- J'he remaining ltatients thetl shotrltl cotr- s:ltion n'hich gave sornervhat clifferent re- stitute a sample of reasonable senetical sults. Some 30 \'ears ago three clifferent homogeneity. The senetical anall'ses clearlr inclicated that tl'reir tnental retartlation :rs North Srveclish lralishes \vere investigatetl b1,Sirieren (1932, 1935). The registration of well as other signs ancl s1'rn1)tomsprimalilv all lou,-gracle rnentall,v clefectives cor.'erecl clepencleclon homozrgositl for a rnajor le- the per-ioclof 1900-1930ancl was basecl on cessivegene rnutation. entries in parish resisters,hospitals, institu- The identification, in this isolate, of a tions for the mentalll'retarclecl as well as on practicallv aclinical fonn of rnental retarcllt- personal ficlcl studies.A total of 180 patients tion of the rnajor gene tvl)e also carries belor-rgine to 134 families lvere tliscoverecl some general significance. The results strp- in the tltree parishes with total popularion port those of Roberts (1952) inrlicating thc of 10,400in 1900 ancl I5,900 in 1930. existence of rnajor senic as r'vellas poll'genir' in the reeion of overlaplling IQ's It was I'ounrl that 1-11of these ltatients causation were clistributecl on 102 diffe''ent I'arnilies, for the mentalll' retarcletl t'eurainitlg a[ter' of no\\' l'ecogniz:rble sllecifil' all rlescenrling from a ferv ancestral ltairs tl're exclusion living in the rnicklle of the lTth century, cliagnoses which \,\':lsas far back as their genealogies Further investigations o[ rtuselecterevalenttvl)es of nletr- seste(l a simple recessivetr,pe of inheritance. tal retardation are likelv to provicle aclcli- f-his "X-sjii oligophreni:r" lvas a congenit:rl tional samples o[ patients of consirlerallle ancl stationary, conclition. JIost p:rtients etio-genetical houroseueitr'. Strch samllles were rrnable to rearl, rvrite or count. Their shoulcl be rvell suitecl for ftrrther biocherni- speech r,v:rsrlysarthric. Their 1;h1'sicalclevel- cal, o'toeenetic'al ancl other special stuclies ol)ment was remarkably normal, in partic- b1' r.r'hich more knor,r'lerlsec:ottlcl be eainecl ular when cornparerl H'ith the c:ornrnon al)- abotrt specific sertetical catls:ttiotto[' ment:rl pearance of lorv-gracle clefectives. None of retarclation. them hatl malformations. I have linritecl this clisctrssiot'tto some ex- Neurological examinations \\'ere normal arnples of a spec'ial ty'1>eof epiclemiolosical lrr-rtc:huracteristic for most l):rtients \4'aslr investigation in tnental retarclation. Hotv- pithecoirl l)osture. ever, I shoulcl like to conclutle by acltling Drrring the last {'er,r'},ear.; :r lollon,-u1t in- that epiclemiological surve,vs of mental re- vcstisation has been macle in this isol:rte tarclation are of crucial importance in orcler :rnd thc fiekl rvork ancl clini<'ul examina- to ltrovicle :rrlerluate care an

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il Vol. 3, No. 4, 1966 GENETICALINVESTIGATIONS 407 the mentally retarcled in any population. har,e an interesting obserr,ation in their studics in Such surveysshoulcl preferably be concluct- Brazil, nanely an increased frequency of matcrnal inbreecling, an association bctween inbrecding antl ed by teamsof geneticists, psychiatrists,ps1'- earlv fetal loss u'ithout an association bctwccn pater- chologists, social workers an(l other sl)e- nal inbrcecling ancl early fetal loss.I think this raises cialists. the interesting possibility that the inbred mothcr REFERENCES might not be a very good host for fetal dcvclop- rncnt, possibh' bccause of homozygosity for rarc rc- Book, J. A., 19ir3. A gcnetic and Neuropsvchiatric Investi- gation of a ,t-orth-Suedish I'opulation, u'ith Special Re- cessivegencs. I u,onder r'r,hctherin your stttcliesvotr gard to Schizophrenia and Nlental l)eficiencl . Actir genet., have obserl'ed a historv of matcrnal inbreerling lnorc Basel 4:1-100. A Genctir: :rnd Neuropsvchiatric Investiga- tion of a North-Srvcdish population. I,arr lI. N{ental Dc- frequcntlv than paternal inbrccding in tl'rc parents ficiency and Convulsive Disorders. Acta genet., Bascl of mental clefectircs? 4:345-4r4. Bdok, A., Shut, and S. C. Reed, l9r'r3..A. Clinical and J. J. think I calt alls\\'er that onc. Genetical Strrdv of Nlicrocephalv. Amcr. Journal Ilcnt. nn. SOOX I

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il ClinicalGenetics At A PopulationalLevel The Ethnicity of Diseasein the UnitedStates*

Victor A. McKusick,M.D.**

I have chosen to alter the title of this simpif inheritecl clisorclers,is likely [o t'ar;' presentation to reacl: Clinical Genetics at among clifferent srouPs ancl indeerl lor a a Populational Level, with a subtitle: The number of examllles is knorvn to clo so. Ethnicity of Disease in the United States. Furthermore, clifferences in the frequency Lest anyone think he has been clrau'n here of commolt clisortlers of multifactoriirl on false pretenses, let rne hasten to state causation, that is, those in which genetic that what I am going to talk about is the factors are involvecl as contributir-rs or same as it rvoukl have been uncler the susceptibilitv factors, are to be exllectecl, previous title. I hope tl-rat talking about although the eenetic basis of interethnic the etl'rnic rlistribution of cliseasein the clifferences is harcler to establish in these United States is not too parochial an ap- cases because it is never certain th:rt ell- proach for this international seminar. vironmental factors, rvhich are also in- ('oltl- Except for the Inclians arnong uS, we volvetl, are itlentical in the grouPS Americans have come here from all parts parecl. of the globe, in the last four or five cen- I am usius the rvorcl cthnic here in thc turies, more from some parts than from getreral serlseto inclicate both racial groul)- others. Furthermore, clespite the melting ing ancl socirrlgrotrltine. Since the tu'o, otre pot metaphor lve ltave remainecl to :r con- biologicallr brtsetl, one culturallv b:tsetl, siclerable extent in separate groups accord- :lre essentiallv aht:n s irtclistinstrishable,otre ing to etl'rnic extraction. Ir-r part this is :t terrn uav be ir-rclic:rtetl. Coming from the consequence of the I'act that insufficient ()reek rvorcl I'or n:rtion, r:tltrlic furtherlnore time hzrselapsecl for mixins, but itr an ap- avoirls the ocliun-tol race atrcl has suf{'icicnt- preciable rlegree is attributable to religious lv general c'otrtrot:ttiotrsto be a1ll>rollriltteitt anrl other social impediments to mixins. the usage here. That people of clifl'erent extr:rctions cliffer --\ stuclv of the cthnicit,v of cliseaseh:ts in many physical characteristics anrl that pr:rctical usefultressin cliagtrosisancl rnatr- many of these clifferences are based orl agement antl it has usefulness,for examltlc, differences in genetic constitution are mat- in the clesignol'screening prograrns. It h:ts ters that no one can clispute. Genetic dif- potential usefuluess to genetic tlleon' b1' ferences extenrl, of course, to clifferences in provicling tl're points of cleparture for stutlv the frequency of pathogenic senes. Because of factors rvhich influence the frequenc\' of of the genetic cliversity of the Anrerican ger)esin poptrlutiotts. people, clifferent groups are likelv to shorv Research on the ethnic clistribution of clifferences in frequency of given cliseases. cliseasecan Lake three or four different a1t- The frequency of N'Ienclelizing, that is, proaches. In the first place a specific ethnic group catr be surveyetl for all disease. This * Prcscntcd art tlrc Intcrnational Seminar on lfedical is the ap1>roach,for exarnple, that lr,'ehal'e Ccnti

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il Vol. 3, No. 4, 1966 CLINICALGENETICS 4W group from most other American ethnic several times in this country, involves com- groups. In the second place, total ascertain- parison of an ethnic group in this country ment or random ascertainment of casesof a rvith close relatives, or at least the same specilic entity can be attemptec\ tor a siven ethnic group, who remainerl in the country area, and the ethnic extraction of the pa- of origin. Thus, .|apanese in the United tients cleterminecl. This approach lve have States and .fapan have been compared, used in connection with homocystinuria Irish, Norwegians, Italians in the United ancl with Riley-Day dysautonomia, and I States and Europe, and so on. This ap- will describe those studies to ),ou. proach has been used in connection with A thircl approach combines the first two. common disorclers, which ar the best have i\ senetic survey of a given population com- complex genetics, but one can point to posed of several genetic stocks, such as r,r'e even more useful stuclies such as those in- have in most are:ls of this country, can be volving the Nlecliterranean and African macle, ancl the frequency of cliseasesin the types of G-6-PD deficienc,v, rhe hemo- various components of the population com- globinopathies, the recently studied adult parecl. This approach we have initiatecl on intestinal lactase deficiency, a srate of high :r moclest scale in Washington County, frequency in the American Neero, all these X'Iarylancl. This county has been the sub- I-ravingbeen studiecl in both the parent and ject of various epiclcmiologic stuclies for the American population. Acatalasemia or close to 40 years. It has a relatively stable at least I'rypocatalasemia,rvhich has been so population of about 100,000, about half nicely stucliecl in .|apan by Hamilton and of which is in the urban county seat of his colleasues (1961), coulcl perhaps be Hagerstolvn, the other half living in small stucliecl n'ith interestine finrlines in Japa- villages ancl on farms. Ach,antages of \{,rash- nese-Americans. ington County for total survey of this type I shall confine the rest of my remarks to inclucle the fact that one hospital provicles illustrations of tl're first two approaches: the majoritl' of care. An early result of a the total stucly of a specific ethnic sroup survey of neuroloeic disorclers in \Vashins- ancl tl-restucl1' of a specific entity as to ethnic ton County is the fincling of a relatively tlistribution. larse number o[' cases of a clominantly in- The heritecl t1'pe of amyloiclosis, perhaps iclenti- Old OrderAmish cal to that clescriberl by Rukavina ancl col- \\'rhen one stuclies senctic disease in a leaeues (1956) in an enclave of Srvissorisin specific sroup, one shoulct familiarize one- in Incliana. The \\rashington County cases self r,t'itlr the char:rcteristics u'ltich Professor are of lrre-rcvolutionary Gerrn:rn irnmigrant Cavalli referrecl to as the eenetic structure extraction. In connection rvith tl'resturly of of the population ancl n'hich Dr. Witkop amyloiclosis in the \\rashinston County :rlso nicelv illustratecl. One should be fa- groul), r,r'cltave been impresseclrvith the fact miliar with the demoerapliic history of the that a total population survev has broueht eroul) in the Old \\rorlcl ancl with the to lieht cases lvhich clearlv represent the history of nrieration (both the history of same entit)' but r,t'ere consirlerecl sel)arate immieration ancl that oI internal mi- entities ltreviously. Becausc of the rather eration). One should be fanriliar with the variable m;rnifest;rtion of rlominantly in- sociologic f:rctors such as relieious ancl heritecl amyloiclosis of this tvpe, these cases others which make either for clispersion or previously existecl uncler tlte suise not onlv cohcsion of the group anrl of course one of a proeressive treurol)athy but also of shoulcl be familiar rvith the current clemo- arthropathy because of the pain ancl stiff- graphy c.f the sroul). The sroup that I ness of the hancls resultine fronr the czrrpal lt'oulcl like to tell y'ou a little bir about-ir tunnel syndrome. n'ill come as no surprise to you-is the Okl A fourth approach ro the studv of Orcler Amish. ethnicity in rlisease,lvhich has been usecl I woulcl like to beein rr rlescription of

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il 410 McKUSICK Ala. J. Med.Scr

cation; they educate their children only to the minimum required by the state, and are opposed in general to consolidated schools, preferring the one-room schoolhouses, many of which are operatecl as parochial schools. All the Amish norv live in the United States or Ontario (Fig. 2). Flowever, the sect originate(l in the Canton of Bern in Sr,vitzerland in 1693 as an offshoot from the olcler antl more extensive llennonite move- ment. .facob Ammann, the Mennonite bishop who founclecl the sect, pickecl up converts among other llennonites, particu- larly those who hacl left Bern to go to Alsace ancl Lorraine ancl the Palatinate. The Amish movernent was a lnovement within a movement ancl geneticallf its adherents were mainly Bernese Su,iss. The Amish began coming to this coun- try about 1720. A consiclerable portion of them, although the absolute numbers were small, came betu'een 1720 and 1770; essentially all the Amish of Eastern Penn- sylvania are of pre-revolutionary ancestry. Fig. l. A pictorial essay on the sociology of the Amish. Later waves of imrnigration continued until about 1850, the later immigrants seeking the group by showing )'ou m?v most prized residence to the \\'est. This difference in picture (Fig. l), which is a sort of pictorial the details of the migration history prob- essay on the sociology of the grouP. The ably has somethins to clo rvith the fact that group is well known, or at least wiclell' one today can recognize separate clemes known, because of their peculiar sociologic within the larger Amish sroup. and technical practices in the miclst of modern society (Hostetler, 1963). The married men wear beards but clo not have mustaches. They \ 'ear a stvlized form of dress. All the Amish are rural living, and most of them are farmers. They do not use moclern mechanized agricultural equipment but use horse-clrawn equipment for the most part. They use the horse ancl buggy for transportation. If there is one characteristic rvhich dis- tinguishes the Old Orcter Amish from other conservative Protestant sects u'ith lvhom they are often confusecl, such as the con- servative Mennonites, the Dunkards, ancl so on, it is the practice of the Old Order Amish to hold religious services in the home. They do not have meeting houses but hold religious services in the homes by Fig,. 2. Distribution of Amish settlements. From rotation. They are opposecl to higher edu- Hostetler.

1-

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il Vol. 3, No. 4, 1966 CLINICALGENETICS 4tl

Over 80% of the Amish live in three clannishnesscan tell you other Amishmen states,in Pennsylvania,Ohio and Indiana who have an ailment similar to the one and over half the Amish live in three coun- you may be dealing with. The con- ties, Lancaster County, Pennsylvania, sanguinity rate, which we are in the process Holmes County, Ohio, and LaGrange of quantitating, appears ro be high. Il- County in northern Indiana. The two legitimacy rate is low. There is a certain counties we have focused on most exten- amount of premarital conception, but the sively are Lancaster County and Holmes biological father, by all indications, in the County. These have been very useful for overwhelming majority of instances,is the comparison purposes. The total Amish legal father. Families are large, averaging population is estimated to be in excessof about eight chilclren per family and the 45,000 now. Each of these two counties, grolrp is immobile. The families do not go Lancaster and Holmes, has about 9,000 off to cities to seekemployment and so on. population. LancasterCounty is closeto us These are great aclvantages in genetic in Baltimore, so that we can get there in studies these days when so frequently about an hour and a half, put in a good families are scattered hither and yon. If clay'swork and be home by u reasonable one is studying mental retardation, as we hour in the evening. have been, it is an advantase,too, that the Table I presents a list of some of the Amish take careof their own at home. They characteristics of Amish society which do nof institutionalize their retardecl and render the group useful for some types of other defectives. genetic study. In the first place, it is a de- I shall next show you the eviclencethat fined population, a self-definedpopulation. in the overall Amish group one is dealing In the seconclplace, it is a closed popula- with a number of separatedemes. I think tion. The Amish do not proselytize and this word "deme" is a rather useful one. for practical purposes no new blood has It was introduced (in recent times, at any entere(l the groups since the immigrations. rate) by George P. N{urdock (1948)to refer Origins in Western Europe are well known. to consanguineal kin groups, or local Genealogical records are delightfully ex- endogamousgroups and that is what these tensive. The standarclsof living and the several Amish groups represent, the Lan- standards of meclical care are relatively casterCountv group being one, the Holmes high. There is a greater uniformity of County group being another, ancl so on. socioeconomic circumstances than one The eviclencefor the existenceof separate usually encounters. There is a great, some- demesis of at least four types: In the first times almost morbid, interest in illness. place the rnigration history ancl the history They are interested in what ails other since the migration; in the second place, Amishmen and parrly because of their the clistribution of surnames; thirclly, the clistribution of diseases,parricularly the fre- cHARACTERrsrrcs.;:fi;"t socrETyFAvoRABLE quency of rare recessivesin the separate FOR GENTIC STUDIES groups; and fourthly, blood group differ- ences. Table 2 compares the name fre- I. Dcfincd population 2. Closed population TABLE 3. \\Iell-known origins in wcstern Europc 2 4. Extcnsive genealogic rccords IrA\III-Y NrllIES 5. High standard of living 6. Rclatively high standarcl of mcdical care Holntes Cottnty,Oltio I.ancaslerCounlt, Pa. 7. Uniform socio-economic, including occupational, factors lf -26% -23% 8. Grcat interest in illncss iller Stoltzfrrs Yoder -17 -12d. 9. High consanguinity' rate % King Tro'r'cr -ll% -12% 10. Low illegitimacy rare Fishcr - -12% I l. Clannishness Hershbcrger 5% Beilcr - 12. Immobility Rabcr 5% Zook - tt% - - 13. Large families Schlabach 5% Lapp i% 14. Institutionalization rcsisted 6W" i2%

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il 412 McKUSICK Ala. J. Med. Sci. quencies in Holmes County and Lancaster evident at birth ancl may be quite severe, County. Six names in each case account for requiring exchanse transfusion. Probably 701" of. the population, but they are differ- all casesdie by age three years if not treated. ent sets of six names. When one gets down Splenectomy has clramatically beneficial to names of lower frequency, there is some effects, convertine the process into :r com- duplication in the two groups, but in the pensatecl hemolytic state which cloes not re- high frequency names, there are these clif- quire transfusion and is otherwise not in- ferences. It is of interest that the name capacitatine. The other cases of pyruvate Stoltzfus accounts for one-fourth the Lan- kinase cleficiencl' that have been clescribecl caster County Amish-interesting because have, in many instances, been quite milcl there was only one Stoltzfus, Nicholas ancl not cletectecluntil arlulthoorl. Stoltzfus, who immigratecl in 1766 ancl who In Hohnes C.lounty, Ohio, hemophilia B, must have contributecl clisproportionately or Christmas clisease,is quite frequent. This to the present sene pool, just as he gave his is an X-linkecl recessive, of course, but the name to an unusually large proportion of arsument here is the same. Hemophilia B the present population. has not been iclentifiecl in eastern Pennsyl- The followins are examples of cliseases vania Amish. which have relatively high frequency in ln '\clams ancl Allen Counties, Incliana, four Amish eroups which appear to repre- the autosomal recessive limb girclle muscu- sent separate demes. The Ellis-van Crevelcl lar clystrophy is quite frequent. This has syndrome (McKusick et al., 1964), wl-rich is been stucliectbv Dr. C. E. .|;rckson of Bluff- a form of chondroclystrophy lr,'ith pol,v- ton, Incliana (.[ackson ancl Carey,, l96l). dactyly, dystrophy of the finsernails, ancl It is of interest that whereas in most series in about half the cases laree atrial septal of cases of mtrscular cl1'strophy, the sex- defect, occurs with unprececlentecllr,' hieh linkecl form has been most frequent, such frequency in tlte Lancaster County Amish. is not the case in the Canton of Bern, where There we have 33 sibships in which at least the most frequent form is tl'ris limb girdle one case of this conclition has occurrecl ancl rnuscular cl,vstrophy. I am sure the signifi- at the time these studies were reportecl there c:lnce of this fact is eviclent to vou. were more casesof this conclition identifiecl \\'e clicl a bloocl group frequencl, survey in Lancaster County than hacl been re- in the Lancaster County Amish, studying ported hitherto in all the liter.arure pur to- the blooci groups of a ranclom sanple of in- sether. However, this condition has not clivicluals rlrau'n from the census of the been identified in Amish except those of population. This was a ranclom sarnple of eastern Pennsylvania. marriecl couples, these being taken to be In Mifflin County, Penns,vlvania, lvhich as r-epresentative of individuals as little re- is another Amish group at the center of the latecl as one finds in this group. Ho\,r'ever, state, pyruvate kinase cleficiencl' hemolvtic lvhen householcls rvere visitecl to get blood anemia (Bowman et al., 1965),about lvhich samples, anyone l,r'ho woulcl give a sample you also heard yesterclay, is unusually fre- r,vas:rlso blecl, so that the total sample u'as quent. We now have l2 sibships r,vith this in excess of 700. The figures in Table il condition. I think that a point th:it Pro- are on the ranclom sample, although the fessor Bodk macle is illustraterl rvell by each total sarnple rlid not cliffer greatly. For of these conditions: the aclvantase of knolv- comparison rvith the Lancaster County ing that one is rlealine with the same entity sample, lve hacl ABO ancl Rh clata on ir in each and all of these cases. There is an eroup of Holmes County Amish. It may indication from the study in the I'fifflin interest ancl amuse you to know how the C-,ounty Amish of heterogeneity in pyruvate Holmes Cotrntl' clata r,r'ere collectecl. Be- kinase deficiency. The cliseasein this group cause of the hieh frequenc,v of hemophilia is very severe and rather stereotypecl in its B in the Holmes County group, the Amish severity. Jaunclice ancl anemia are alreacly appreciate the importance of bloocl trans-

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TABLE 3 generational data, but I shall not take the ARO PHENOTPE ]IREQUENCIES time t-o go into those now. IN TWO OLD ORDER ANTISH DEMES You have heard reiterated several times, Canton Holmes Lancaster So. and very nicely illustrated, the fact that Bern Co. Co. Englanrl isolated inbred groups represent a happy N : 1027 N =215 hunting ground for medical geneticists be- U .19 .34 .106 .4+ ;\ .r0 .:)'t . t-40 .45 cause of the increased frequency of homo- B .0'4 .06 .047 .08 zygotes for recessive genes. There is a popu- AI] .03 .06 .r07 .03 lar notion that inbreeding causesa build-up "a of fusion and do give blood freely. So the Recl of genes-usually it's said build-up are aware, Cross donor center had an extensive file. bad p;enes." Of course, as you gene Holmes County is largely a rural county inbreeding Fer se cloes not influence fre- ancl the Amish constitute about 35 per cent frequency. It influences eenotype of the population of the county. We hacl quency because it increases the Proportion inbreeding a complete tabulation of Amish surnames, of homozygotes, but I repeat, gene We so in going through the files, we coulcl per se does not change frequency. to experimental pick out Amish surnames; but since some have in many as compared non-Amish have the samesurnames, we hacl species such as the mouse an anomalous clomi- the problem of distinguishing them. The situation, in that known autosomal ingenious methocl which was devised by nants exceecl known autosomal recessives, is the N{iss N,Iinerva Stauffer, who did these whereas in the mouse the converse 285 studies,was to pick those inclividuals who case. Verschuer in 1959 estimate(l autosomal had Amish names and who, on the donor autosomal clominant traits, 89 not too carcl, had no telephone number recordecl. recessives. The precise figures are I am empha- The Amish clo not have telephones ancl relevant; the ratio is what in 1961 essentially everyone else has a telephone. sizing here. Dr. Nlargaret Green the mouse 35 Thus the 1027 Holmes County samples estimaterl for me that in mutations are known (Table 3) are on personswith Amish sur- autosomal clominant recessive muta- names and no telephone,if you will accept and about 123 autosomal morrse those as Amish! tions. There are perhaps 300 ratio remaining The blooclgroup data in LancasterCoun- mutations now known, the ty show rather peculiar ABO frequencies the same. with a very high type A frequency, three- If I may be permitted to tlo a little ad- fourths of the group being blood type A vertising: I have recently completecl for (seeTable 3). We presume this represents publication catalogs of autosomal dominant clrift but this is only a presumption. The and autosomal recessiveand X-linkecl traits Holmes County group has an ABO blood in man. Two "classes of citizens" have group frequency closer to those of the been maintained in the catalogs: first, RerneseSwiss shown on your left. I might those in which the particular mode of in- say that Rh negativity in both the Lan- heritance seems aclequately establishecl, ancl casterCounty Amish ancl the Holmes Coun- secondly, those in which the particular ty Amish is relatively high, being about 25 mode of inheritance has been suggested but per cent in both groups. There are many as yet not proved. The inclusion of the other interesting ramifications of the Lan- latter seems important for heuristic Pur- caster County bloocl group study. There poses, so that we will keep our eyes open were somerare blood groups and one could, when similar cases come along. In recent by genealogical tracing, iclentify the proba- years, the total number of autosomal re- ble point of introduction of thesegenes into cessiveshave been "coming up fast," maitr- the population. There were other interest- ly through the burgeoning of biochemical ing aspectswith referenceto the correlation genetics ancl the description of new inborrt between blood type ancl family name and errors of metabolism, but autosomal clor-ni-

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fl 414 McKUSICK Ala. J. Med.Sci. nants still exceed autosomal recessives.I the Ellis-van Creveld gene or the Pyruvate list 269 dominants and 237 recessivesas kinase deficiency gene, it is logical to pre- "proved." It is interesting to note that I sume that only one of the founding fathers was willing to accept fewer traits as proven was a heterozygous carrier for that gene. autosomal dominant than was Professor Of course. a consideration that modifies Verschuer in 1959, but the figures are not that presumption is the frequent practice very much different. of brothers and other close relatives to im- The studies in the Amish, like the studies migrate together. This would increase the of Dr. Witkop in tri-racial isolates and the chance that more than one funding father studies of Professor Bcidk in Swedish iso- was a carrier. In the caseof a number of lates, have uncovered new recessives.For the conditions that we have studied in these example, we have a form of dwarfism called populations, we have tracecl back the an- cartilage-hairhypoplasia (McKusick et al., cestry of each and every Parent of all af- 1965), a chondrodystrophy in which the fected individuals, and have been able to hair is fine and sparse. We have found a identify that only one founding father (ancl fatal variety of intraheparic cholestasisin mother) was shared in common by all par- the Amish group. In a survey of major ents. This is a matter that one can do very neurologic diseaseand mental retardation laboriously by hand but which is aided in the Holmes County group, two recessive- greatly by the computer. For example, in ly inherited major neurologic disorders the 33 Ellis-van Creveld sibships this in- which by all indications have nor been volvecl tracing back of the ancestry of 66 previously identified, have come to light, parents to iclentify the fact that only one and there are other "new" recessivesthat I immigrant ancestral couple, Samuel King could cite. I think this type of pursuit re- and his u'ife, was shared by all these par- quires no defense,but if it does, let me say ents. In the caseof pvruvate kinase defici- that this should not be considered merely ency, it u'as again possibleto trace it back stamp collecting, that description of "new" to one ancl only one immigrant ancestral entities provides information on what couple, "strong .|acob" Yoder ancl wife. Lewontin referred to as "the mutational This strengthensthe idea that the conclition repertoire of man." These abnormalities is a recessive.It certainl)' strengthensthe are the indications we have of what the idea that one is dealing rvith one and the normal genetic constitution of man is like. same entity in all the cases.(The chance that 66 LancasterCountl' Amish would by In both the Lancaster County and the chance all trace their ancestry to Samuel Holmes County groups, total censuseshave King or that 24 N{ifflin County Amish been assembledand in conjunction with r,vould all trace their ancestry to Stronp; these,total genealogiesare well on the way is thought to be very lorv, but quanti- to completion. By total genealogywe mean |acob tation of this chance should probably be a tracing back to the immigrant ancestors attempted by computer using the total as completely as possible of each and all genealogies.) members of the community. The purposes to which the total genealogy has been put A study attempting total ascertainmeut are mainly two. One is in the calculation of neurological disorders of major nature of coefficients of consanguinity and the and mental retardation in the Holmes second, in the determination of common County group lvas conducted. There were, ancestorof all parents of sibshipscarrying in addition to the forms of mental re- a given recessive disorder. In an inbred tarclation that have certain clinically dis- group such as the Lancaster County deme, tinguishing features, although thus far no which is a closedpopulation and of which biochemicalones, some instances of familial, the number of founding fathers (and nonspecificmental retardation of severede- mothers) was relatively small, when one is gree. As mentioned earlier, "nevy'" neuro- dealing with arr ordinarly rare gene such as logic disorders were uncovered. Mongolism

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il Vol. 3, No. 4, 1955 CLINICALGENETICS 4t5

was also studied in this grouP with The patients with homocystinuria, like karyotyping of all cases. Sixteen were the patients with the true Marfan syn' found in the population and we estimate drome, have cardiovascular problems of that this is not an abnormal frequency. It major grade, but they are of a different agrees closely with that found in outbred character entirely, being thrombotic in populations. nature, thrombosis in arteries and veins. Common diseasescan be studied usefully We have seen thrombosis of both internal chil- in closed populations such as this, and we carotid arteries in 1Z-and l3-year-old have under study in the Amish l) hyper- dren, death from coronary occlusion in per- tension-that is, blood pressure,2) diabetes sons,.evenfemales, in their teens or early mellitus, 3) congerrital heart disease, and 20's, thrombosis of the inferior vena cava, legs 4) cervical cancer. Cervical cancer came to and recurrent thrombophlebitis of the light as a matter for special study because complicated by pulmonary embolism. of a Papanicolaou program which has been Homocystinuria was first described in under way in Holmes County for about 1962 by a group in northern Ireland who twelve years, and in which an impressively had detected it in the course of a urine lower frequency of cervical cancer was chromatography survey in institutions for turned up in Amish women. This has been the mentally retarded. Simultaneously it looked into further and seemsto be a "true was described by Dr. Waisman in Madison, bill." Wisconsin,who had studied the single case Homocystinurio of a severely retarded infant. Because of background, mental retardation was I shall move now to a consideration of a this a hallmark of disease.This, in study of the ethnic distribution of two spe- considered fact, not to be the casewhen a more cific diseases.The first one that I shall talk proves selected series of cases is about is homocystinuria, which is an inborn appropriately Among homocystinurics ascer- error of metabolism, simulating in many of studied. casesof ectopia lentis, its features the Marfan syndrome. In both tained by screening have unequivocally normal the Marfan syndrome and homocystinuria over 40 per cent Even in the same family re' the patients are often unusually tall and intelligence. homocystinurics may have ectopia lentis and chest deformity. tarded and unretarded Take, for example, the family The cyanide nitroprusside screening test be observed. brother and sister reported was used as the primary mode of ascertain- with affected al., 1965). Both have ment in the survey I will describe to you elsewhere(Schimke et the brother in (Schimke et zl., 1965). This test is, of dislocation of the lensesand tall and has chest de' course, also positive for cystine, so other particular is quite l.g after relatively means rnust be used for distinguishing the formity. He broke a thereafter developed two. High voltage paper electrophoresisof minor trauma and embolus. the urines was the method used to dis- thrombophlebitis with pulmonary graduated tinguish cystinuria from homocystinuria. He is of normal intelligence, college and now We think that all patients with homo- from a state teachers is moderately re- cystinuria have ectopia lentis, by age l0 teachesschool. His sister high at any rate. The dislocation of the lenses tarded; she was unable to complete enzyme assays seems to be progressive. We have a num- school. In both of these sibs by Dr. ber of experienceswhere a patient has had were done on liver biopsy material the demon- competent ophthalmologic examinations Harvey Mudd at the NIH, with low levels early without this being detected. The pro- stration that both had the very i.e., low gressivenature introduces an aspectof hope characteristic of this disease, very is as far as being able to influence the disease levels of cystathionine synthetase,which in by treatment. All the patients seem to have the enzyme whose activity is deficient osteoporosis. They are prone to fractures this condition. also. Facial flushing has been quite impressive,

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il 416 McKUSICK Ala. J. Med. Sci.

W I oh ,l oo

11 n-n ul- W "r* "1" I "r" I * I oo oa O Ol.q" o 5a / / l/ ,a / sTTZ / 14 17 "t.-lFt wrytl I Jn.+ tl. 5'ET-1-o a a*b* // /l / o-ooaJo

20 m i ryrytl I i-i-i-j - =ra ---o --tffi. ,ar:r t Fig. 3. Twenty families with homocystinuric members. From Schimke et al.

particularly in tl-re Britislt cases. I have tlrine samples coulcl be easilv seltt in bv wonderecl, not entirely facetiousll', whether rnail. Arrangements h'ere matle lvith a large the lack of central heating l-rad anything to number of eye clinics over the country to do with the lesser impressiveness of flushed screen cases in the categories mentioned. face in l'rornocl'stinurics in the U.S., but To rlate we I'rave screenetl something in n'ith at least we have at least one Patient with a very recl excess of 700 farnilies, each face. He is a clairl' farmer ancl has exposure one case of this t)'pe, itncl h:tt'e uncoverecl to the elernents, but I think his face is 35 sibships rvith patients rt'ith this con- is redder than 1'ou woulcl expect from that clition. The total number of c:ases 62, alone. He has dislocation of the lenses. I believe. In Fig. 3 :tre frasmentarl' pecli- He is six feet three inches tall, has chest grees of the f irst 20 I'arnilies. Homo- deformity, hy'pertcnsion, hacl hacl a myo- cystinuri:t :lppc;lrs to be a sirnllle recessive, carrlial infarction ancl is fibrillating. This like all other inborn el'rol's o[ ruetabolism man's parents rvere both born r,r,'ithina few of the Garroclian Lyl)e, autl segresatioll miles of each other in the Dalarne area of analysis proves that this is the case. Sweclen ancl althoush thev were not knowll The following is an analysis at the ltoint to be relatecl, lve have suspicions. I woultl when we hacl 50 crrtsesclistributecl in 26 be intereste(l to know u'hether Professor kinclreds, lvith 28 sibshiPs, nvo o[ the kin- Book knows if homocvstinuria has been tlrecls havins t'lt'o uffectecl sibships. Thirtl' detectecl in Su'eclen. I am nof aware of re- of the 50 were male. The agesof the living ports of this conclition emanating frotn patients variecl from 6 to 45 years. Ten hacl Sweclen. rliecl, all of thrombotic complications, \'ary- Norv I arn :rt last gettins to the popul:r- irg in age at death from 3 to 28 )'ears. tional aspect o{' this clisease.\\re untlertook tJsing rigorous criteria ancl throlving into a screening l)l'osram of all cases of clis- the retardecl group an)'one lvho lt'as even "clim," locatecl lenses ancl/or presumecl Nlarfan suspiciously it \t'as founcl that 22 of synclrome fronr lvhich u'c coultl get urirte. the 50 inclivirltrals u'ere of normal intelli-

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C Vol. 3, No. 4, 1966 CLINICALGENETICS 417

TABLE 4 CON{PARISONOF HONTOCYSTINURIAAND THE, NTARIANSYNDROME

Homocystinuria IIarf an Syndrome Inheritance Recessive l)ominant

Skeletal abnormality Osteoporosis,f ractures, Arachnodactyly ancl occasional arachnodactyly loose -j oi n tedness more striking

Pectus excavatum Irequent Frequent or calinatum

Ectopia lentis Prcsent Present

Vascular discase Dilatation witlr tlrrombosis Dilatation and/or in medium-sized artcries dissection of aorta and veins

Skin l{alar flush, livedo Striac distensae reticularis

Mcntal rctardation Frequent Absent

lack gence. Consanguinity was either Proved or of the respiratory mechanism to oxygen suspected in a number of instances, and the and CO2 excess, so that these children can ethnic background of these caseswas quite literally make themselves Pass out by hold- diverse. We were not impressed with any ing their breath. The children should not evidence of heterogeneity in ethnic back- be allowed to swim under water, it is sround. Many northern European nation- thought, because they may, as it were, for- alities, southern Italians, Nep;roes and Jews set to come up. They have episodic vomit- were representecl. We hacl no cases of ing, unexplainecl high fever, skin blotching, Oriental extraction. On the other hand, we excessive sweating, motor incoorclination, screened very few Oriental families. 1;ostural hypotension, and paroxysmal Table IV compares the features, clinical hypertension such that a number of these and genetic, of homocystinuria with those patients have been explorecl for suspectecl of the true Marfan syndrome. pheochromocytoma. Progressive scoliosis is a feature. Among the clinical finclings is FomiliolDysoutonomio also absence of the deep tenclon reflexes. The other condition that we are atteml)t- The facies is rather characteristic with ing to stucly on a total zrscertainment basis transverse moutll ancl tentlent'r'Io facilrl is farnilial clysautonomia, an(l in tl'ris in- drooping. stance the ethnic homoeeneity observecl N,Iental retat-clation is llot lll) illtcsral with the previous cronclition was'not ob- feature of this condition, zts llettl'lv as rvc served. This con(lition u,'as first clescribetl can cletermine. There ma)' be solrle re- in 1949 by Riley, Da,v, ancl colleasues, tarclation, but rve think that it is set'ontlarv pediatricians at Columbia University. Thel to clehyclration, the hieh fever, or tlte: notecl early the strikins preponclerance of breatl-r-holcling attacks rvith ttncotlsciotts- tliseltse. the disease in persons of .|ewish extraction. ness antl not tt ltart of the ltrimarv The features of this disease are listecl here. Some lratients have large, sn'ollell ktlces. The babies often sholv abnorrnality right n'hich :rre quite ltainless, antl reltresetlf, bv from birth with trouble I'eerling ancl sr,r'al- all inclicatiot'ts,Charcot neurol)itthic joints. -I-his trotetl, lowine. Partly because of this, l)erhaps feature has not been previousll' mainly because of this, they get recurrent partl), becauseolcler srouPS of llalients hltvcr bronchopneumonia {rom aspiration. Lack not been stucliecl. Tl'rese patients, I repeat' of tears is an imprcssive feature. Insensi- are relativelv insensitive to llain ltncl lt;tvc tivity to pain, emotional lability, irn(l absent cleep tenclon reflexes. X-rav short's breath-holding attacks with loss of consci- clestruction in the epiphl'sis; the x-r'rr\ ousness occur. It has been clemonstrated in changes o[ Charcot joint clevelol>ins be{'or-c this condition that there is an insensitivity c:losureof thc epiph,vsisis qtrite tlill'erclrt

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il 418 McKUSICK Ala. J. Med.Sci.

9 -r 'r' "1" "T" "l'I I "T'I -l-t TT" lo on l* aTt fr* fla fl1.

Dp *"l- I ryI T + ,1, , ,t T T . .lA T 581 5T F? ol 'frTa ?? ad--a'a ti TJ *l- TTtl ry I ryI oro I F-q flT-l o I m 6-516Tr -T- T d-lt" 55Et + *+ + + "p| "t""ib| | | l- I l= 5a I a 5-,r 3 r offi- ooz=- Fig. 4.-A 164 families in which one or more member has familial dysautonomia. From McKusiek et aI. from that of Charcot joint occurring in an dition is absenceof the fungiform papillae adult syphilitic, for example. from the tongue. The circumvallate A consistent diagnostic sign in this con- papillae are also absent in most, Perhaps

39 ry ";;I +++++I I I I ! J- f d;t -flfrtir1* ..4 5-F T.l nlTTT nl .O

"it +++llllll ++++ A+A . - ++ + l. a t r a al* n a a a a I 5U-T+ dzff!

-: -r -r "l' "t- "t" "]t + T*Ti 5rTAI q-aI q I 6Ta* ffift*l l'- o-"-o I I o-o

tl' I T -:L I T "r"r 5ft Tol* 5Z .oar'n loJI o 5-_A 5. I I 5TE

ryqo + + tt ",.lttttl "-1. "it "it l+ .il1- T oD o not- oltla i*i-lr 1.. noa' I

ae I O DisAUToNor4rA ry . ABoRTToN "T" I a s PR.BABLEo/sAuTottot4tA + I + DEAD L l oo aia i t fT5-6T ? POSSISLEDYSAUTOTDT\,IIA Fig. 4.-B

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all of these cases;the filliform papillae are 4A-D presents fragmentary pedigrees of normal. thesecases. The ascertainment of families with this The Lenz-Hogben analysis on these fam- disorder was accomplished mainly through ilies, which contain 200 cases,shows close the Dysautonomia Association,which has an agreement of the observed number with aggressiveinterest in this condition and for that expected of an autosomal recessive that reason an aggressivecase-finding pro- disorder assuming either complete or ran- gram. In addition, we communicated with dom ascertainment of families. a considerable number of pediatricians and In these 164 families there are 200 af- pediatric neurologists to learn about other fected, 9l male, 109 female. Sixty-sevenof cases whenever possible. We have infor- the casesare dead. In 162 of the families, mation about 220 families and thus far we both parents are of .fewish extraction. In have had a chance to visit and interview two of the families, the mother ostensibly 164 of these families, and it is the findings has no Jewish ancestry; the father is Jewish. in these that I shall tell you about. Figure Among the .|ewish parents, there were only 90 9l 9? 93 TO TO rJ_tt "1" o-o- : o #1

94 95 96 97 "1" *f" T" T I t I fr* il.

99 too lol rp *i" I "1"ll -J-l t 'i tr ol' I c o *Ptoz I aoo Fig. 4.-C

-3-

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il 420 McKUSICK Ala. J. Med. Sci.

two who could not be tracecl to eastern cently Nlyranthopoulosancl Aronson (1966), Europe. These other two cases had an- in connectionwith Tay-Sachsdisease, which cestry in the Rhineland and in Austria. Thc showsa rather similar picture although less parents were related in nine of the families; strict limitation to Jews, have presented in five, the parents were first cousins. eviclencewhich does not quite reach sta- It is the area of the so-called fewish Pale tistical significance but weakly suggestsa in Lithuania, Poland and Russia where the heterozygous advantage for the person Ashkenazic .|ews who constitute the great carrying the Tay-Sachsgene. Possiblysome- majority of the .|ews in this country livecl thing like this is occurring in the caseo[ in earlier centuries, ancl it is this same are:t (lysautonomia, but it is very clifficult to where the ancestors of the clysautonomia know. Talking earlier today to Dr. Motul- cases livecl. The question is how one ac- sky, it seemsthat it would be useful to try counts for this. I have been inclinecl to to get information on the sizeof the Jewislr think that this was a matter of clrift, that groups that movecl from the Rhineland to one of the founders who movecl east from the area of the subsequent Pale. It is the Rhinelancl in the 1200's, clriven by the plannecl to check on whether any concen- l)ressure generatecl by the fervor of the tration of ancestorsis clemonstrablein par- Crusacles,and drawn by the invitation fronr ticular areasof EasternEurope, as has been the King of Polancl to settle in that area, clemonstratedfor Tay-Sachsclisease (1966). carried this mutation. Perhaps this is not Our method of ascertainmentmight be an aclequate explanation. It presumes that. criticized as biasedtoward |ewish cases,but the founcline sroup was quite small. Re- I doubt that this is a very seriousbias. It

1.O SURVIVORSHIP

J m c0^ E. o_

AGE (YR) F-ig. 5. Survivorship in dysautonomia. From Mcl(usick et al.

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TABLE 5 THE tsTHNICI.IY OF DISEASE: SIN{PLYINHERITED DISORDERS

Ethnic Group Relatiaely H igh Fre qu,ency Relatiaely Low FrequencY AshkcnazicJens Tay-Sachs disease I'henylkctonuria Pentosuria Gauctrer's disease Familial dysautonom:a Stub thumbs Bloom's disease Factor XI (PTA) deficiency Niemann-Pick disease Abetali poproteinemi a Dystonia musculorum deformans Spongy degeneration of brain trIcditerranean Peoples Thalassemia (mainly B) Cvstic fibrosis (Italians, Greeks, G6PD-deficiency, Mediterranean Sephardic Jews) type Familial Mediterranean fever Africans Hemoglobinopathies, esp. HbS, Cystic f ibrosis HbC, a and p thal, persistent Hemophilia HbF Phenylketonuria G6PD-deficiency, African type Wilson's disease Japanese (Koreans) .{catalasia Oguchi's disease Dyschromatosis universalis hereditaria Chinese c[ thalassemia G6PD-deficiency, Chinese type Armenians Familial Mediterranean fever

is true that this has been considered a Jew- nuria is essentially absent in American ish disease from the beginning, and that Jews. Jewishnesshas been used almost as one of Fig. 5 presents a survivorship curve on the criteria of diagnosis,but I think doctors the dysautonomia cases.By age l0 over one' in this country tend to be, by and large, fourth of the casesare dead. By age 20 over iconoclastsand nothing would they delight half the cases are dead. This gives a in more than to be able to describe non- minimal estimate of the gravity of the con- Jewish casesof this disease.I simply do not dition becauseit is possible that our ascer- believe that many non-Jewish casesof this tainment missed some of the more severe diseaseexist. casesthat died very early in life. We estimate the frequency of this con- In Table 5 are listed some simply in' dition as between I in 10,000 and I in herited disorders that have relatively high 20,000 births in American Jews. The fre- frequency or relatively low frequency in quency of Tay-Sachs diseasehas been esti- Americans of particular extraction. Cystic mated at I in 6,000. Pentosuria has been fibrosis is rare in persons of African ex- thought to have a frequency of one in every traction. Phenylketonuria is rare in the 2,500 births in American .Jews. Tay-Sachs same group. Cystic fibrosis may be rarer in diseaseoccurs in non-Jews but is about 100 persons of Mediterranean extraction than times rarer. Pentosuria for practical pur- in those from northern Europe, but this posesoccurs only in Ashkenazic Jews, show- requires systematic study. ing as rigid limitation to this group as does One can, of course, study the common dysautonomia, perhaps more so. On the diseaseof complex genetics from the point other hand, some recessive genes are less of view of ethnic extraction and some of frequent in Jews. We know fewer that are these conditions are listed in Table 6. less frequent because these are rare con- It follows from what has been saicl that ditions and it is difficult to distinguish clinically it is diagnosticallyuseful to focus "very rare" from "rare," but phenylketo- on the ethnic extraction of patients.

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Furthermore, it is important in connection is existent but is widely scattered. Giving with the description of any new recessive thought to the best mechanism, I had disease-any new inborn error of metabo- thought the objective might be best lism, for example-to record the ethnic ex- achievedby a conferencein which plenary traction of the patients. Finally, with sessionswould be devoted to overall con- conditions such as cystic fibrosis, phenyl' siderations and section meetings to indi- ketonuria, galactosemiaand so or, it is vidual ethnic grouPsor to individual topics. useful to scrutinize, clinically and bio- I have a comprehensive and necessarily chemically, the casesof various ethnic ex- large conference in mind. It might be a tractions as separate grouPs. Genetic seriesof conferences.The objective of the heterogeneity, emphasized already in this conferencewould not be fulfilled without conferencein the caseof G-6-PD deficiency a written record. Indeed, a multi-authored and phenylketonuria, is likely to come to book on the American people from the light when one keeps in mind the many broacl point of vierv rve are discussinghere ethnic groups that comprise the American is a possibleway to clo it' It has the dis- population. advantage that no feedback and other in- There is a need for the collation of teraction is possibleand such would be im- historical, sociologicand biologic informa- portant in an interdisciplinar,vundertaking tion on the groups which constitute the of this type. American people. A wealth of information I am disturbed by the statement that

TABLE 6 THE ETHNICITY OF DISEASE: DISORDERS WITH CO,\IPLF-X GENETICS OR I^T' WHICH GENETIC FACTORS ARE NOf' PROVED

Ethnic Group High Frequency Low Freqnecy AshkenazicJews H ypercholesterolemia Cervical cancer Diabetesmellitus Tuberculosis Polycythemia vera Alcoholism Hyperuricemia Ulcerative colitis and regional enteritis Kaposi's sarcoma Pemphigus vulgaris Buerger's disease Leukemia Northern Europeans Pernicious anemia Chinese Nasopharyngealcancer Japanese Cleft lip-palate Ot.osclerosis Cerebrovascularaccidents Acne vulgaris Gastric carcinoma Breast cancer Chronic lymphatic lcukemia Filipinos Hyperuricemia Polynesians(Han'aiians) Clubfoot Africans Polydactyly \Iaior CNS malforma- Prehelical fissure tions (anencephaly, Sarcoidosis encephalocele) Tuberculosis Skin cancer Hypertension Osteoporosis and frac- Esophagealcancer ture of hip Uterine fibroids Poly'cythemia vera Corneal arcus Pyloric stenosis .A.inhum Gallstones Cervical cancer Keloids American Indian and Gallbladder disease Mexican Tuberculosis Am. Indian, Lapps, Congenital dislocation No. Italian of hip Icelanders Glaucoma Eskimos Salivary gland tumors

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is a sensitive subject to Rukavina, J. G., W. D. Block, C. E. Jackson, H. F. Falls, ethnic extraction J. H. Carey and A. C. Curtis. 1956. Primary systemic a majority of present-dayAmericans. It is amyloidosis: a review and an experimental, gentic and clinical study of 29 caseswith particular emphasis on the said, for example, that ethnic extraction is familial form. N{edicine 35:239-334. Schimke, R. N., V. A. McKusick, T. Huang and A. D' a topic so taboo that it woulcl be political- Pollack. 1965. Homocystinuria: a study of 20 families ly inexpedient to propose an ethnicity with 38 affected members. .l.A.lI.A. 193:7ll-719. I question for the 1970 censusschedules. Questions: am not convinced that ethnic extraction is DR. ARNO MOTULSKY: I would like so sensitive a subject. Although one may to make a couple of comments and I have raise questionsas to the valiclity of the in- one question.Sometimes these can are uninformed Problems formation-many people be more complicatedthan initially thought. their ancestry-or about how on questions For instance, recently the rheumatology the information would be collected and group in Seattle have been restudying the handled, there can be no question that problem of hyperuricemia. They Previous- ethnicity is an important variable in many ly found that Filipinos living in Alaska had sociologic and biologic considerations. a higher uric acid level than white Cau- To paraphrase Dr. Dudley Duncan, we casian people in Seattle. However, it ought to recapture the spirit and language turned out that PeoPleof higher socialclass of earlier decades.The grand tradition of apparently had higher uric acid levels, so America of which we can justly boast is the it appearsthat there is a genetic difference creation of a nation from peoples literally which only comes out under the environ- of essentiallyall backgrounds. ment of a high diet. The high social class Books such as Brown and Rouecek's One people and the workers who lived in Alaska Arnerica and Witke's IUe Who Built ind ate better than their comPatriotsin the America present very well, although neces- Philippines did in fact have hyperuricemia. sarily briefly, the history of migration and You would not detect it in the Philippines. assimilation and the contributions of the Another examPle of this sort might be component groups the of American people. something we have been studying some I would like to see popular magazines years ago in an experimental model. We devote more discussion to the marvelous have been struck by the rarity of hereditary cultural and biological heterogeneity of our spherocytosis in American Negroes. This country. Politicians should find the topic never has been really carefully studied and an advantageousone. may not be true, but it at least has been many have R,EFERENCES claimed, and clinically PeoPle said that they haven't seen very many Bou'man, H. S., V. A. McKusick and K. R. Dronamaraiu. 1965. Pyruvate kinase deficinet hemolytic anemia in an casesand I haven't seen many cases in Amish isolate. Am. .J. Hum. Genet. l7:l-8. Damon, A. 1962. Some host factors in disease; sex. race. Negroes. I am wondering how this might ethnic group and body form. J. Nat. N{ed. Assn. 54:424- 43r. come about. We thought that maybe since Hamilton, H. B., J. V. Neel, T. Y. Kobara and K. Ozaki. the red cell in this diseaseis sensitive to 1961. The frequency in Japan of carriers of the rare recessive gene causing acatalasemia. J. Clin. Invest. heat, possibly this disease might be at a 40:2199-2208. Hostetler, J. A. 1963. Amish Society. Baltimore: Johns greater disadvantage in a hot environment Hopkins Press. Jackson, C. E. and J. H. Carey. 1961. Progressivemuscular and therefore might not reach the frequency dystrophy: autosomal recessive type. Pediatrics 28:77-84, that is reached in Caucasian populations. l\{cKusick, V. A., J. A. Egeland, R. Eldridge and D. E. Krusen. 1964. Dwarfism in the Amish. I. The Ellis-van So at that time we came acrossan animal Creveld syndrome. Bull. Johns Hopkins Hosp. ll5:305- 336. model for this disease,a deer mouse with I\fcKusick, V. A., R. Eldridge, J. A. Hostetler, J. A. Egeland and U. Ruangwit. 1965. Dwarfism in the Amish. II. hereditary spherocytosisand put this to a Cartilage-hair hypoplasia. Bull. Johns Hopkins Hosp. I l6:285-326. test. We put normal deer mice and deer ]\{cKusick, V. 4., R. A. Norum, H. J. Farkas, P. W. mice with hereditary spherocytosisin some- Brunt and M. Mahloudji. 1967. The genetics of the Riley-Day syndrome with observations on survivorship. what elevated environmental temperatures An interim report. Israel J. Med. Sc. in press. I\{urdock, G. P. 1948. Social Structure. New York: Mac- and found that within two or three weeks millan. Myrianthoupolos, N. C. and S. M. Aronson. 1966. Popula- half of the spherocytic deer mice were dead tion dynamics of Tay-Sachs disease. L Reproductive fit- severehemolytic crisis, while the sli.ght- ness and selection.Am. J. Hum. Genet. 18:313-327. of

.b-- provided by the Maternal and Child Health Library, GeorgetownUniversity 424 McKUSICK Ala. J. Med. Sci. ly eievatetl temperature rlid not bother at they both have increasecl heat sensitivity all the normal mice. So here we hacl again in uitro, so from €iross pathophysiological an example of the interaction of the criteria they are the same. However, the genetic trait that clicln't bother the human diseaseis dominant, and so far the spherocytic mice at room temperature- specific clefect, as far as I know, is not clear. they were quite well, and you hacl to clo It seems to me, putting all the many data special tests to show that they had spheroc),- together, that the most likely possibility is tosis-but if you just raised the rempera- some tlefect in the red cell stroma, the red ture a little bit, they became severely sick cell membrane, ancl there are some sugges- and half of them diecl within a few weeks. tions of that. There are no ideas at all of The question I have is: In your non- what the clefect mieht be in the mouse. If .|ewish cases of clysautonomia, \{'ere they one woulcl generalize, as ),ou did, maybe consansuineous as was shown in Tay-Sachs it is an enzyme clefect, but it coulcl be a clisease? Since that is more common in stromal clefect too, that takes a clouble .|ews, the consanguinity rate woulcl be ex- sene to procluce the disease, so I think we pectecl to be higher amons non-.|er.r's be- n'ill have to wait until we know more about cause there the trait woulcl be rarer ancl it that. is more likely that two consanguineous I)R. ELLIOT VESSELL: What was the people rvotrlcl inherit it from the same an- geographical distribution of the caseswith cestry. Irrmilial (l)'sautonomia?

DR. NIcKUSICK: I think 1'ou misuncler- DR. IIcKUSICK: Very rvirle incleecland stoocl my statement of the data. There \,vere it follorvs very closely the clistribution of two fanrilies in which the fatlter was in- .|ervrf in this country. \\'e probablv have deed.[ewish ancl the morher lvas non-.]ewish ten casesin the Los Angeles area. We have and they were apparently nonconsanguine- c:lsesin Toronto, Ilontreal, tliami and so ous. I think the Filipino erperience with on :rncl other sczrttere(l cases all over the hyperuricemia illustrates rvhat I lvas com- tlnitecl States map. The largest concentra- menting on, one of the approaches to the tion of cases,of course, is iu the New York ethnicitv of disease,a comparison of eroults area. I might sav that the 164 that we have in this country with the homelancl, ancl this been able to sturh' so far is a pretty wicle particularlr, obtains with multifactorial geographic samplins of the some 220 that conclitions. I am not prejuclicing the vien' rve have leaclson. as to rvhether hyperuricemia is multi- DR. IIARGAREf- BATSON: How con- factorial, but in those conditions it lt,ill be sen'ative, hou'othoclox are these people? useful to use this approach. to sort out the I)R. IIcKLISICK: I think they cover the environmental factors as )'ou har.e pointecl rvhole sljectnlm irr this regarcl. out. In tlte c:rseof hereditary splterocl'tosis, woulct )'oll agree, Arno, that the mouse DR. DAVID SNIITH: There secmetl to spherocl'tosis probably is not a very eoorl be in the rapirl view of the pecligrees quite replica of the human clisease;in view of its :r feu,' :rbortions. Coukl y'ou comment on recessive inheritance, it probably has arl that, please? enzynre rlel'ect when the truth is known, DR. NIcKLISICK: Yes. one of our fielcl whereas it begins to look, cloes it not, as rvorkers rvas rnuch irnpressed with that and though hereclitary spherocytosis in man, a thought that she hacl a finrl. Perhaps she clominant conclition, has a structural clefect rloes. On counting them ul), they aren't too of the retl cells? irnpressil'e. I am sorry I can't eive you the DR. N{OTULSKY: Well, parhophysio- precise figures. We clidn't have goocl figures logically, the two diseasesare exacrly alike, to compare it with. I think this is an indi- since they both have the similar shortened cation of very goocl interviewing and not an survival, enlareed spleens, splenectomy inclication of any increaserl frequency of cures the disease in man ancl mouse. ancl abortion in these families.

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Gene DosageEffects In Manl

tdr6me Lejeune, M.D., Ph.D.'l

Human cytogeneticsis still in its infancy this general law also applies to chromosotnal and its constant progressprevents making changes. The question is now to know why, in 40 minutes a review of what we know ancl and how this genic closage effect can be of what we would like to know. So let's achieved. discusswhat we can consider as being in- Unfortunately, it is only too easy to shor'v teresting in chromosomal clisorders as far that excess of an autosome is cleleterious. as autosomesare concerned. Trisomy 21, which is relatecl to the fact I think we better not discuss the sex that one of the tiny 2l chromosomes is in chromosomesin which so many different excess (Lejeune et. al., 1959) cloes proclr-rce rliseasesare known. The particular interest the classical picture that all of you knorv. of chromosomal clisorclersis that they are If I recall this trisomic conclition, it is only introducing a new concept in the uncler- to consider lvhat kincl of mask the tlisease standing of genetic cliseases.In most of the is able to throlv on the personal lcattrres of well-known genetic afflictions, a genic the chilcl, so as to make thetn :tllpar ently changeis involved. There is an error in the like each other. As 1'ou kno'rv, the briclee of DNA molecule which procluces,by com- the nose is very flat; there is epic:rnthus, " plex mechanism that you know, a kind of microcephaly ancl so on. This particular misprint in the enzyme controlled by the mask is the same for all chilclren :rtrcl is gene.This misprint changesthe behavior of superimposecl over the partictrlar seneIic the enzyme ancl proclucesa biochemical or makeup of the inclividual. morphological variation recognized as a The same features of trisorny 21 llre disease.Most of the genetic diseasesare found in Whites, Negroes, ancl in Asiatic in thought of in terms of an error the babies. Although vaguely reminisccttt at are dealing genetic information. When we birth of Asiatic traits, thcse features are not with chromosomalimbalance, the situation at all racial. The characters change lr'ith the is Not in anima uiti entirely different. only growth and in the aclult, only tlle cl1'srnor- but also human beings we know by ex- in phy persists, the same for all raccs' I rvill perimental demonstration, that the genic not go very far into the clescription of this involved is en- content of the chromosomes synclrome of trisomy 21, for it is very rvell tirely normal. It is only a quantitative known to everybocly. I lvoulcl just remincl change which produces cliseases.When a you that together with many cleforrnities of is excess,as in trisomy, or chromosome in internal organs, the cliseasehas a sign:tture is lacking, in monosom), we know that as on the hand. the genetic information is unchangecl;but normal riclge patterns the repetition or the omission of this per- Instead of havine havc fectly all right messageis the sole causeof as everybody has, 2l trisomic people that the tri- the disease.Redundancies and shortcomings the ridges abnormally set so a convergence of the have always been the pitfalls of talks and radius in which there is in the root conferencesand it is interesting to seethat ridges, which is normally locatecl of the palm' -OI"n,"U of the hand, is in the middle at. the International Seminar on l\{edical The two flexion creases, (let's say the "line Genetics, University of Alabama Nfedical Center, Birming- "line fusecl ham, Ala. September 2, 1966. of heart" and the of head") are sProfessor, Chaire de Gdndtique Fondamentale, trIedical together in a transversal crease. I will not Faculty, University of Paris.

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discuss further the dermatoglyphics because togeneticists are really reacling the destiny Dr. Uchida rvill speak about rhis, but I of people on the hancls ancl these minor wantecl to inclucle it to give you the idea stigmata are a great help in the cliagnosis. that the u'hole individual is shifted from From just the remembering of those three normal bv the presence of the extra chro- well-known synclromes, we can draw the mosome ancl that we can predict just by first conclusion: If the excess of a given clinical inspection what chromosome prod- segment of the genome is able to produce uces the rliseases. a specific clinical entity that we can recos- In other trisomies, like trisomy lB, in nize, it follows logically that genes locatecl which a rnenrber of the pair number 18 is in this chromosome control more or less in excess (Erlwarcls er. al., 1960) the picture clirectly the manifestation of those traits. is typical but very clifferent from trisomy 21. If we find somethine wrons rvith an enyzme, The chiklren al'e rather small and many this does not prove that the structural gene traits allor,r'the clinical diagnosis. They are of that enzyme is surely on the chromosome microcephalic; they have a very small chin, involved, but at least that something con- a short sternum and a narrow nelvis. The trolling this thing is locatecl on that chro- hands are heltl in the surrencl-erllosition mosome. All of the chilclren carrf ine an ancl the first and the fifth finsers cover extra piece of an autosome har.'ein common respectively the thircl and the fourth. In a very clramatic s)'mptom-all of them are the external ears the scaphoid dimple is feebleminclecl. For the rnornent this rule rathcr flat ancl the helix is poorly rolled. has no exception. \\'e rnllst then ask our- Those things are very small symptoms but selves why it is so. The stlaishtforl,r'arcl we will see later thar rhey have possibly a conclusion that there arc "genes of intel- very sreat senetic interest. The dieits are ligence" locatecl in chromosonle 2I, chro- very short, have only one flexion crease in- mosome 18, chronlosome l3 ancl evel')'other steacl of t\vo; ancl most of them exhibit autosome is entirely un\,rarralltecl. The best arches on the fineer prints. representation of the situation is that in In trisomy, l3 a big acrocentric of the D an extremely complex ancl highlf integrate(l sroup is in excess (Patau er. al., 1960). system, any change is likell' to ltrocluce, be- Here also the clinical picture allows diag- siclesits specific aspects,some cleeraclation of nosis, ancl one can briefly remember the the best performance of the s),stem. Thus, deformities of these children. All of them human intelligence is ah.va;,s suffering if are microcephalic and have a very peculiar some of its substraturn is abnormal. Incleecl, disturbance of the cephalic development: it seems possibly a little too anthropomor- the eyes are poorly cleveloped rangins from phic to state bluntly that human intelli- pure anophthalmia to simple microphthal- gence is the top performance of livine mia. Harelip ancl cleft palare are common, systems but cytogenctics cloes not rule out and the ears are abnormal. Among many this idea. other congenital cleformities, the most typi- Now let's see lvhat not excessbut lack of cal is an apl:rsia of the olfactory lobes, which chromosomal material, like in the "cri clu classify the cliseaseas arrhinencephaly. This chat" clisease coulcl clo (Lejeune et. al., disease, clescribed in this country rather 1965). All the chilclren affectccl by this clis- recently, was in fact entirely and perfectly ease have a rather peculiar face. They have describecl 300 years ago in Denmark by wide-spaced eyes, epicanthus, rather small Bartholin who founcl in one srillborn all chin, and are very microcephalic. Otherwise, the characters of the disease.This historical their morphology is not greatly abnormal. reference shows us that these "new diseases" In common, all the children have .a very have always existed and are not an inven- particular cry which is the "symptome tion of cytogeneticists. There is also a sig- d'appel." In early infancy, they have a high- nature of the disease on the palm prints pitched, prolonged cry, which is by its plain- together with extra digits in hands and tive tonality, very reminiscent of the cry of toes. As Dr. Uchicla will discuss later, cy- distress of a young, suffering cat, hence the

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n:lme of the clisease"cri tlu cltat" clisease. then the chilcl wus a typical cri clr"rchat syn- I his is related to abnomalit,v of rnatura- clrome. ln another chilcl, she transmittecl tion of the lar,vnx. Recent spectlal anall'sis the normal 5 but the bis l3 and then the of the sound has confimrecl the sirnilrtrities chilcl became trisomic for this little seg- between thc cry of the :rffecteclchilciren ancl ment, because she hacl two normal exem- the cry of kittens. The cliseaseis r-elateclto plars ancl an extra one, stuck on the 13. This the loss of part of thc short arm of chromo- is then the reciprocal of the cleletion, that sorne 5. To my knorvleclge there :lle more is trisomy for the very same fragment, n'hich than 70 casesnow entirelv inr.estigateil ancl is monosomic in cri du chat clisease.Another all of them have a cleletion of a portion of girl was entirely normal because she re- chromosome 5. The signature of the clise:rse ceivecl a normal 5 ancl a normal 13. The nvo in the palm prints is essentially a moderate sisters lvho hacl receivecl the extrzr ltiece elevation of the axial triraclius :incl a ancl who were trisomies were l'er1, severely straight, short, clistal f-lexion crease. The mentally retarclecl. The olclest eirl has a problem was to knon' lvhether those chil- particularly big ancl long nose, rather con- clren were really sufferins from genetic loss trastins lvith the sharl> little nose in cri because it rvas generallv believetl that tnon- clu chat. osonricshar,ing only one exenrplar of a siven The voungest cliecl at seven months of segment of chromosonle shoulcl be lethal age anesancl countertyPcs

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il 428 LEJEUNE Ala. J. Med.Sci. have pure monosomic children, that is, trisomy, was very high in this child com- haplo 21, having only one 2l chromosome pared to normal. We have then the notion given by the father. Although more than that when one chromosome is absent, or 200 children born from such mothers have partly absent, the cleformity is contrary to been recorded, no such case is yet known that produced by the excessof this parricu- (Lejeune, 1964).It can be consideredthat lar choromosome. pure monosomy for chromosome 2l is not I would just discussanother example of viable. But it is possible that situarions this type and countertype pair which is a very closeto pure monosomydo exist. deletion of part of the long arm of chromo- A child was observed to be a carrier of some lB, a diseasewhich has been recently ring-Z1 chromosome (Lejeune et. al., 1964). individualizecl (Lejeune, er nl., 1966, de One member of the Zlst pair, instead of Grouchy et al., 1961). Affected children being shaped like a rod, was a little ring. have an underdevelopment of the middle Somematerial was probably lost cluring the part of the face with slanting eyes and formation of the ring. More interestingly epicanthus.Also they have abnormality of the child had lost the ring in many cells the superior lip and of the insertion of the which were then entirely monosomic for nose. The features are the same no matter chromosome21. In the blood 95 percent of what the race of the chilcl; the retraction the cells had forty five chromosomeswith- of the middle parr of the face,and also the out the ring; thus, the blood was quite little jugal nodule and the special feature purely monosomic 21. In other tissue, /, of the lip and of the nose are founcl. of the cellswere monosomir,/u having kept The syndrome is also producing a kind the ring. All the abnormalities exhibited of "hyper normality" of the ears.The helix by the child were exacrly the conrrary of is overrolled, the anthelix is prominent and the stigmata of trisomy 21. The child was the scaphoid dimple is extremely deep. very hypertonic instead of hypotonic like These are minor points but they are just the 2l trisomies.He had very big ears and the contrary of what we have seen in tri- they are small in trisomy 2l.The ear chan- somy lB in which the helix waspoorly rolled nel was very broad but it is very narrow and the scaphoid dimple was quite flat. in trisomy 2l.The noseis extremely salient Those children have also frequently a (and that was calculated also in radiogra- little dimple on the shoulder and on the phy) and as you remember it is very flat in dorsal part of the phalango-metacarpal ar- trisomy 21. Insteadof having the epicanthus ticulations. The morphology of their fingers in the inner part of eyelids,he had a kind is exactly the contrary of what was seen in of "hypocanthus" on the lower part of the trisomy 18. The digits are long "fusi-form," eye. A second case was recently described and fingerprints show a great excess of in this country (Reismanner al., 1966).The whorls. All of the children have lost about child also had one little ring of the 2l and one-half of the long arm of chromosome 18. lost it in many cells; and he had also the This diseasewas describedin three casesbut big ears, the prominent nose and an enor- there are now other casesknown, not yet mous rigidity. published. Two caseswere observed in Eng- As I told you, for biochemical traits, the land by Dr. Insley who kindly sent me the child examined had also the contrary of karyotypes and the picture. Other cases trisomy 21, that instead of having elevated have been detected, in Boston (Dr. Park alkaline phosphatase of polymorphs, he Gerald) and another instance in Paris, had a low value. Instead of having hypo"- which are mosaics. All together it can be osinophilia, he had an excessof eosinophils said safely that this new syndrome is an in his blood. And also for the tryptophan entity clinically recognizable. metabolism, which is abnormal in trisomy We could now try to discussthe interest 21, the ratio of 5-H.I.A.A. excretion in the of this concept of "types and countertypes." urine compared to kynurenin excretion, First it makes an immediate improvement which is very low compared to normal in of semeiology: observing a symptom which

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is shifted one way by excessand the other morphological traits there is some degree way by loss and allowing to determine what of liberty in their expression,which is ob- is the precise point of action of a particular vious by the differencesoccurring in normal chromosome. The second interest is pos- beings. It is this possibility o[ variations sibly more general. To understand how which make possible the recognition of chromosome dosage can operate we have types and countertypes. to build a logical model. The basic rule in One gene-one enzyme, three genes-three genetics is that one gene controls one enzy- enzymes is extremely naive indeed. Never- me or one specificprotein. If we just quan- theless,even if not exactly true, this model tify this rule, we can figure out a first does have a great heuristic interest. If it hypothesis to explain these types and coun- represents a part of the truth, it would tertypes. In monosomy one gene produces mean that in chromosomal imbalance, chil- in the cells one unit of enzyme; in trisomy, dren have the whole biochemical machinery three genes being present, three units of right at hand with just some stepsrunning enzyme will be produced. Then compared too fast or too slow. To control the speed to normal with two genes the shift will be of a biochemical reaction is surely much in different directions in monosomy and in more feasible by metabolites or antime- trisomy. Thinking in these terms of quan- tabolites than to replace an entirely lacking tity of enzyme is quite different from what enzyme. Indeed, these crucial biochemical is the biochemical results of point muta- steps have yet to be discovered, and this tions. construction is for the moment purely Let's take for example, PKU: one enzyme speculative. But if you remember that at does not function, the biochemical step is birth one child in every hundred is severely stopped and then the diseaseoccurs. In the affected by such a chromosomal imbalance caseof excessof enzyme, the chemical reac- and will suffer in his mind and his flesh tions are entirely normal but just going too for all his life, you will find that those fast. And if there is not enough enzyme, it speculations are not academic but just an is going too slowly but in both casesthe immediate challenge. This way of research quality of the reaction is normal. We can must be investigated for it is our only hope then understand why a reaction going fast that someday we will be able to do some- would produce the contrary of a reaction thing for those children who, having not going slow. Still it remains a paradox: if received an equitable patrimony, are in the types and countertypes are really mirror true senseof the term, the most disinherited images for minor symptoms, they are alike of the children of man. for major disasters. All the children are REFERENCES feebleminded. To understand that, we can Edwards, J, H., D, G. Harnden, A. H. Cameron, M. V. take an example and suppose that genes Crosse and O. H. Wolff. 1960. A New Trisomic Syn- are like the musicians in an orchestra, each drome. Lancet l:787. Grouchy, J. de, P. Royer, Ch. Salmon and M. Lamy. 1964. of them playing his personal partition. Now D6letion partielle des bras longs du chromcome 18. Path. et Biol. 12:579-582. if some musician decides to play his parti- Lejeune, J., M. Gauthier and R. Turpin. Les chromosome humains en culture de tissus. 1959. C. R. Acad. Sci. tion slower than the rest of the orchestra Paris 248:602. Lejeune, J. 1964. The 2l-Trisomy. Current Stage of or if he clecides to play faster than the Chromosomal Research, ed. A. G. Steinberg and A. G. others, in both result is Bearn. Progress in Medical Genetics, Vol. III, New York: casesthe bound to Grune and Stratton, Inc. pp. 144-177. be cacophonic. But if the musician is chang- Lejeune, J., J. Lafourcade, R. Berger, J. Vialatte, M. Boeswillwald, Ph. Seringe, and R. Turpin. 1963. Trois ing his tempo, not during a tutti but during cas de d€l6tion du bras court du chromosome 5. C, R. Acad. Sci. Paris 257:3098-3102. a solo, then the fact of changing moderato Lejeune, J., J. Lafourcade, R. Berger, and M. O. Rethore. 1965. Maladie du cri du chat et sa r€ciproque. Ann. to pretissimo will produce one symptom Genet. S:ll. and the change to a largo will make the Lejeune, J., R. Berger, M. O. Rethore, L. Archzmbault, H. Jerome, S. Thieffry, J. Aicardi, M. Broyer, J. Lafourcade, contrary. It is probably because the enzy- J. Cruveiller, and R. Turpin. 1964. Moncomie partielle pour un petit acrocentrique, C, R. Acad Sci. Parir matic processesare so much intricated for 259:4187. Lejeune, J., R. Berger, J. Lafourcade, and M. O. Rethorc. building high functions like human intel- 1966. La d6l6tion partielle du bras long du chomosome lt. Individualisation d'un nouvel 6tat morbide. Am. Genet. ligence that any shift is deleterious. For 9:32.

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l-- il 430 LEJEUNE Alz. J. Med. Sci.

Patau, K., D. \V. Smith, E. Tl.rerman, S. L. Inltorn ancl than thc othcr, that its th,vmidine incorporation is 4. P. -\\ragncr. 1960. \Iultiplc Congeniral ,qnomilv Caused by an Extra Chromosome, Lanccf l:7g0. not cractlv the sarncand so otr, that is for thc bcttcr; Reismarln, L. 8., S. Kasahara, C. y. Chung, A. Darnell but therc is no rcason at all to changc thc etiquettc and B. Hall. 1966. Antimongolism. 'n'ith 'the Studics"i,., i.,far.,i a I'artial \Ionosomy of 2l Chromosome."r., Lancet u,c have put in it.'lhat is thc samc for trisomv l8 l:394. or trisornv 13.\\'e have to make the blunt _ to knon' hou' it shorrld appl)'. Obviously rcsrrlation prt'ciatr. r't.r'yrrruclr )our cotnlneltts. plars a trcnrerrclotrslolc in thc explanation; it is DR. thc nrairr rcasorl l'hv I saitl that thc simple arith- LEJlitrNE: My coltultents n,ill ltc r.cn. ltricf. \Vhcn wc rnctic I l'as giviug rvas jtrst rxtremely naivt:. Iior alc giving a ntrnrber, a ntrrrreral to a chrontosornc, 'I.hcn thc nronrcnt thclc is no logical model w.hicl.r rvcr it is purcly a cortvcniece. the bcst is to say can safelv procluccanrl trY to tcst by cxperinrcnts. by convention that nc call 2l that chromosorne which produces the svndrorne rvhcn in DR. LE\IONE YIF-LDING: I think it is espc- triplicate. If wc can say later that this chromosome cially interesting \\'hcn rvc considcr that a nror'(l Iurs srrch an

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If repressors of various sorts play a large role in rcsemble the mother lnore than thc father? differentiation, thcn if rve have a gene which pro- DR. LEJEUNE: That is an old question that rluces a large conccntration of rcpressors, then it is has been especially investigated by British u'otkers, casy to see how u'c could disturb the whole process by Dr. Penrose in particular. Hc founcl that there of differentiation. rvere, as far as blood groups are concerncd, large DR. LEJEUNE: That is obvious. It doesn't mat- similarity with the mother, but this is a very tricky tcr in fact to the theory whether the gene is a problem. Susceptibility to a differelce in blood structural gene or a rcprcssor sene. The genic dosage groups between fetuses and thc mother is great; then cffect 'rvill be clifferent on thc cncl result but the it could be just a selcction cffcct. If thc trisomies mcchanisr.nwill bc thc satne. rvhich are weaker than nortnal fctttses do not re- DR. FREDERICK HECHf': In Down's syndrome, sernblc their mother, thcy are possibly discarded. as wcll as in thc othcr autosomal trisomics, thc There is no clue as far as I ktton' to tell that rnean maternal agc is incrcased. lfhis is usually trisomy l8 or 13 are more alikc thc t.nother than taken to be evidence that thc rnajority of casesarc they should bc. The notion that thc accident is

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il 432

Dermatoglyphicsand Chromosomes#

IreneA. Uchido,Ph.D.t

Early work on the dermatoglyphics of radial loop on the fourth antlior fifth fin- mongolism was initiated by Cummins in gers, the latter being a rare phenomenon in 1939.Since then detailed investigationscar- normal inclivicluals. A single crease on the riecl out over many years on this common fifth cligit, r-eplacing the norrnal two creases, abnormality have led to a clear demonstra- is present in 20 percent of these patients. tion of the associationbetween dermal con- This single crease,rvhich is exceedingly rare figurations and mongolism. Thus an objec- anong normals, is probably relatecl to the tive criterion for the diasnosis of mongol- shortness of the finger and the very small ism had been found and the verv old prac- micldle phalanx. An interesting observation tice of fingerprinting took on new status ir-r is that 70 percent of the mongoloids with a medical circles. single crease were males. The only inst:rnce Blincl hunts for similar correlationsrvith of a single crease amons 1,000 controls was other groups of various diseaseentities were also a male ancl among normal relatives of made. The resultswere frustratingly unim- patients with chromosomal abnormalities, pressive.fn some instancesthere seemedto tu'o males wel'e found rvith a single crease: be slight correlations of cloubtful signifi- one was the father of an l8 trisornic and the cance. Mongoloids surely dicl not hold ex- other the father of an XO. clusiverights to distinctive dermal confisu- A clistal axial triradius is usually found rations, but it was beginnine to look that on the palms of rnongoloids. A simian crease way. also occurs frequently: abotrt 35 percent It was a fortuitous set of circumsrances comparecl r,vith three percent in the normal that led to my being on hancl rvhen the first population. There appears to be no sex dif- casesof trisomy for chromosomesin the D ference here. ancl E eroups were demonstrateclin 1959 at An arch tibial pattern in the hallucal the University of Wisconsin. Reing rvell area of the foot is most c'haracteristic since aware of the associationbenveen monsol- it occurs in ahnost half of the mongoloicls ism, dermatoslyphics anrl trisornl', Drs. but rarely in normals or even other abnor- Patau ancl Smith suegesterlcxanrining the rnalities. An equal numbel of rnoneoloids clermalpatterns of their new trisornics.For- have a loop clistal lvhich :rt times mar,'bc so tunately there happenecl to be several of srnall as to make it ahncst inclistinguishable these patients on hancl. Similarities in the frorn an arch tibial. clermal configurations among the infants The dermatosll'phics of the orher t$'o within both groups were immecliatell'obvi- r,r'ell known autosornal trisomy s1'nclromes ous. The missing piecesof the puzzle fell are as diaenostically' r'aluable as they are into place; dermatoglyphics and chromo- for rnongolism. Characterisric of the I8 tris- someswere inevitably linked. omy syndrome is the presence of arches on The characteristicpatterns found in mon- most fingers and toes. This observation was golism are well documented. Lllnar loops first made on a sample of l5 patients gath- are found on the digits with the occasional ereclin Wisconsin ancl Nfanitoba. The series has now been increasecl to 30 and the fre- - 6l..r"rrt"a before the International Seminar on l\Icdical Genetics, September 2, l9OO. University of Alabama licaiiii quency of arches remains high: 20 had eight Ccnter, Birmingham, Alabama. __fDirector, Department of I\{edical Genetics, Children's or more arches and of these I I had arches I:Iospital of - Winnipeg, and Deparrmenr of paediatrics, Univcrsity of Manitoba. on all ten fingers. In no instance were arches

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il Vol. 3, No. 4, 1966 DERMALOGYPHICSAND CHROMOSOMES 433

completely absent. In a control samPle of city of thesepatients theseconclusions were 500 females,eight or more archeswere Pres- drawn on a small sample and need to be ent in only one percent while none of the verified. Similar to XXY patients, XXYY 500 males had so many arches.A single flex- individuals have low ridge counts. ion creaseis frequently founcl on the fifth The prints of patients with other combi- digit ancl sometimeseven on all four fin- nations of sex chromosomes,such as XYY, gers. The absenceof the distal crease is XXX, etc., are being accumulated but the probably related to the flexion deformity. seriesso far are too small for any meaning- On the palms there is a transverseorien- ful quantitative analyses. tation of ridges often with fusion of these Dr. M. Alter, of Minneapolis, made an ridges across the pattern areas. In general interesting suggestionthat an inverse corre- there appears to be an absenceof distinct lation may exist between the number of X patterns, other than arches, in all areas chromosomes and the total digital ridge where patterns are usually founcl.A simian count. This observation,though intriguing, creasehas been found in half of the Patients hasyet to be confirmed with adequateseries examined. of individuals with more than two X N'Iostcharacteristic of D trisomy is the chromosomes. distal location of the axial trirad- extreme Recently much interest has been focused ius the A distal triradius was on palm. Pres- on the deficiency syndromes. There is ^ ent in all of ten patients examinecl.A sim- suggestionof associatedPatterns but their ian crease was observed in almost all of reii significanceis still somewhatin doubt' these patients, a much higher frequency Loss of parts of chromosomes aPPear to than for mongoloids. The most frequent have less effect on formation than pattern in the hallucal area of the foot is Pattern has extra chromosomalmaterial. the arch fibular or a derivative thereof. No deficiency syndrome, characteristic pattern has yet been found A well clocumented loss of part of the short on the digits. characterized by in the B group, was Abnormalities of the sex chromosomesclo arm of a chromosome and his collea.ques not have as mnch influence on ridge forma- first describeclby Lejeune callecl "Cri du tion as clo the autosomes.XO patients fre- ancl is now appropriately of this s1'ndrome quently have large loops or whorls on the chat" synclrome.Typical crease'A simian' cligits with high ridge counts. These pat- is the presenceof a simiall parallel transverse terns are usually elongatedwith ridges that partiai simian or t\vo b,v Dr. Lejeune in his are packeclclosely together. The mean axial i..ur., were founcl creaselvas triraclius is slightly more distally located original five cases.A sirnian Pres- l','ehave seen' The than in normals but not so distal as for ent"in all nine casesthat displaceddistally' mongoloids. A simian or partial simian axial triradius is stightly 50 cases)has been creaseis frequently present. The largestseries (almost in Nelv York In contrast to patients with Turner's syn- by \\rarburton "..r*.riutec1 findings' No strik- clrome, XXY indiviciuals have an increased r,vhohas confirmed these found in the cligi- frequency of arches or small patterns on ing consistencyhas been the digits 'n'ith correspondingly low ridge tal patterns. counts.It is interesting that normal females Patients rvith l8 short arm deficiency are have lower clisital ridge counts than normal more ctifficult to identify clinically. It is males but XXY patients have even lower probably for this reason that only a few counts than normal females and XO pa- ."r.t have been reported. In a small series tients have higher counts than normal of sevencases no obvious deviation in thc males. dermal patterns or flexion creaseshas been The XXYY variant of Klinefelter's syn- noted. drome often have an ulnar triradius which From this resume it is apParent that the is associatedwith a loop carpal, loop radial strongestassociation exists between clerma- or arch radial pattern. Becauseof the scar- toglyphics and the atttosomal trisoml' s1n-

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il 434 UCHIDA Ala.J. Med.Sci.

tlromes. The sex abnormalities have some- cannot be easily identified, it might be dif- r,r'hatless characteristicpatterns and defi- ficult to decide whether a patient is an cienciesappear to have the least influence XXYY or an XXY-mongoloid. The clerma- on the dermal ridges.Certain characteristics toglyphics could give the definitive cliag- have been firmly establishedand are clini- nosls. cally of diagnostic value, but larger series Recently there have been many reports must be accumulated in order to evaluate of patients with an extra small acrocentric rnore carefully featureswhich are still some- chromosome.Much discussionhas arisen as rvhat only suggestive. to the identity of this chromosome.Since All three autosomaltrisomies are charac- the clinical phenotypeshave not been con- terizecl by clermal confieurations that are sistent,it is suspectedthat the extra chrom- clistinctivein that they occur consistelirly osomeis not the samein all cases.Chromo- with a very high frequency. Not only I ti some 22, d,eficiencyof a member of the D trisomics but monsoloids, D trisomics ancl sroup, Ioss of both enclsof a chromosome normal inclivicluals,too, may have arches 18,have all been suggested.If the dermato- on many of their digits. An occasionalD or glyphicsof a large number of theseparients lB trisomic may have a larse number of could be gatherecltogether, it may be pos- ulnar loops on his cligits, blt this shoulcl sible to classifythese parienrs on rhe simi- not confuserhe clinical picture. Similarities larity of the patterns. in someof thesepatterns amons indivicluals All patients rvith an)' t,vpe of chromo- are like similarities in orher clinical fea- somal aberration should be printed no mat- tures. Holvever, thesepatterns form a com- ter how rare or how insienificant the aber- pletely objective criterion thar is reaclily ration ma?vappear to be. Sinceit is difficult measurable;it is difficult to obtain an ob- for one centre to accumulateseries of suffi- jective measureof the slant of the eyes,the cient size,particularly of rare abnormalities, set of the e:rrs,or the size of the jaw. But pooling of clata will be the logical proced- neither clermatoglyphicsnor chromosomes ure. The I'altreof such prints may only then are meaningful without a good clinical cle- becomeapparent. scription. For instance,an extra small acro- Interest in clermatoely'phicshas increased centric coulcl be either chromosome 2l or considerablyover the past few years.Where 22; the dermal patterns of a monsoloicl are at one time it was consicieredof mythical occasionallyatypical. In the total picture, value, it has now become quite a respect- however, an evaluation of the clinical signs able science.This is the resulr of its link llong with the affay of dermatoglyphic rvith cytogenetics.Xluch prosresshas taken findings will, in the majority of casei,leave place in onr understanding of clermatogly- no cloubt as to the diagnosisof mongolism. phics, but there remain many unknowns Ideally all testsshoulcl be carrieclout for a such as the role of hereclity and environ- complete picture. ment in the development of dermal ridges In adclition to its clinical value, derma- and horv in a particular environment an toglyphicscan help in the interprerarion of abnormal chromosomecomplement affects cytoeeneticfindings. If the Y chromosome pattern formation.

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il Reflectionson 20 Years Experienceof GeneticCounsellingl

l. A. FroserRoberts#

How many people need genetic advice? known to ever)'one, is likely to get in the Experienceat the City of Bristol givessome present state of knowleclge. indication. We establisheda clinic for ge- The next point is the kincl of person who netic advice some 12 yearsago, and this ex- is referrecl. In out- expet-ience more than 90 perience is easier to interpret than the per cent of all enquiries come from couples findings at The Hospital for Sick Children, who have had a chilcl r,vho is abnormal in Great Ormoncl Street, although the num- some way ancl rvho rvant to knolv rvhat the bers there have been much larger. At Great risk of recurrence may be shoulcl they have Ormoncl Street,it is impossibleto estimate another. Of course, we clo get some en- the effectivepopulation from which we are quiries prior to marriage or the birth of clrawing enquiries, and the caseshave been children. There are the sisters of bo1's suf- referred in a variety of ways. At Bristol fering from haemophilia or muscular clys- things were different. At the outset we trophy. There are the people who them- brought the existenceof the clinic to the selves suffer from a defect, say, a limb ab- notice of all the specialistsin the area, and normalitl', or epiiePsy, or r,vho have had a also took stepsto inform the general prac- retinoblastoma. There are those with a titioners aswell. I think that for many years family histor,v of Huntington's chorea, or the great majority of doctors in the area of deaf-mutism. There is the very small but have known that the facilities exist. The steady flo'lv of enquiries from first cousins population is about 500,000,and for a lone who rvish to marry. But, as I have men- time the number of nelnrcases referred has tionecl, more than 90 per cent of all en- been running very steadilv at about 50 a quiries come from the parents of children in some way or other. I ,vear.So we are p;etting one new enquirv u'ho are abnormal I clo not cleal with the per 10,000population per )'ear.It is not a shoulci aclcl that large demancl. If enquiries r,vereto come senetic problems of mental disease (Mental in at the same rate in the Unitecl States, rleficiency, of course, is not excluded). It provision for 20,000enquiries a year woulcl is mv belief that advice in cases involvins cover the whole country. I am not saying mental illness must be given by ps,vchiatrists. that everyonewho would profit by genetic In London lve are fortur-rate in having a advice is being referred. And, of course,' clinic for this specific purpose, establishecl some specialistsand general practitioners by Dr. Eliot Slater; so any cases involvine will be giving advice themselves.But I do mental illness that come my lvay go straight think that the figure of one per 10,000 to him. population per year is a reasonableestimate Although it is only iI relatively srnall of what a genetic clinic, whose existenceis proportion of people u'ho neecl genetic atl- vice, those who do neecl it neecl it baclh. The clecision as to whether to risk hal'ine further children is an anxious ancl crucial *"u,"U before the International Seminar on \Iedical one. N'Ioreover, the whole subject is rich irl (lenetics, September 2, 1966. University of Alabama ltedical Clenter, Birmingham, Alabama. superstitions ancl olcl wives' tales. Doctot's #Pcdiatric Research Unit, Guy's Hospital lledical School, can be sure that if they

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il 436 ROBERTS Ala. J. Med.Sci.

to the best of their ability, the unfortunare small risks no one woulcl ever have a child. couple are onlv too likely to get plenty, ancl Before turning once again to the main from very ill-informed sources.And as we theme of this talk I should like to digress, all know, old wives' tales seldom err on the and say something about the caseswe do side of foolish oprimism. It is my belief that not seeat a clinic for genetic advice,and I useful advice can be given in the grear am very thankful for it. This relates to majority of instances.Sometimes we know queries about the risk of recurrence of some the odds precisely. Often we cannot quote of the common, chronic diseasesof adult exact risk figures, but we have useful em- life, which have, it is fairly evident, some pirical figures for the chancesof recurrence. genetic component in their causation, and Sometimes,and I admit this at once. we possibly on occasion quite a sizable one. are uncertain and have to give a distinctly Now I do not mean to say for a moment qualified estimate,or even perhaps cannot that genetic studies on the causation of give one at all. But when this happens,and common diseasesare not important. They it really is not very often, we should be able are extremely important, and I only wish to send the couple away in the full convic- I had had the opportunity of taking part in tion that no one else knows either; they are more of them. It is when we turn to the at least saved from the old wives' tales. outlook for the individual that doubts arise. Experience shows that the giving of ge- Possibly the genetic history may sometimes netic advice is facilitated by a supreme ia- be useful in diagnosis,though I have many vantage, so that in practice it turns out to reservationshere. Perhaps occasionally pre- be an easier task than, I imagine, anyone cautions might be taken by those specially who has not tried it would think. The great at risk; but again I have my doubts. advantage is that there is a direct relation When we compare the magnitude of the between the simplicity of the generics and extra risks with those involved in genetically the seriousnessof the outlook. In general, determined conditions, or, say, with those when the genetics are simple and straight- congenital malformations which evidently forward the outlook is bad; but as the [en- have a fairly strong genetic element in their etics become progressivelymore obscure-,so aetiology, we can seethat the order of mag- do the empirical chancesfor the individual nitude of these risks is entirely different. couple improve. Indeed, often enough, Thus, the risk of recurrence of phenylke- when we have only the vaguest ideas about tonuria, or galactosaemia,in the subsequent the geneticbasis of the condition, the actual children of parents who have had an af- chance of recurrence is verv small. fected child is 10,000times the risk in the An allied advantage is tirat risks for re- average pregnancy. The sister of haemo- currence in subsequentchildren tend strong- philic boys is 2,500 times more tikely ro ly to fall into two groups, the bad and the have a haemophilic son than is the average good. If we say rhar a bad risk is worse than woman. Parents who have had a child suf- one in l0 and a good risk better than one fering from fibrocystic diseaseof the pan- in20, we find that in practice very few esti- creas are 500 times more likely to have a mates fall between theselimits. I call one in recurrence in a subsequent child than is 20 or better a good risk, becausewe always the random couple in the population. Even have to remember that the chance that any with the partly inherited congenital defects random pregnancy will end with ro-. ,e.i- the risks are still large. A parent with hare- ous abno_rmalityor other is probably about lip is 35 times more likely to have a simi- one in 35. If the special risk for the in- larly affected child than is the average dividual couple is not very much greater person. A woman who has had congenital than this I think we can conclude thit they pyloric stenosisis 30 times more likely to really do not have very much to worrv have an affected son than is the average about. We find that this view is indeed woman. A couple who have had a child :haredAy many couples.After all, if people with spina bifida are 25 times more likely \r'er€ deterred from parenthood bt uiry to have a similarly affected subsequentchild

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than are coupleswith no such history. But ful. Another woman was convincecl for with the common cliseases.even thosewhich many years that she would never live to be admittedly have some genetic element in 50. This was becausea rather remarkable their causation, the order of magnitude is number of close relatives had died of vari- totally different. The brother of a man who ous cancersat a relatively early age; she is suffersfrom duodenal ulceration is 3 times now in excellent health at the age of 75. more likely to develop an ulcer than the As I have mentioned, family histories of averageman. It is possible that the sister common diseasesmay be of some help, and of a woman who has a cancer of the breast may be given some weisht, in individual may be twice as likely to develop cancer diagnosis and possibly in the institution of the breast as the random woman in the of preventive measures, but morbicl fears general population (and even this is should not be fostered.If it is not one clis- cloubted by some). With some of the com- easeit will be another. The personrvho has mon cancersany increasedincidence in close a family history of this or that common relatives is very small, ancl u'ith some of cliseaseshould be encouragedto rise above them it may not be increaseclat all. it ancl reflect that it is much more likely For the incliviclual these are very srnall that he or shewill be stricken in somequite extra risks and they are reallv rather l;es- different way and die of something quite ligible. It is much more likely that t'losc: unconnectecl.We all of us tend to be over- relativeswill be affected by quite different impresseclby the occasionalstrikins coin- cliseases,and cliefrom quite different causes. cidence ancl fail to recollect the far more Yet the ideas of hereditary predestination usual negative finding. Only systematic, ancl doom are far too common in the minds large scalestudies are to be trusted, ancl as of the general public, and incleecl in the has been mentioned, thesewiil usually show minds of doctors as well. "His mother died a very moderate measureof family iikeness, of a stroke, so did his aunt, and another which though indeed very important in relative; he will probably go the sameway." stuclieson causation, mean little for the One patient was sent to see a surgeon be- inclividual. It is fortunate that at clinics cause of a lump in her breast. She asked for genetic advice practically no enquiries, him bluntly w6ether she hacl cancer. F{e, at leastat present,come from sufferersfrom rightly judging her to be a courageorls common chronic diseaseor from their rela- woman, said that she had. But at operatiorl tives. it turned out to be a mastitis. Here I think I have stressedthis rather inciclentalpoint that both family doctor and surseon had becauseI believe that with common clis- been unduly impressedby the fact that an eases,when one is thinking in terms of in- older sister had died of a cancer of the clividuals, the danger is not of paying in- breasteighteen months previously.It might sufficient attention to family history br.rt, be added that this mistakecaused no trouble on the contrary, of overvaluing it. But I at all; everyone was delighted. In another must return to my main subject, the clinic family a husbanclclied at a relatively early for genetic advice. There are a number of ug. oi the result of a peptic ulcer. Hls wife important factors which enter into the had had a partial gastrectomy.Their claugh- provision of genetic advice.First of all, giv- ter was normal, but their son, aged 18, per- ing geneticadvice depends on three factors: forated while ricling his bicycle, and died diagnosis; the individual family historyi in hospital. But so what? This is a very and the background of the literature. There common condition. There might have been are complications of many different kincls some special inherited predisposition, but to be considered,for example, when lvhat quite possiblyit lvaspure coincidence.I am appears to be the same encl result mav not impressedby such histories unless it is clepend on different genes,sonletimes ciif- demonstrated in large, properly collectecl ferently transmitted, or when the effectsof seriesthat they do in fact occur with a fre- geneson the one hand, or of environmental quency which is appreciable ancl meaning- influences on the other, cannot be tiis-

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il 438 ROBERTS Ala. J. Med. Sci. tinguished. There is the use of overall em- Here are the results: pirical estimates of risk. There is reassur- Parents Subsequent Children No. of No. per ance about nolmal children and other nor- couples couple having No. of (all mal relatives, when this can properly be No. % childrcn children couPlcs) siven. There are the psychological factors Bad risks 28 involvecl, ancl these are most important. Decided to lnsteacl of clealing with these topics in a go ahead 6 21 4 B r.33 general ancl formal way, however, I thought Doubtful 4 14 I | 0.25 1'ou mieht prefer another plan. I have with Against lB 64 5 5 0.28 me a file of my rough notes of cases seen Good risks 47 at the Bristol clinic. The file is alphabetical, Reassured23 49 2l 26 r.l3 so it is constantll'changing. \\'hai I thought Doubtful 14 30 6 7 0.50 we might clo is to select some number, sa1,, Not re- anything betrveen 4 ancl 10, ancl then to assured l0 21 2 2 0.20 turn up the case notes in this ranclom wa1'. If we clo this (ancl I am sure some awkwarcl As you lvill see, the attitucle to the aclvice ones n'ill turn up!), I hope it u'ill be pos- is incleed closely reflectecl in the number of sible to sive some of the kind of Picture subsequent chilclren born. What is rather queries that have come our way, ancl as we disappointing is that only half the couples r:onsicler the inclir-iclual casesit lvill be pos- given a goocl prognosis were in fact com- sible, I hope, to brins ollt the various points pletely reassured. The other half were either that to ltl)I \vay of thinking are imPortant cloubtful or else frankly' did not believe rvhen ure se[ out to git'e eenetic acivice. rvhat r,r'etold them, or thought even a very I should like to encl with a few worcls on small risk too big to run. I think one im- a follolr'-up r,r,hich lve carriecl out on cases portant lesson is that whenever Possible seen at (ireat Ormoncl Street. Couples were couples shoulcl be seen again after an in- visiterl b), u social r,vorker, my former col- terval. Thel' n'ill then have hacl time to league NIiss lluck. Thel,had been seen from think things over and one can try to make two to sevell )'ears previously. \\re attempted sure that thel' really' unclerstoocl rvhat they' to cliscovenvhat the,v had thought of the lvere told. I norv tr), to arrange a seconcl adr.'ice thev lvere siven ancl then r,l'e found inten'ielv lvhen possible. I shoulcl like to what subsequent rhildren they hacl actuallv aclcl in closine that the intervieu,s are sel- hacl ancl lt,hether ther, r,t'erenormal ol' not. clom as clifficult as might be imaginecl. Peo- The figures quotecl refer to rather a small ple referrecl for genetic aclvice are, on [he sample, but a larser sample clid in fact zlvel'age, much above the general run of give just the same sort of rcsults. The cases hospital patients in intelligence ancl in a are clivirlecl into bacl risks and soocl risks sense of reslrcnsibilitl'. Thel' are goocl citi- on the lines I nrentionecl earlier. llere are zens lvhorn \\'e shoulcl like to rlo all \\'e carl the fisures: to help. Subseqrrcnt thildrcrr

No. of Too I'orrng Questiorts; couples Norrnal .,\ffccted to classifr DR. H,{NS ZELL\\'EGER: Dr. Roberts,I havea Bacl risks 28 7 4 3 qrrcstionconcenring Duchcnnc muscular dy'strophr'. Goocl risks 49 33 I :1 You lravc indicatedthat $'ith a safetvfactor of 90 perccllt, \'ou can sav l'hcthcr the mother is hetero- The object of the follorv-ull was not to scc z)'gor.lsor not, and u'c think about thc same thing. r,r'hether our preclictions hacl been corrccI But no'rv,if I'ou havc these chanccs of l0 percent for this mother l'ho has otherwise no other family or not. This rvoulcl require far larger sarn- historv, thcre is still a risk of ten percent that she 1rles,ancl concentration on particular con- coulcl be a carrier. Is this ten percent probability in clitions. What \,\'e \.\,'anteclto fincl out rvas such a horrible disease as Duchenne type muscular clsi'trophynot sufficient to advise against further rc- the :rttitude to the aclvice siven ancl the production? If this should be correct then all our :I('tion :rr:trralll'takt'tr. heterozvg<-rsitvstudies in Dtrchenne type muscular

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dystrophy are really vain becausewe find a high cre- of caution about the creatine phosphokinase. At atine kinase or a few hypertrophic fibres or what not Great Ormond Street a lot of work was done with in these mothers or in the sisters and then rve knorv this procedure and a lot of data available. Frcquent- that they are carriers or we find it negative and ly I have had experience with laboratories u'ho just there is still this ten percent chance and that makes started out doing this test and who weren't doing it the great difficulty at the prcsent time. I wonder too well. So my strong feeling is that in such an how you get over that hurdle. important decision where a woman may or may not DR. ROBERTS: I am verv glad that lou have be a carrier based on very small differences in a few raised these interesting points. Thc thing is that 90 points in a laboratory tcst, only highly specialized percent accuracy, a figure vou agrce with, isn't laboratories u.'ho have a lot of experiencc u'ith nor- really as bad as that in practice because in the lab- mals and with knorvn carriers such as yours should oratory at Great Ormond Street the cutoff point do this. Unfortunately, it is being thrown around which best separatcs the heterozysotes from thc and lots of laboratories are setting rtp these tests normals is 1.9. Whcn you gct a woman rvith a value and genetic counseling may be clone that is poor of say 1.2 or 0.9, thc risk of her being a carrier is becauseof the test being not donc propcrly. This is exceedingly small. Or vou rnav get another rvoman true for this disease and for hemophilic globulin who gives )'ou a fistrre of 2.5; she is certainly a car- assa,vand some of these other tests. rier. It is only ruhcrt vou gct a value, let us say 1.7, DR. ROBERTS: Yes, I agree entirely with you, 1.8, 1.9, 2.0, or 2.1 vou are urlcertain. You see the Dr. ilIotulsky, because the results differ even at uncertainty of tcn perccnt cloes not rcfcr to every good laboratories and ,vou get diffcrent stalldards at individual case. different laboratories. I shoultl mention onc thing, DR. ZELLWEGER: \\'cll, what would be nonnal that the Great Ormond Street figtrrcs are the when you accept it as a dcfinite answer? averageof three indcpendent detcrminations, which DR. ROBERTS: \\'ell now, I mustn't ride an- of course raises the accuracy. But I quitc agree, it other hobby horse. It seems to me that the clinical is very dangerous if laboratorics sct ollt to clo these pathologists very often throw away a lot of infor- tests rvithout standardizing their proccdurc against mation. They get a nice accrlratc reading and they other laboratorics rvith more cxperience. Very dan- say it is normal or abnormal, instcad of looking at gerous but I hope that in time it will all settle the figure. Here, it is not a question of what is nor- dorvn to something mrrch more accurate. mal and what is abnormal, it is the statistical fact DR. ELLIOT \/ESSEL: What about the combina- that if you gct a crcatine kinase value of l, then the tion of LDH-5 values u'ith CPK values?It has been chance that that woman is a carrier is hundreds to reported that carriers of muscular dystrophy have one against. If you get a creatine kinase value of 2.5, a decreasein LDH-5. it is almost certain that she is a carrier. It is only with this doubtful section in the middlc, so you see DR. ROBERTS: I am afraid I arn talking lvith- it is not an uncertainty of ten percent rvhich applies out first-hand experience hete, but my impression to every case. It is an uncertainty u'hich applies to is that that rvork has nol really becn confirmcd by ten percent of cases.I hope that ansl'ers the ques- others 'n'ho have tried to rcpeat it, but it may in- tion. deed be that rvith improvements in techniquc, a DR. N{OTULSKY: I u'oulcl like to put in a rvord double test of that sort u'oulcl improve acctlracv.

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il PANEL I: GENE I UNCTION N'Ioclerator: K. Lrl,roNn YInlorNc, N'I.D. Thomas W. Feary, Ph.D. Bacterial Coniugationas a L[ethod f or the Studyof Gene Expressiorz Gerald L. Carlson,Ph.D. RoIe of wRNA in Gene Expression Marshall W. Nirenberg, Ph.D. Manuscript not submitted. f)n. Ylr:r.orxc: It is rvith a great deal of pleasurc that I introcluce thc pancl rvho rvill discttssgcne functiou today. Two of the panelistr ut. from our own institution, Dr. Torn Fcary of the Dcparttnent of Microbiologv, ancl Dr. Geralcl Carison of the Departrnent of Biochcrrristry. \Ve are especially huPPy to welcotne Dr. Marshall Nirenberg, who visits us today from the National Insfitutes of Health. I thi;k that perhaps one of thc most exciting insights that has dcvelopcd in motlertr biologv and tncdicitre 'w'ay is the elucidation of the chcmical nature of gencs ancl th.e in which the fortnation contaitred it'r gelres is translatcclinto biological evcnts. It is obvious that thcsc advanccshave conre front a varictv of clisciplines and may be brought to bear clircctly in understancling hurnan diseascs.I think this rvill bccotlreollr'iotts as our panclists discuss differcnt aspectsof p;eneftrnctior.r. \\'e rvould likc to start our clisctrssiotrrtith some lclrarks frol'r l)r. Fearv, rvho rvill icldresshirnself to thc role of microbial geneticsin cluciclatir-rggcnc fttrlctiou.

BacterialConiugation as a Method for the Studyof Gene Expression

ThomasW. Feory,Ph.D.*

-fhe conccpt of a futrctiotral rclationship bct.rvcctr Sttrclics of thc tttcchauisttrs regttlating cllzl'llrc syll- gene s atrtl protcins dcvcloped f ronl thc sttrdY of thcsis in bactcria havc rcstrltcd in tlte conccpt that gcttctic biochcrnical rnutants iu Neulospora. Thc one-genc- protcin srnthcsis is srrltjcct to a tlottbic dc- one-enzvrnc hrpothcsis of Bcadle and Tatutn (1941) tcrrninisrn: orr thc onc hatrhcnotvpic groups accordine (Nircnberg antl \Iatthaei, l96l). to rvhich cllzvlnc in a particular biochcrnical sc' quence is clcficicut. It is of ittr.r"r, to notc that in *,Arrirr"r,, I'rofcssor of l{icrobiologr', Universitl' of Ala- is, banra trIe

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il Vol. 3, No. 4, 1966 STUDYOF GENEEXPRESSION 441

structural gcnes specifying the synthcsisof cnzymcs rurating process at cliffcrent titnc itrtcrvals after mix- in single bio-s1'nthctic or dcgradative ing donor and rccipient cclls in a nutricnt medium. involved a 'l'he pathway, are usually clustered in a small region of results of experiments of this type showed that thc chromosornc and that frcqucntly, the order of chromosome transfcr from donor to recipient occurs gcncs within this region correspond to thc order in an oricntcd fashion and that for a givcn Hfr of biochemical rcactions involvcd in that particular donor onc could preclict the time of entry of a pathway. 2.) A seconcl gcneral category of genetic given marker. As a control for interrup.ted mating charactcrs in bactcria concerns their susccptibility' expcriments, mixtures of multiply auxotrophic pa- or rcsistance to toxic agents such as antibiotics or rental strains werc plated on selcctive media so as bacterial viruses. Such mutations serve two useful to sclect for thc inhcritance of singlc biochemical purposes; first, as unsclected markers in bactcrial rnarkers. When the frequency of trnselcctedmarkers crosses,and secon

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il 442 FearY Ala. J. Med. Sci. sythesize p-galactosidase bccause of a mutation in siderable support as the resttlt of iutensive investi- the z gene. .'\fter transfcr of the lvild type markers gation of metabolic pathwa-vs and finc structure to the recipient cells, synthesis of p-galactositlasewas analysesof small regions of the chromosotne within measured in the absence of inducer; under these the related group of enteric bacteria. In addition conditions neither parent is able to synthesizethe the elucidation of mechanisms of repressor action cnzyme. The results of several experinrents of this and attempts to isolate aucl characterize the chemi- type shorvcd that the introduction of the wild type cal nature of the repressor tnolecule are also under structural gene z resulted in an immediate synthesis current intensive investigation. of B-galactosidase in the abscr.rccof inclucer. Ap- Clearly, the operon is the genetic unit of regula- proxirnately sixtv minutes after the introduction of tion of protein synthesisiu organismsin rvhich func- the wild type i gene, the rate of p-galactosidasesyn- tionally related genesare clustered;but as yet, there thcsis fell to zero. but could bc restored to its tnaxi- is no clear idea of how the operon hypothesis could mal rate by addition of inducer. be modified for related gerres rvhich are scattered. The interpretation of the results obtained from Recent reports (Fargie and FIollorva,v, 1965) sug- experirnents of this type werc crucial to thc devel- gest that for sotne bacteria, clustering of related opment of current concepts concerning the genetic genes may be the exccption, rather than the rule. regulation of protein synthesis. Thc observation Other reports sllggest that the nature of coutrol that the structural gene for B-galactosidascwas im- mechanisrnsfor inducible (Hegeman, 1966), or re' mediately expressed upon entr_v into recipient bac- pressible (Crawford and Gttusalus, 1966), enzvme teria suggested that the prior synthesis of stable s)'stemsdiffer markedly in certain bacteria from the templates was not essential for the synthesis of spe- well characterized coutrol tnechanisms itr the cn' cific proteins. Speculation concerning the mecha- teric bacteria. Further cltrcidation of regulation nisrn of information transfer from gene to sitcs of mechanisms exerted ovcr tnetabolic sequences in protein synthesis led to the concept of unstablc other organisms, or the discover,vof presently un- "messenger" molecules and to the subsequent denr- knou'n regulatory mcchanisrns, coulcl bc a reflectiou onstration of a messengcr RNA species. On the of a different chromosomal organization and could other hand, the delayed expression of the regulator lead to a greater ltnderstauding of the rlegree of gene in recipient bacteria clearly dernonstrated that rclateclnessbctweeu different organistrls. such genes control the svnthesis of a cytoplasmic repressor substance which, lvhen prescnt in suffi- REFERENCES cient concentration, inhibits the expression of the Beadle,G. W.. and E. L.'fatum. l!i41.Genctic control of structural gene. Speculations about the site of ac- biochemicalreactions in ll-eurrt.sltora.Proc. Natl. Acad, Sci.,U.S. 2t':499. tion of the repressor then led to the concept of Yanofskl', C., and I. P. Crarvford. 1959. Thc effects of de- operator genes and the isolation of operator mu- letions, point mutations, reversions and suppressor mllta- tants. tions on the nr-o components of the tryptophan s,vn- tlretase of E. coli. Proc. Natl. Acad. Sci., U.S. 45:1016. In the enteric group of bacteria, it is non' gcn- Ingram, V. \I. 1957. Gene mtttations in httman hacmo- erally accepted that the chromosome is organized globin: the chemical difference between normal and sickle-cell haemoglobin. Nature, 180:326. into units of regulation called operons (Jacob and Nirenberg, N. \I., and J. It. Nlatthaei. 1961. The depend- I\{onod, 196l). The essential feature of the operon ence of cell free protein slnthesis in E. coli tlpon natu- hypothesis is that several structural genes related to rall-v occurring or sl nthetic polvribontrcle otides. Proc. Natl. Acad. Sci., 47:1588, a single metabolic pathway are clustered together Jacob,- F., and J. Ilonod. 1961. Genetic regtrlatory-qgqhan- isms in the svnthesis of proteins. \Iol. Biol., 3:318. on the chromosome and are organized into a single J. 'I'he llonod. 1959. genetic unit transcription and regulation. The activitr' Pardce, A. B., Ir. Jacob, and J. of control and c-vtoplasmic expression of " inducibility" in of the operon is coordinately controlled by the op- tlre s-vnthesis of p-galactosidase bv E. coli' J. lIol. Riol.' erator gene which is located at one extremity of the l :165. Fargie, B., and B. \l'. Hollon'a-v. l96ir. Absence of clustering operon; the operator is defined not only as the of frrnctionall-v related genes in Pseudomonas aeruginosa' initiation site for transcription of the operon bttt Genct. Res., Camb., 6:28'1. enzl'mes of specific Hegcman, G. D. 1966. Svnthesis of the .the also as the receiver of the repressor. The mandelate pathrva-v by Pseudononas putida. J. Bacteriol., repressor for a given operon is produced by a regu- 9l:1140. lator gene which is located outside of the operon. Crarvford, I. P., and I. C. Gunsalus. 1966. Inducibilitl' of llrptoplran svnthetase in Pseudontonastnttida. Proc. Natl. Currently, the operon h,vpothesis has received con- Acad. Sci., U.S., 56:717.

Dn. Yre1uNG: I think Dr. Fearl' has illtrstrated hon' the study of microbial svstems has revealed how genes worli; and how such studies lvill continue to yield ncw understancling. Dr. Carlson u'ill now discuss some of the biochemical events involved in gene expression'

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il Vol. 3, No. 4, 1955 mRNAlN GENEEXPRESSION 443

Role of mRNA in Gene Expression

Gerold L. Carlson,Ph.D.*

In continuation on I)r. Iiearr"s rcrnarks, I am TABLE I* going to emphasizc thc rolc of rrrRN.{. in genc ex- pi'ession. Leuels of Genotype Enzyme Activity in Cells If control gcnc cx1;r'essionis in the of of process Llninduced Induced transcription of rnR\.\ florn the sequenceof bases TSS i+ z+Y 0.020 0.5 in DNA of structural gcncs, an esscntial feature of .I'SS mRNA is that it harc a half life short enough, that i*TSSz*Y-li* z+Y 0.0015 1.0 slowing the ratc of nrR\.\ procluction u,ill result +From 1965. in curtailment of thc rate of svnthcsisof the eltzvlnc Sadler and Novick, J. Xlol. Biol' 12' 305, if = repressor gene or enzymes by thc agcncv of thc messenger. I arn z = structure gcnc for p-galactosidase not going to clwell on the l)NA-dependent RNA y : gene for "permeasc" for galactosicle TSS - mutant in wlrich svnthesiso[ rcpressor is tempera- polvmerases thought to producc rnRNA, instead I turc scnsitive $'ill consider possiblc modifications of the operol-t systernof Jacob and Nfonorl (196I). rnal level by a nunrbcr of clrugs. \Vhen induced with -{ feature of the p-galactosiclasesvstem not di- allylisopropylacetamide, the half-life of the enzyme rectly applicablc to adult manrmalian tissue is that is foun

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il M4 Carlson Ata.J. Med.Sci.

TABLE 3 MODIFICATION OF EXPRESSIONOF UDP.GALAC'IOSEI'OI,I'SACCHARIDE TRANSFERASEGENES IN Dictyosteliurndiscoidezrn FR-171

Time Vo of lllaximum SPecilic Time of Addition Enzyme Fir.st Actiaity of Enzyme of Actinomycin D Obserued Witltout Actionomycin D 2 Hours 6.5 Hours 0 Control 4 Hours 6.5 Hours 50 llxperiment 6 Hours 6.5 Hor-rrs i00

Cycloheximide 5 Hours 9.5 Hours 5-lc Present in 2-5 7 Hours 9.5 Hours it0 Hour Period, Hortrs 85-90 Then Removcd 9 Hottrs 9.5 By Washingt --.Rrni.,., acid incorporation resllmcs at once at nearlv normal rhte lIirom data of M. Sussman l(P. N. A. S.,tJ, 813 (1966)1.

a protein. At fertilization, the protein is apparcntly the pcriod of nrRNA production for the transferase. 'fhe removed by action of cathepsins activated by sperm transferase used in rnaking the polysaccharide entry and protein synthesis then commences rapidly is fornred after all of the rnRNA spccific for the (Monroy et aI. 1965). However, only maternal tlansferase has been rnade (Table) 3' In other mRNA is used until just before gastrulation when rvorcls,the mRNA svnthcsisis ottt of phasc with en- about 4oo/nof the mRNA is replaced by a different zvrne synthesisas in the caseof the sea urchin egg' sort of mRNA, and significantly, new types of pro- Normally, rnesscnseris produccd for a limited time teins are made. At first glance the inhibitory pro- rrrrly irr Dict'tosteliurn discoideunt and its turnover tein or proteins in the complex with nRNA ancl is evident in that transferase activity disappears ribisomes in the sea urchin egg is not a repressor rapidlv after reaching a maximulu level. In con- in the sense of Jacob and Monod, but Sadler and trast to the sea rtrchitr data, experiments with Novick havc suggested repressor acts directly rvith Dictt'osteliun?seellr to inclicate control of gene ex- a region of mRNA made by the operator gene in pression by control of ntRNA svtrthesisas such' cxplanation of thc requirement for growth in B-ga- Cvclohexir-nidc, rvhich tenrporarily intcrrupts pro' lactosidase induction. There may be a strong for- tein svnthesis,also clisrupts svnthesisor function of mal resemblance in fertilization and induction rvhen transferasernRNA in the normal period. However, the dctails of the phenomena become clear. the event lcadirrg to mRN.A. production is either not In another differentiating system, the cellular a transient onc or is repeated as transferase messen- slime mold Dicytostelium discoideum, part of the ger is again producecl in a belated period of time. process of conversion of a portion of the cells of Lastly, let us consider an instance of non-chromo- the maturing organism to spores involves formation sonral inheritance in the mold N. crassa as an in- of a mucopolysaccharide which is a structural com- stance of modified gene expression in cornplex or- ponent of the spores. Actinomvcin D, rvhich blocks ganisms. f,ocatiottal specificity refers to roles of DNA dependent RNA polymerases,was used to find arnino acids not in the active sitcs of enzvmcs.

TABLE 4 GENETICS OF ENZY\IE LOCATIONAL SPECIFICI'|Y1 rN t'Et'RosPo R.1 (i11,^l.ts.-l

- - Nlitochondrial DNA (p l.?01) Ntrclear DNA (p 1.712) N{ i trr}rondrial Structural Protcin Association o in \Ialate Dehydrongenate (t\rsP) ][itocirondrion (asE)

\lichaelis Constant for llalatc (m,\I)3 MSP Added PrototroPlt l[utartt No. t0 20 None 0.72 1.1 2.1 2.9 1.4

Prototroph 0.72 J.J l0 Ji) Mutant m-l 500 6 3 it 1.4

m-3 40 a/ r.7 t.2 4.5

irritctretf, P., Ann. Rev. Microbiol., I), 407 (1959). i\Voodward and Munkres, P. N. A. S., t5,872 (1966). :\{unkres and Woodward, P. N. A. S., 55, t2t7 (1966).

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Non-chrornosornal inhcritatlce implicating rnito- talk about this coding prpblern I attt overrvhelmed chondrial DNA has been indicated in the gene for by the progress he reports. I think it is fair to saY, the rnitochondrial structural protein of N. crassa- then that wc pretty rvell know rvhat gencs are in Structural protein has no known ettzymic activity' terms of biochemical structure. Gencs are nlost cer- but is found in all mitochondria. As shown in tainly DNA (or RNA for some viruscs). We krrorv Table 4, rnitochondrial mutants m-l and m-3 each in general how gcnes rvork in a gross wav, and in have a structural protein with a single amino acid fact we are now finding out preciscly horv they tvork changeBruce Ames first proposctl inlportant lolc sible for gene translation into phenotvpic cxpres- haps codon neighbors u'ould play an to con- sion. I think it is the ultimate aim of any biologist in regulation. This is a very real possibility is that to takc a livine svstcrn or takc thc components of sider. One vcry important point, I think, corrcspoucling any biological svstet.trand ptrt thetn toeether in a svnonyms, that is dcgctrcrate codotts Thcv tcst tubc and have thctn work, and this incleed is to thc same amino acicls are not identical. Ofterr prccisclv rvhat L)r. Nirenberg and his associateshavc cliffcr markcdlv in t'uauv, lnany propcrtics. spccics tlone for tlte gcnctic information of the ccll. That tl'rey are recognized bv completely tliffcrerlt is, that thcy have assembledit'r an in vitro system of s-RNA also, so that one can spccifv a u'ortl' or the rnachinerv for protcin synthesisand using sytr' rathcr amino acid in complctely diffcrent rva-vs,an

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il 446 Carlson Ala. J. Med. Sci.

Down's synclrorue u'hich has becn rvorked out. The just have to u'ait and fiud out. eviclencesfor this are two-folcl. The first is that the DR. J-{N{ES PITTNI-{N: I u'ould like to ask onc enzyme changes of Dou'n's syndrome seen to be of the panelists, anv of the panclists, maybe Dr. conlined prinrarily to n'hite cells and very possibly Nirenberg or I)r. Yiclding, altout how horrnone to recl cells, but I think quite clear eviclence has action might fit in here. It has becn very much in becn prescntcd showing that there is no altcration vogue recently to invokc actions on RNA activity, of enzyme levels in Dor,r'n'ssyndrome in either fibro- as a rnethod, hormonc activity or horrnone action blasts or platelets and this work on fibroblasts has in turning on or turning off rcactions. How u'oulcl now bccn demonstrated by several cliffcrent labor- this tie in and horv tvould the reccnt ideas about atories. On the other side of the fence there is in- altering rates of transcription tic in with thc turning creasir.rgevidence to suggest that in Dor.r'n's syn- on and turning off of protein synthcsis? rlrome there is an abnormal rate of turnover or re- I)R. YIELDING: I think thar the problem of hor- production of the white cell cycle itself. I think Dr. trtone action is a very' complex one becausc we havc Mellman has shown this by traccr studics. I think to consider horrnones possibly operating at several the plevious paper by Dr. Motulsky on G-6-pD be- ler,els. First of all, thcv most ccrtainlr. do have an ing abundantly present in young cells is certainly influence at the genetic level in ternrs of modifr,ing also suggestivc evidencc to the effect that there the rates of protein svnthesisof various sorts altd at probably is an abnormality in the stmcture ancl this levcl could operate either at the levcl of regu- function of onc ccllular system rather than a gen- lating the rate of messcngersvnthesis or ntesscngcr- cralizeclgenetic altcration. formation. It could operate at the ribosome level DR. YIELDING: I think we are caught up here itr terms of regulating the reacling of the rnessage, in the problem of thc complexity of the mamrnalian or coulcl opcrate at the lcvcl of rnessetrgertuntor.cr. systern bccause rrc have the very real problem of C)n the othcr hand, rve certainlv recognizc sor-necx- knou'ing rvhich enzymes to look at and which corn- arnples,at least in ritro, of horrnones having an ef- ponents of the cell to look at. In looking for ab- fect on existing cltzvnle molcculcs, ancl this reprc- normalties in the trisomy state, and I rvoulcl ccr- scnts quite a different mechanisrnof action because tainly have to agree rlith l'ou that therc is no evi- no change in genetic ntatcrial is inrolred in this dencc for such an idea operating, and can only effect. I could conunent on that simply. to say that comment that it may sirnply mean that u'e haven't thcv could rvork anvrvhere,at our prcsent statc of lookcd at a sufficicnt numbcr of s\.sterns. \\Ie rvill knorvledge.

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il PANEL 2: DISEASESAT THE TIOLLCL]L,\R LL,VEL

Nloclerator: J. Creuor Bexxnrr, NI.D. Ernest E. llcCov. lt.D.: Induction of Leukoc'1,tr:Alkalinc Plrospltatasaby Str:roidsitt D(,non's Syndt'orne Richmoncl S. Paine, II.I).: Enzymatic Studies in Doiurt'sSyndrornc \\'illiam L. N1'han, l[.D., PIr.D.: Hy pertrriccttt ia

Dn. Cr..rurlr.lllrrr,r'r.: I think it is interesting to rccall that it lras norv bccn sornc l0l vcars sincc N{cttclcl lttrblisheclhis papcr on inhcritancc in peas.You u'ill also rccall that thc functional trrtits that hc clescribeclat that time, u'hich \\'c ltor{ understand as gencs,rvcre littlc rnore than tnathcmatical abstractions. But now \{e knorv thcse things in tcrtns of their chenristr,v,somervhat in ternrsof thcir orgauizatiott,thc rvay iu lvhich they act, and evcn a littlc altout tl-rcircontrol. It is vcry in"rportant for thosc of us interestetl in discasetncchanisms at the molecular lerel to holcl thesc things in nrind. Is there sorncthing rvhich \{c carl clo or catr etrvision cloing at a future date to tnoclifv tnolecules in diseasc states in a predictable antl

Induction of LeucocyfeAlkalinePhosphataseby Steroidsin Down'sSyndrome

ErnestE. McCoy,M.D.*

lt is one hundred years since Langdon Don'n de- this enzyme rvoulcl indicatc that the increascs of scribed the clinical condition knowr-r as mongolisur multiple enzynlcsvvoukl be of a non-specificnature. or Do$'n's Syndrome. Although studies on the inci- If one cxamines the data for increascsof leucocyte dence and relation to maternal age rverc known for alkaline phosphatascbctu'cen I)own's Syndromc pat many years, the discovcry by Lejeune of trisomy for tictrts and controls, one carl sec a considerable over- chromosome 2l focuscd attention on the tme ctiol- lap in the values, despitc the fact that frequently ogy of the clisease.A number of studies have becn overall there is a one-third greater incrcase in en- undertaken in an attempt to relate chen'rical ftrnc- zvrnc activity in one group compared to anothcr. tion to trisomy for chromosome 21. Several papers This perhaps shorrld be expccted, and especially have been published lvhich showed that the alka- with our incrcasein knorvledgcof biological control line phosphatase of polymorphonuclcar leucocvtes nrechanisms,that perhaps it rvould bc unlikelv that from Down's Syndrome patients showed greatel ac- the additional chrornosorlre 'rvould tlirect the s1'n- tivity than those of the controls. It I'as also noted thesis of materials abovc the physiological requirc- that leucocyte alkaline phosphataseactivity was de- rnents of the organism rvithout being subject to creased in chronic mvelogenous leukemia n'hich is control mechanisrns.\Vith this in mind we lookecl associateclwith deletion of a portion of chromosorne for a rnethod by rvhich thc potcntial biochemical 21. T'hese tlvo pieces of information have been pre- cliffcrences resulting from trisomy for chromosome sented as evidcncc for localization of leucocyte alka- ?l n"raybc accentuated.f'he studics to be presented line phosphatasc to chromosome 21. It u'as shon'n here todav shorv that when the production of leu- that in acldition to an increasein LAP activity, in- cocvtc alkaline phosphatase is stimulatcd by thc creases in activity of glucose 6-phosphate deh,vdro- acltninistration of steroid hormones. thc Dorvrr's genase, gal l-P uridyl transferase and acid phos- Svndrome patients have a greater increasein cnz\,rue phatase also tvere present in the leucocytes of activity than thcir controls matched for agc an

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il 448 McCoy Ala. J. Med. Sci. there rvas a dccrcase in Al, activity. These studies When 2 mg./K prcdnisolonc was injected intra- showed that stcroi(ls will increasealkaline phospha- muscularly into l)own's Syndrome patients, the aver- tase activity. age base line values increase from 1.5 to Ii.6 at 3 hours and to 6.6 p.M p-Naphthol/rng. prot./hr. 6 Dr, DiCarli has shown that an increasein numbcr hours later. The average base line value in control of the G group chromosornesof the heteroploid cell patients incrcased from I .l to 2.7 at 3 hours and to line EUII is associatedwith increased activity of 2.6 pM p-Naphthol/mg. prot./hr. 6 hours afrcr alkalinc phosphatasc. The grcater number of G prednisolone. The greatcr incrcases above base line group chromosorncs,the greater activity of alkaline 'I'hcrc levels in Down's Syndrome patients was significant phosphatase. would appear to be some rela- at p(.005 at both 3 and 6 hours. tionship bctrvccn thc numbers of G group chromo- somesprescnt ancl the level of alkaline phosphatasc To aid in determining whcther the greater in- activity. crcases in LAP activity following prednisolone was a specific or a non-specific everlt, a simultaneous We used thcsc two previous findings to determine mcasurement of the rate of increasc in whether in vir o steroids would affect leucocytc alka- activity of I AP and the X-chromosome linkcd cltzyme line phosphataseacrir,'ity in rabbits. In these stuclies C6PDH was carried out. Following the intrarnuscular rabbits rvere injectcd intraperitoneallv with various injec- tion of prednisolone 3 rng./K, average types of stcroids susltended in glycerol. The steroid LAp activitl' increased in Down's Syndrome paticnts front which provcd to bc the most effcctive was proges- a base line value of 2.6 to 8.6 pM p-Naphrhol/mg. tcronc. This perhaps should have been anticipated, prot./hr. at 6 hours. The differencc was as it n'as knorun that during pregnancy lcucocytc significant at p(.005 level. The average basc line values alkaline phosphatase increasesand then clecreascs of G6pDH in Dorvn's Syndromc patients \,vasl.l TI)NH/10(i after thc presnaltcv is terrninatccl.The incrcase in ,,rM leucocytcs/hr. ancl in conrrols leucoc,vtealkalinc phosphataseactivitv is mcasure

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taurinc in urine. This is a fairly large list of ab- drome patients following steroids is further sugges- nonnalities but in none to date is there sufficient tive evidence but not conclusive that the locus for data to link any to trisomy for chrornosome 21. leucocyte alkaline phosphatase is located on chromo- The greater increase of LAP activity in Down's Syn- some21.

Hyp"ruricemial

Williom L. Nyhon,M.D., Ph.D.*

.l' We were'askcclto consider with vou toda,vone of this who while restrained is happy and comfortablc, the inborn errors of metabolisnr as illustrative of he becomes terrified, and although he can't seem in rnolecular diseasc, and u'e have clcctcrl to discuss any way to prevent this kind of activity, his hand observations on a group of paticnts u'ith a newl-v goes to the mouth, and he indulgcs in destructivc recognized disordcr of purinc nretabolisrn. biting, screaming all the time as if in pain. The paticnts n'ith this particular disorclcr arc A little boy u'ith this disorder showed a partial rather well-developed and wcll-nourished btrt arc arnputation of one of the fingers and x-rays of his quite mentally rctardccl. Our first paticnt u'as four hand indicate that this involved even some of the ycars of age and hc could neither sit nor rvalk ancl phalangeal bone (Figure 1). Somc of these children had virtuall,v no speech. Sincc then, we have had have destroyed parts of their fingers. N{ost of them an opportunity at one place or another to stucly have cvidence of loss of tissue about the lip, and, some two dozen patients with the abnormality and if you look closely at this boy's face, you notice that I.Q.'s where tcstable, seemeclto run in the range of he has destroyed his lower lip to a point where it is the severcly retarded, under 50. Our first patient now no longer accessibleto his teeth (Figure 2). showed marked hypertonicity of the legs indicating A patient that we studied with Hocfnagel at that we were dealing with a problcm of cerebral l)artrnouth at age I I u'as thc oldest paticnt wc palsy. Indeed severe cerebral palsv has becn regu- studieclwith the abnormalitv and ccrtainly the rnost larly found in this syndrome. scvere example of this kind of sclf-rlcstructivebc- Another of our patients was a little bov l'horn rve havior. have secn recently at the Sunland Hospital in Or- The clinical history of the first paticnt was that lando and like the othcr patient, had mental retarcl- hc carne to the hospital alrcady retanlcd and shorv- ation ancl ccrebral palsy. The choreoathetosiscoukl ine all the clinical manifestationsr\re have lust bccn almost be obscrved fronr thc bov's posturing in a talkine about but presetrting tvith het.uatttria. .{ still picturc. Wc are now building up then a clinical verv good housc officer cottfrotttetl rT'ith this pcr- picture of mental retardation, verv scvcrc spastic fornrecl a carcftrl cxat.trination of the ttritlc. Hc ccrebral palsy and chorcoathctosis. .Somc of rnr founcl that it ruas loaderl rvith crystals, :rtr

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il 450 Nyhan Ala. J. Med. Sci.

:1:'llf,6. |iffi

Figure 1. Patient E. W.-Roentgenogram of the hands illustrating loss of ciigital tissue which followed biting. and levels over 6 are consistent with a diagnosis of adults who are gouty and who are known hyper- gout. The patients we studied with this condition excretors because they have more than about 600 have had some variability in the plasma uric acid milligrams of uric acid in a 24-hour urine. In control levels, but most of them have had levels at most children, of about the size of the patients we have times in the vicinity of l0 milligrams per 100 ml. studied who weighed about l5 kilograms, less total Therefore, a distinct elevation of uric acid in the ttric acid is found in the urine than in adults, as plasma is characteristic. 1'ou might expect from their size. In this disease The data on urinary excretion of uric acid (Table excretion of uric acid in the urine would put the l) were obtained on a fairly rigid diet which ex. patients in the hyperexcretor range even if thcy cludes purine intake. Under these conditions an rveighed 70 kilograrns. Nearly all of our children adult has an excretion of uric acid that is under have had uric acid ercretion over 600 milligrams about 500 milligrams per day. The vast majority of TABLE 2 adults who have gout have uric acid levels that are GLYCINECONVERSION TO URINARY URATE in the same range. There are a small number of

TABLE 1 No. "/o Conaersion Range URIC ACID EXCRETION Control Adults" 7 0.17 0.1I -0.22 Goutv Adults* 9 0.29 0.06-0.36 ( Normal Excretors) No. trIS./Day Range MS./KS./Day Gouty Adultst 3 0.49 0.25-0.73 Control Adults* 4 370 325-455 5.3 ( H_vperexcretors) (ioutv Adults* 14 408 253-514 5.8 ControlChildren** 3 0.012 0.011-0.013 ( Normal Excretors) Patients** 2 2.25 2.12-2.39 Coutv Adults* 4 J54 587-1054 10.8 ( Hvperexcretors) {ry-lln"-1-6'n P.O.-From published data assembled by Control Children 3 233 l 76-289 10.3 W.vngaarden, J. 8., in The Metabolic Basis of Inherited Disease, edited by B. Stanbury, B. Wyngaarden antl Patients 2 690 669-712 45.3 J. J' D. S. Iredrickson, 1960,p. 712. *From *Glvcine-U-C11 published data assembledby Wyngaarden, J. 8., in " I.V. The trIetabolic Basis of Inherited Disease, edited by R. J. Total CPNI in 7 Day Urinary Urate Stanbur-v, J. B. Wyngaarden and D. S. Fredrickson, 1960, 7o Conversion = x 100 p. 712. Total CPII Glycine Administered

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il Vol. 3, No. 4, 1966 HYPERURICEMIA 451

Purine is synthesized de novo from small mole- cules such as glycine, and the labeling on the gly- cine molecules indicates glycine is incorporated into the uric acid molecule whole, making up the 4, 5 and 7 positions in the uric acid molecule. We have undertaken studies in which patients and controls have been given glycine and then we have estimated the rate at which uric acid is formed out of this glycine administered. Glycine was administered; all of the urine pro- duced was collected over a period of seven days; thc uric acid isolated and purified and its radioactivity assessed.In the patients there was a very rapid peak of conversion within the first 24 hours. In controls, the peak is not so readily appreciated, but probably someplace between one and two days the specific activities are a bit higher than at other times. At the peak, it is easy to distinguish the two populations with different ranges from that of 30 in control to from 100 to 250 counts per minute per milligram i of uric acid in patients. Studies of this sort have q been done in adults with gout, in trying to compare them with normal adults' It has never been possible \ to distinguish the two populations, so that bio' iii chemical rheumatologists hal'e tended to do this kind of experiment and express the data in terms n of the cumulative percent of the glycine that was then converted to uric acid (Table 2). a- {'ir? dd -* * L In seven days of study our control individuals hundredth of a per- Figure 2. Patient E. W.-Loss of tissue of the converted approximately one lower lip followed biting. cent of the g\cine administered to uric acid, where' same period of time the patients converted per day. \\'e have rnade a transforrnation for size as in the of the administered glycine to uric expressing excretions of uric acid in milligrams per up to 2 perceni a difference of approximately kilogram and we have expressed it in milligrams fnit represents "iia. is considerably in excess of anv per milligram of creatine excreted. These patients 200 times, *tti.h could establish among adult in these terms, have anyrvhere from four to eight difference that one model again of the gouty adult times the values of any other population group. populations. In the vs.^the normal adult, the biggest difference in those These findings of increased excretion and in' two populations is of the order of two times differ- creased plasma level suggest the possibility of a ence. rnetabolic defect, and a certain number of studies have continued in this condition' wc have been carried out which are consistent with As studies few more patients' The earlier experi- that idea. One of the classicways of studying gout have seen a suggestedthat this was a familial is a turnover study, in which thc patient is injected ence with brothers We have studied a family in Michigan with uric acid labeled in the 2 position with carbon condition. ancl reported recently in the Journal 14 and then the uric acid of the urine is crystal- rvith Dr. Oliver This mother had a son who had diecl lized, purified and its specific activity determined. of Pediatrics. symPtoms precisely like those rve In this wa,vor)e can get a figure for the total amount earlier and hacl She had another boy u'ho of uric acid in the body and the rate at which it have been describing. months, which is, too earlY to kno'rv turns ovcr. The slope ,rf tn" line, r,r'hich indicates died at four he had this condition' She then rc- thc rate of turnover in the patient, was much whether or not the propositus whom rve studiccl' stceper than that which one would expect even in married and had both clinically and chcnricallr' overproduction gout in the adult. He has the syndrome suggests that there rnight be sotnc- These chilclren have been making over a gram of This kindrecl that mother. uric acid a rlav. This matches, without expression thing special about has been rcportctl llr for size, that scen in gouty adults. In terms of the A somewhat larger kindred that this is a discaseof rate at which thc whole pool of uric acicl,or all thc Hoefnagel. His data suggest through the fc- uric acid in the body, gets rejuvenated, this hap- the male ancl that it is transmitted large kinrlrccis has pens among adults at about one-half pool a day. In male. Experience with two othcr is car- this condition turnovers iu the range of one and a led to thc conclusion that the abnonlralitv half to two pools a clav are found and considerably ried on the X-chrolnosomc. exceed any of those seen in any othcr condition. In summarv, rvc have describcd a llc\r clisoltlcr of

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fl 452 Nyhan Ala. J. Med. Sci. the type that Dr. Motulsky spoke this morning and that the disease may'be just as bizarre from thc of which Dr. Garrod wrote in the early 1900's.This biochemical point of view as it is from the clinical abnormality is unusual in its clinical characteristics, point of view. From the point of view of genetics, rvhich include mental retardation, cerebral palsy it appears that it is transmitted on the X-chromo- and choreoathetosis as well as bizarre self-destruc. some. tivc behavior. Biochemical studies have indicated

EnzymartcF ri Studiesin Down'sSyndrome

RichmondS. Poine,M.D.*

The first enzyme in which an abnornality was uridyl transferase in white or red cells was elevated discovered in connection with mongolism, as Dr. in the trisomies but not in the translocations as McCoy pointed out was alkaline phosphatase. u'erc alkaline and acid phosphatase in leukocytes, O'Sullivan and Pryles in 1963, studying some pa- glucose-O-phosphatedehydrogenase in the rcd cells tients at the Wrentham State School near Boston and ir-nucleotidasein the white cells.But in the case mentioned that the alkaline phosphatase levels as of creatine phosphokinase, rather to orlr surprise, judged by the Gomori stain method were not ele- the blood levels were elevated in both types. vated in the 3 translocation mongols which were A number of cnzymes did not show elevated ac- included in the group they studied. It seemed logi- tivity. These were all in the plasma. In general the cal to study more extensively the chemical differ- conclusion would seem to be that of the limited ences, if any, between trisomic and translocation number of enzymes tcsted, out of hundreds which mongols. Finding a sufficient number of transloca. would be possible, it is the cellular enzymes,par- tions rvas not casy. After making a hundred routine ticularly those in red and white blood cells which karyotypes of mongols rve had only one new trans- are likelv to be abnormal, and the plasma cnzymes, location in addition to the four or five we had except for creatine phosphokinase,have not bcen. before, but thanks to the help of a number of col- In Fieure l, we sce one cxample. This is acid leagues who made their patients available, rve rvere phosphataseexpressed in Sigma units pcr milligram able to collect 12 translocation mongols, eight of of protein: the patients are trisomic mongols repre- which were of the D/G type and four of rvhich rvere sented by circles, translocations by triangles, and G/G's: 12 trisomies were matched to them by age controls by solid squares. There is quite a neat and sex and other factors insofar as possible. These separation, and regardless of age, there is no over- were examined "blind" by an experienced pedia- lap betrveen the translocations and the controls on trician rvho also hacl neurological training and thev were also tested, completely blind, by our chernical staff, an

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- il Vol. 3, No. 4, 1965 ENZYMESAND DOWN'SSYNDROME 453 the one side and the trisomies on the other. In the tt caseof alkaline phosphatase, the separation is much l''t--r M (r) to, l!. ta,a r2.0 6.1-12. t better. All the translocations and all the controls l'' -1 M (r) 26. 26, l0. a 2!.0 I 2a r. t-1,9 were well below 2.0 units per milligram of protein l^rr. 5t 0.4-o.a rvere above it. Very similar l*t and all the trisomies crx..68 !$- 2&, t5o. !t6 l!0-a2l I graphs were obtaincd for galactose-l-phosphate l'" 5,4 24. o-t2 uridyl transferase in the red cclls, for 5 nucleoti- FIGURE 3 des in the white cclls, and for G-6-PD. This, as Dr. McCoy has commented, is an enzyme, for which the kinase in the plasma, are not significantly different. gene is generally accepted as being on the X-chromo' The range of variation of normal will be presented some, since the lack of it is a sex-linked recessive later, but the increase in the trisomy is in only one inherited trait. But there is an equally good separa' instance (galactose-l -phosphate uridyl transferase tion with all the trisomies being above and all the in red cells) increased by approximately 5070 $9To translocations and controls below 500 units. Sero' in this case). All the others are increased by con- tonin, another substance we have studied, is in con- siderably more than that, and it may be more attrac- trast depressed. Serotonin is measured in whole tive to think that we are dealing with a secondary blood-I shan't give the details of the method here, or tertiary effect, either a regulator gene of some since it has been published (Rosner et. al., 1965). sort, or perhaps we are measuring a more remote Again, if one draws the line at just about 100 metabolic or biochemical consequence of whatever pg./100 ml. all the trisomic mongols have levels genetical material is involzed. below this and all the controls and translocations The next logical approach was to study the par- have levels above it, regardless of age. ents who carried the D/G translocations. In Figure A number of other enzymes have been studied. 3 we see the results for five Di G carriers, four Hsia and his colleagues have reported several to be females and one male. We had already mentioned normal. Webler has reported elevated acetylchol;n- that except for creatine phosphokinase in the esterase in the red cells. The question now arises as plasma, nothing was abnormal in the translocation to what the explanation might be for the elevated mongols. These figures fall within the normal levels in the enzymes of the trisomies. It is possible range by the methods we used, but creatine phos' and conceivable, if one wants to take the "gene-dose phokinase would normally range from almost zero hypothesis" that one could postulate that the tri- up to 12 units (in the caseof the normals the mean somic mongol might have three "doses" of the gene was 5.4. whereas the means of the translocations present on the short arm of the 2l chromosome, and was well above this). We found, however, that the that this material might be lost in the process of mean level of creatine phosphokinase activity in translocation and that the translocation mongol the plasma of the maternal carriers was well down might then have only two of this particular gene. in the normal range. In the case of the single pa- Equally, one could speculate that the effect was ternal carrier, it was a little bit on the high side modified by some neighboring genetical material but much below what the translocations them' in the new locus of the translocated DNA. If either selves had. I don't know, and don't pretend to hypothesis were correct, one would suspect (and know, why the enzyme which we associate with particularly if it were a triple versus a double gene pseudohypertrophic muscular dystrophy should be dose effect) that the elevated level of enzyme, if it abnormally high in patients with mongolism. Per- were a direct effect, ought to be 50/" greater in the haps it indicates a greater wasting of their muscles trisomy than in the translocation. But we see that but, in any event, whatever the mechanism is that this is not the case. In Figure 2 we have the means produces this elevation in the translocation, it ap' for normals, the means for the translocations, and pears not to be present in the balanced carriers. the means for the trisomies expressed as percentage We also studied a patient with some additional of normal. It will be seen that the differences be- genetical material on the short arm of number 21. tween the translocation and the normals in terms This woman had elevated levels of acid and alka' of means, except in the case of creatine phospho- line phosphatase and this is of some interest, as perhaps, even though she was clinically normal involved; and Normls Tran!l6alron! I rigomi6 herself, some chemical effects may be it reminds us that the clinical findings don't neces' t{ea n Mean Mea t ol l{ormel sarily correlate with the biochemical ones. Gal.-l PUI llrEC l0.l D.l ?r.5 252 We have been extending this approach to other G.l.-l PUI lRSC ?6.) 26.9 4t.7 159 chromosomal anomalies, and this is an obvious next Alt. P'.se IIVBC l.l4 |.2t Llt ?)l move. Hsia has already reported elevated activity Ac. P'rs€ lW8C 0.{a 0.{6 0.t? tE6 of alkaline phosphatase in trisomy 18 but not of a

6luc. 6 PD IRBC ll6. 260. t?0. ?11 number of other enzymes. We are faced with the why this same biochemical change shoulcl t'ilucl'.se IWBC 20.2 20.2 6?.1 t0t question: be noted in two entirely different chromosomal Creat. Pl rPlri 5.4 26_2 2l.8 >1> anomalies. Our own particular patient rlas a FICURE 2 routine retarded child who came from a consicler'

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il 454 paine Ala. J. Med. Sci. able clistance for investigation. The only reason the on thc f rcsh blood and for this reason all the chromosomes were studied at all was for complete- studies rvcre performecl on fresh blood right at the ness, since she had no remarkable physical findings time, for both controls and the trisomics. aside from mental deficiency and a narrow anterior DR. HECHT: \\Iere the controls clrawn at the same fossa of the head with a metopic ridge. She had a place? sort of marker chromosome with extra material on DR. PAINE: Normal controls rvere always tested number 13. She also had elevateclgalactose-l-phos- at the same time as the translocation mongols. The phate uridyl transferasc activity in both red blood control trisomic mongols were located in thc Wash- cells and white cells. The patient's father was ap- ington area, for the most part, but all were studietl parently the carrier of this little marker chromo. fresh. some. He was clinically perfectly normal and the DR. BENNETT: I have one question for Dr. Nr:- enzymes were normal in his blood and I suspect han regarding the ntetabolic phenomena in these that we are dealing here with an entirely coinci- patients. The drug used now quite a lot in treating dental marker chromosomc with a little additional adult gout is allopurinol to block the conversion of material which is perhaps not clinically significant. hypoxanthine and xanthine uric acid. What effect I think one could sum up by saying that it is if any does this have on your children? probably feasible to carry out enzyme studies as a DR. NYHAN: I am delighted that you askcrl. \Ve relatively cheap and efficient means of differen- have been working with allopurinol for about two tiating trisomies from translocation mongols, but I years now and have had a fair experiencc with the should not have confidence in it unless a number use of this in chiklren rvith this discase and in of enzymes were measured. We have had occasional gouty adults. The drug is, of course, very cffective exceptions to the general statement that the en- in lowering the concentrations of uric acid in the zymes mentioned are all elevated in trisomies but blood and urine of the paticnts with gout. One not in translocations, but I have not seen any cases rnust use relatively high closcs for the size of the where as many as two or three were crossing us up. patient, becausethis is a competitive inhibitor. We If onc has to do complicated enzymatic chemical have treated a number of patients with allopurinol determinations of this sort on fresh blood gotten for over 12 month periods now, and there is no right at the laboratory and involving skillecl ancl question that n'e can control their levels of uric expensive technical help, it may be just as cheap acid. Various individuals in looking at them have with the advances now being made to study chromo- been somewhat encouragedby their behavior, but if somal karyotypes and I think I should have more vou look at them critically, it is hard to seea differ- confidence in doing this. We certainly are not map- ence clinically or behaviorally. However, if phenyl- ping the genes on the 2l chromosome. I think ketonuria or maple s)'rup urine disease was our rather that we are nleasuring more remote effects, tnodel, ,vou would expect that. In other words, you and the fact that this sort of approach has raisecl rvould expect that their abnormality would be more questions than it has supplied answers, lnav treatablc only during the first year of life. We are be a very healthy situation, which I hope rvill be now excited about the fact that two boys had been illurninated further in the discussionswhich will born in families under stucll'. Male children have follorv. been found now in Dallas, Tcxas and in Clevcland, BIBLIOGRAPHY Ohio; and I think wc can sa)' quite clearly that we

Rosner, F., B. H. Ong, R. S. paine and D. \lahanand. are able to make the diasnosis in the first 24 hours 1965. BIood-serotonin activity in trisomic and transloca- of life and we norv have two chilclren who hare tion l)own's syndrome. Lancet l:llgl-1193. becn treated with allopurinol sincc at lcast dal threc. One of these chilclrcn is thrce months olcl. *,i,,'i, Tirne will tell whether this is a wav of prcvention of defect, but n'c are larrnchecl. DR. FREDERICK HECHT: This quesrion is di. rected to Dr. Paine. I n'ould be interested to knon. DR, BENNETT: I l'raveone other rluestion in re- about geographic distribution of the translocarion garcl tc metabolisrn and that is that Sorenson in cases.\Vere the bloods shipped to you; ancl if so, Chicago has now shorvn that the antirnetabolite u'ere they shipped in n,ith controls? azathioprine or Irnrrran can bkrck the rapid conver- DR. I'AINE: The question concerns the geograph- sion of Cu glvcine to uric acicl in gouty adults. Does this ical locations. The patients were, I believe, 21 alto- same thing occur in vour patients or have l'ou gether; there were three located at the Wrenthanr lookeclat that? State School in Massachusetts,and I think four at DR. NYHAN: \\'c have not yet looked. the Lynchburg Training School, Lynchburg, Vir- DR. -\NDREW LORINZ: I think we ought to con- ginia. In each case our chemical team and also a gratulate the panel for bringing us up-to-date as to physician went to the institution itself and the rvhat is up in Down's and gonc bevond thc p;out. laboratory work was done on the spot until the This is an obvious question to Bill Nyhan about enzymes were to the point where we knew them to the hcterozygotes.Can yon do loading studies or are be stable from previous work on controls. .{. num- there any biochemical tests for detecting carriers of ber of the chemical determinations hacl to be done this trait?

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il Vol. 3, No. 4, 1966 ENZYMESAND DOWN'SSYNDROME 455

DR. NYHAN: I really don't know the answer to not appear to be too rnuch variation, but I am not that, although I would hopc that in a couple of entirely satisfied on this point, and this is indeed a n'eeks I will have it. Wc hal'e been chasing it ver,v very preliminary sort of report. hard and I think that we can tell the heterozygotes. DR. N{cCOY: Just one other comment, Dr. Yielding, How best we will be able to tell depends a little bit and that is that there appears to be a continuing upon the fact that u'ith this type of study, controls toxic process which is going on in the Down's syn- are a little hard to conre by. We have been accumu' drome patient after birth. This is evidenced by the lating them this summer. I think we can, but I don't fact that punctate cataracts are not visiblc at an think we are in a position to answer yet. early age, but by about age 20 almost 67fo of the DR. PAINE: I think both these points are very Down's syndrome patients will have this type of u'ell taken and all the comments extremely valu- cataract. The second bit of evidencc for a continu- able. The main thing that it proves probably is that ing toxic process is an increased rate of destruction n'e know more about the gencs of rnicroorganisms of neuronal cells. This is chicfly the work of Dr. than we do about the genes of rnongols or of nor- Benda. So that those two things would make me mal people. The selcction of rvhat enzyme to study somewhat encouraged, although I agree with you is indced a vexins problern. .{s we proceed from wholeheartedl.y that we don't knorv what changes the study of mongols, on which there is a certait-t are occurring. lVe may be able to detect some essen' amount of published information ,much of it ad- tial derangement although here again I am sure it mittedly wrong, about biochemical changes of one is not going to bc a single enzyme or disorder but type or another to the study of 13-15 and 17-18 probablv a multifaceted situation. trisomies and other chromosomal anomalies, we DR. L. L. CAVALLI-SFORZA: I *'ould like to men- have no guidelines at all. The guidelines we have tion in addition to those data stated in alkaline bcen trying to use are to study enzymes which phosphatase, there is some el'idence on tissue cul- might plausibly be rclated to the tissuesin which trlres, that points ottt that alkaline phosphatase is n'e know anatornical abnormalitics are present, but probably on chromosome 22 or 2l after all. By this is merely an educated guess and it is going to selecting alkaline phosphatase negative mutants on be a long, slow process because of the amount of tissue cultures they shon'ed a reduction in the num- time required to study each individual enzyme in ber of chrom<-rsomcswhich are presumably of grouP even a few patients. As to the ntatter of variation 21. They cannot obviously be distinguished from within an individual patient, u'e did not find anv 22, and tissue cultures have a somewhat variable significant difference according to sex or according; number of chromosomes but the evidcnce was rather to age, and in the limited number of determina' clear-cut and several strains were investigated, and tions 'rve were able to do on the same patients in the other enzymes u'hich were mentioned were not differcnt hours of the day, different days of the affected under those conditions, while alkaline u'eek, or different phases of the lnoon, there does phosphatase shon'ed a very tlramatic decrease.

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il PANEL 3: DIAGNOSIS AND MANAGEN,IENT OF I,TETABOLIC ABNORMALITIES Moderator: DoNoucn O'BRrnru,N,I.D., F.R.C.P.

Robert S. Krooth, M.D., Ph.D.: Diploid Cetl Stroinsf rom Patients uith Inherited Bio- chemical A bn ormaliti e s. Donough o'Brien, N{.D., F.R.C.P.: some comments on Diagno.sisand Treatment of Meta- bolic Abnormalities. charles A. Alforcl,.1r., M.D.: congenital Rubella: A lr[odet f or chronic in utero Inf ections in Man.

Diploid Cell Strainsfrom Patients\Mith lnherited BiochemicalAbnormalitiest

RobertS. Krootfi,M.D.)

Sotne of thc gcncs rvhich causc nlctabolic abnor- Sclcctive svstcnrs arc nseful, because (at least in nralities confer a distinctive phenotype on seriallv theory) thcy enablc us to detect cells which havc cultured human diploid cells. Unfortunately, hor,r- changed genctically, cven when such cells are very ever, only a fraction of the known loci in the hu- infrcquent in thc population. rnan genontc can lte stuclied in cell culture (Gartlcr I should like now briefly to sumrnarize the two and Pious, 1966; Levintow and Eaglc, lg6l; Krooth, sclectivesvsterns. 1965).Many loci are not cxpressedin cultured cells, As vou know, galactoscmia(Isselbachcr, 1966) is a at least not in a way that we catr detect. Although lare autosornal rccessivedisordcr. l-igurc I summar- culturcd cells carry out a varicty of rnetabolic anrl izes thrcc scqucntial rcactions that are irnportant synthetic activitics, thcy do not usuallv contain thc ir-r the conversion of galactose to glucose. In addi- tissue-specificenzvrncs of the organ from tr,hich thev tion to thc cnzvmescatalyzing thcse reactions,there wcre isolalctl. are at lcast three othcr activitics (Issclbacher,1966; At present, thclcfore, \\,c can studv onlv those loci Cuortccasasand Scgal, 1966; Ohno ct al, 1966)that which affect molccr.tlcsthat thc cell spontaneouslv help in thc catabolism of galactose.Thcse other svnthesizes.For cxarnple, .n'ecaltnot study the loci activitics havc not thus far been identified in cul- affecting hcrnogloltin, becauseserially cultured htr- tttrecl hurnan cclls. Hornozygous patients rvith the n)an cclls, irrcspectivc of gcnotypc, do not s)'n- clinicallv significant forrn of galactosemiahave di- thesizchcrrroglobin. nrinishe

--J-

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TABLE 1 'I'HL, JO\IE GENE,TIC ARNOR\IAI,ITIES THAT AFTI,CT SPECIFIC TIOLL,CULES PRESEN-I IN CUL'TURED HU}TAN DIPLOID CELL

Disease or Variant Enzyme Genetics f)isease or Variant

Acatalasia I (Japancsc variant) Catalase Atrtosomal reccssive {,catalasia II (Swiss variant) Catalase Arrtosomal recessive Familial non-spherocl tic hemolytic Glucose-6-phosphate Sex-linkcd recessive anemla dehydrogenase Electrophoretic variants of G-6-PD Glucose-6-phosphate Scx-linkcd co-dominanl (asymptomatic) dehydrogenase (G-6-PD) G-6-PD deficiency Glucose-6-phosphate Sex-linked reccssive (Mediterranean t-vpe) dehydrogenase G-6-PD deficiency Glucose-6-phosphate Scx-linkcd recessive (Negro type) dchydrogenase Galactosemia UDP galactose transferase Autosomal recessive Hurler's syndrome Uncertain Sex-linkcd rccessive or autosomal recessive Electrophorctic variants of lactic Lactic acid dehydrogenase Autosomal co-dominant acid dehydrogenase N{aple syrup urine discasc Llncertain Autosomal rcccssive Niemann-Pick disease Unccrtain Autosomal recessive Orotic aciduria O rotidi ne-5' -monophosphatc ALrtosomal recessive pyrophosphorylase and orotidi ne-5'-monophosphatc decarboxylase Electrophoretic variant of P hosphoglucomu tasc ,\utosomal co-dominant phosphoglucomu tase Electrophorctic variants of ti-phosphoglucuronic acid Autosomal co-dominanI 6-phosphoglucuronic acid dehy'drogenase dehydrogcnasc

Figure 3 shows a sirnilar experiment, performed synthesizc tn'o of the bascs of RNA and t!{o of the concurrcntly, on a galactosemic cell strain. The bases of DNA, as wcll as ccrtain other necessary growth curves are much the same, except that now compounds. Fluman diploid ccll strains from pa- the 100 milligrarn pcrcent galactose curve does not ticnts who arc r.nutant homozygous at the orotic lie between the two glucose curves. Instead it fol- aciduria locus havc about a fifty-fold reduction in Iows the hexose-free curve. I havc reported else- specific activity for the final two cnzymcs of the where (Krooth, 1966) a more detailed description of pathway (Krooth, 196'1;Howcll ct al, 1966). The these experiments. clinical features of the discase respond dramatically Figure 4 gives the pathway whereby hurnan cells to supplementation of the dict with uri

c U rCCrtxPtnl^{NT lr I t

H.lNmgmlGucose

* . lm mlm * 0dbclole

{]{.5 mgma Cllcos€

E

005

oJlra, ' '! 0 2 l 6 I l0 le l{ l0 ll nz 2t z! a 0 2 a 6 I l0 l? l1 16 t8 ?l ?2 ;1 ?oI ltt rolYsl Tttt IDAY5t Fig. 2. Growth of a diploid cell strain which is homozygous normal (GG) at the galactosemia locus. This experiment was performed simultaneously on both normal and homozygous galactosemic (g*g*) cells, each at three different initial population densities. The growth of the galactosemic strain is shown in Figure 3.

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fl 458 Krooth Ala. J. Med. Sci.

J 0 U 1g'9' txPtRlMIIT l( t rI L

nn2126d l0 l2 t4 t6 1E20 22 U 26A Ttst {otYs)

Fig. 3. See Figure 2.

c00H I \ CHNH^ ASPARTATE H,N CH" lz H2NC00P IRANSCAR EAMYLASE D|HYDRO0ROTASE lr+ - r l. CARBAMYL rl 1"2 PHOSPHATE co cH-cooH I c00H ASPA RTATT I I \-,/ I I I uRilDosucctNtc 0H. I ACID l. I c00H /\ I N CH^ CHNH^+ATP ll' 1', io tH-coog cH^ \,/ aut' t' orHyoJoonorrc c0NH2 ACID GLUTAMI NE I I DtHYDR00ROTtC REDUCTASE I I DNA tt. -5- /'\ OROTIDINE MONOPHOSPHATE /w:, OROTID I NE.5. MONOPHOSPHATE NCH ,. /'t\ --)l DECAREOXYtASE N cH l" RNA PYROPHOSPHORYLAS E ,,' lll I ll I c0 cH c0 c-c00H,! MG". PRPP c0I ll,c-c00H COENZYMES

I I H .5- RI BOSE MONOPHOSPHAIE RI BOSE-5- MONOPHOSPHATE OROIICACID -5'- URI DINE MONOPHOSPHAIT OROTIDINE-5'- MONOPHOSPHAIT

(UMPI (OMP)

Fig. 4. Biosynthetic pathway whereby cultured mammalian cells synthesize uridine -5'-monophosphate.

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il Vol. 3, No. 'f, 1966 DIPLOIDCELL STRA]NS 459

GROWTHOF THE HOMOZYGOUSMUTANT AND NORMALCELLS IN VARIOUSNUCLEOSIDE SUPPLEMENTS

,8

f f ,' ,'tC /A g t" ,,/a ,'/ .,A l'."'E //',i ./' i/ ////,/' ,/ ,// / /'./ / 't/ / ll ./ / .-E l/ o ./ E 200 l/ ',/ o / ,/'"' I oD : ---- .Eo l, ,/

z. ,' / ---F l-ll roo F -''=-':'1" D o l' / ,rt= E 80 ,, ./. (L A=AUTOMEDIUM i/, J 60 B=AUTOMEDIUM + URIDINE J IJ C=AUTOMEDIUM +URIDINE+ADENOSINE O D=AUTOfT4EDIUM +ADENOSINE E=AU TOMEDIUM + GUANOSINE F =AUTOMEDITJM+GUANOSINE + ADENOSINE CELLSTRAIN Ru CELLSTRAIN AUC

Fig. 5. Selective system for the orotic aciduria locus.

rninish bv another three or four fold their specific prcsunrably because thc mutant senc has lcft thc activity for at least one of these enzylnes:orotidine- ccll lvith some residual activity for both thc cn- 5'-monophosphatedccarboxylase (Krooth, 1964).Thc zymes it affects. The growth of the mutant homozy- spccific actir.ity of noimal cclls falls bv only about gous cells is, however, always stimulatcd if uridine l0 perce nt undcr thcse conditions. \Ve still clo is prescnt. not knon' rvhat pregrorvth in adenosinc cloes to If one adtls not uridine but adenosine to thc orotidine-l'r'-rr.ronophosphatepyrophosphorvlase-thc medium, thc mutant homozygous cells becomc un- other enzyme affected by the gene. ablc to sustain growth. The reason for this is prob- Figurc 5 dcscribesa growth experinrcnt pcrfornrcrl ably that the furthcr reduction in enzyme activity on nornral and mutant homozl'gous cells. The cells occasioned by the adenosine is cnoush to slow were gro\\'n in rninimal mcclium alonc and in mini- uridylic acid synthesis to the point wherc cell pro- rnal mcclium supplernented rvith various nucleosides. liferation cannot continuc. This vicw is supportcd Note that the various r-rucleosides,at the concentra- by the fact that the cells are protectcd from the tions used, do not rnatcriallv affect the growth of growth-inhibiting property of adenosinc if uridinc normal cells. The mutant hornozvgous cells are able is also in the medium. So, putting it perhaps a to grow, to some extent, in the abscnccof uricline, little gliblv, one can say the rnutation is thc firing

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*=il 460 Krooth Ala. J. Med. Sci.

squad, and exogenous adenosine is the coup de genetic block, or perhaps the block involved a re- 'Ihere grace. action peculiar to erythrocytes. arc other I should like now to ask the following qucstion: possibilitiesas well. I mention this example because Of what use are cultured cells in the diagnosis and I suspect experiments of this kind may sometimes management of rnetabolic abnormalities? At the reveal the location, and also incidcntally the appro- lnoment the answer is very simple: The cells are of priate therapy, of abnormalities of biosynthesis in no use. The best approach to the diagnosis of these human tissue.The great limitation of this approach abnormalities is by history and physical examina- derives from the failure of the cclls to synthesize tion and by chemical determinarions on thc body tissue-specificenzymes. fluids and tisstres.It is much casier to measure All of these experiments fall under the heading specific enzyme activity on red or white blood cells of research.They have little to do with diagnosis in than it is to develop a diploid cell strain from the the clinical sense. Diagnosis in the clinical sense patient. The rnain use of cultured cells at present rneans finding out into which of a number of pre- derives from their value as research tools. A diploid existing categoriesa particular patient falls. Finding cell strain, biochemically marked by the action of a a new category or learning more about the pre- Mendelian gene, is analogous to a mutant strain of existing ones is, I would say, research. However, in Itleurosporaor E. coli.It allows one to apply to hu- the case of genetic diseascs,this pedantic distinc- rnan cells, some of the methods of microbial genet- tion breaks down. Suppose a patient's cells for ics. C)ne can look for viral transduction, cell hybrid- genetic reasons lack catalytic activity for a particu- ization, DNA-mediated genetic transformation, etc. lar reaction, and suppose also thc patient gets sick Indeed some of these phenomena have already been becausehe runs out of product or accumulatestoxic reported in heteroploid mammalian cells. amounts of substrate. When one has identificd the In addition to helping with experiments aimed catalytic activity which is missing, one can give the at changing the cells genetically, biochemically disease a chemical name-often a !.ery imposing marked cell strains can provide information about one. But uhy is the catalytic activity missing? We the mechanism of gene action. Some of this infor- now know from experiments with conventional mation could not be obtained from clinical studies microorganisms (reviewed elsewhere in this volume) on the whole patient, or from tissuebiopsies either. that the presence of a mutant gene can result in For example, Figure 5 showed orotic aciduric cells diminished catalytic activity even when the struc. growing for almost two weeks in a medium free of tural gene or genes are wild type. For exanrple, in uridine. It would be improper ro wirhhold uridine the case of.E. coli we notv know of several dif f erent from affected patients for this period of time, once ways in which mutation at sites other than the the diagnosis had been established, just as it would structural locus can diminish enzyme activity. More- be improper to raise the blood galactose level of a over, if the mutation is at a structural locus. one galactosemic to l0O milligrams percent, or to lower can ask whether there is an amino acid substitution anybody's blood glucose level to zero. It might also in the protein, and, if so, what the substitution is, be impossible to effect such drastic changes. Homeo- and, even harder, precisely rvhere in the polypeptide static mechanism and even tissue necrosis would chain it has occurred. There are still other questions interfere. The effect of adenosine is also an example one can ask. All the questions have the following of the way cultured cells can provide information two properties: unobtainable by other means. Here the cells were l) Answers to them presumably exist, grown in the absenceof uridine and in the presence 2) They are relevant to exact diagnosis. of l5 milligrams per liter adenosine. The effect of Fortunately, we do not require such precise diag- adenosine was not evident until after the first ?2 nostic classification in order to treat most genetic hours, and then one saw an arrest of net protein diseases with existing methods. Flowever, as our synthesis. One would not be able to detect such a approaches to therapy become more subtle and phenomenon with the classical tissue slice methods powerful, that situation could change. of biochemistry. I believe that cultured cells can help in two ways- Several months ago, Dr. Phillip Becroft senr me a First the cell culture system affords an opportunity 4 millimeter skin punch biopsy from a one-year-old for trying to coax the cell into developing the New Zealand infant who had a severe megaloblastic catalytic activity it lacks because of gene mutation anemia. The known causesof megaloblastic anemia at a control locus. One can remove all product had been ruled out. I developed a diploid cell from the environment of the cell in the case of strain from the biopsy using our most complete biosynthetic enzymes, or deluge the cell with sub- nrcdium. Once the strain was developed, I placed strate in the case of catabolic enzymes. These are sorne of the cells in minimal medium. Unfortu. crude examples, but more cunning experiments nately, the cells grew as rapidly in minimal medium aimed at enzyme induction can also be performed, as in the complete medium, so that my experiments in spite of our ignorance of the natural inducers. s'ere of no help to the clinicians in New Zealand. The second way cultured cells can help is by I could not say what molecule, if any, the patient's creating lots of protein. A "Blake" bottle has abont cclls failed to synthesize. Perhaps there was no 187 cm2 of surface area available for growth. Today

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il 46r Vol. 3, No. 4, 1955 DIPLOIDCELL STRAINS

restriction in the use of the cells for diag' rt is quite feasible (though neither easy nor cheap) sceable Unhappily, this dilficulty is not one that to grow 10,000 Blake bottles from a small biopsy' nosis. to be bn the verge of solution' If, however' it Amizingly enough, it does not require a very large seems is true that the cultured cell descended from the laboratory to do this. Ten thousand Blake bottlcs zygote by mitosis, that no genetic has rvould yield between 50 and 100 grams of cell pro' ,information bin tosi, then the problem is in theory suscePtible tein dry weight, and betwecn 300 and 60O grams of of solution. There may, however, be Processes cells wet weight. This is likely to be enough to operating during cell differentiation that we do not crystallize, fingerprint, and perhaps sequence many know about. Our ignorance of such matters enzymes of ttre cell. Most patients with metabolic Present is so complete, that it poses no barrier to optimism' diseases will relinquish 600 grams of tissue with ln any caie, the study of human mutations at cellu' visible reluctance; some patients with metabolic lar control loci may provide one avenue for learn' diseasesweigh less than 6000 grams, and are in no ing more. position to forfeit more than l0 percent of their REFERENCES cells. If one tried to use leukocytes rather than cul' to tured cells, one rvould have to obtain from tho Cuortecasas,P., and S' Segal. 1966' Galactoseconversion " ttt"ituiiu" .ot't" of galactose metabolism' 100 liters of blood. t;-;;i;i;;;,-i" donor 50 to Science153:549-551. of mam- More work nee

somecomments on Diagnosisand rreatmentof MetabolicAbnormalities

DonoughO'Brien, M.D., F.R.C.P.*

one situation with loss of enzyme function may be I would like to generalize on some of the more by any one of a variety of stearic mis- practical objectives of diagnosis-.a.nd treatment in occasioned it tl"td of metabolic abnormalities' I think that informations. " least as Dr' with inborn errors of metabolism we have at Perhaps we should Pause for a minute' conven- one other objective for diagnosis than the Motulsky inferred. this morning, and say something so as to treat; tional. Obviously, one has to diagnose about what might be done and what is worth do- we need to diagnose and find at the same time ing at the present. Obviously, one population that ways of diagnosing to make what yeu might call is especially attractive to investigation is the new- a stamp collection of some of these Yery rare syn- born because here you have the opPortunity to do dromes. There is a tremendous need to collect and something in the way of treatment where treatment document these very rare syndromes, a Process that is availab-le, even though at the same time you a-r-e made more complex as it becomes clear that any is involved in looking at i very large population with -lD-.o".,*"nt a very small dividJnd. Investigators are now tryine tests of Pediatrics,University of ColoradoilIedi- to define the optimal assembly of screening cal Ccnter, Denver, Colorado'

rL

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il 462 O'Brien Ala. J. Med.Sci.

to which ir is econornically and scientificallv iusti_ cular rearrallfJel)lelttthat destrol,stl"rc cnzvmc's ac_ fiable to strbjcct a newborn infaut, an

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il Vol. 3, No. 4, 1956 DIAGNOSISAND TREATMENT 463 that we may conceivably fincl drugs that will There have been suggestions that administered change it back into the right shape. A drug, R5- nucleoproteins could restore the norrnal chain of 3608, developed for the use of Parkinsonism, was information in the ccll. So far as I know, none thought to have an enhancing effect on phenyl- of this work has been substantiatcd. Finally, there alanine hydroxylase in phenylketonuria; adminis- are some stimulating observations in which ccrtain tration of this durg in large doses resulted in pre- organisms which had been inducecl to develop mu- liminary figures which suggested that thc phenyl- tations in relation to arginine could be induced alanine went down and thc tyrosine levels went up in the prescncc of streptomycin to rctranslate the after its administration. We have just finishcd misinformation back into the correct information. using this ancl have found initially a significant Nothing was said about the mechanism of how fall in serum phcnylalanine in one untreatccl this might be but again it opens an avenuc for phenylketonuric br,rt none in sevcn othcrs. It may' Lls to consider ways in which misinformation can be possible to devise a pharmacological means of bc restored to proper information to supplant thc restoring to normal a misshapen molccrtle. decicledly crttde approach of clictary trcatment.

CongenitalRubella, A Model for Chronicin utero lnfectionsin Man

ChorlesA. Alford,tr., M.D.*

Understanding of the overall role of infectious gestation, being recoverable frorn affected infants agents in production of intrauterine disease in man at and for varying intervals after birth. Thus, in requires specific detection of the offending organism recent years, virus isolation has been introduced as either in the pregnant mother, developing concePtus an important procedure to be employed for the or infant after birth. Currently, clinical recognition r.etrospectivediagnosis and study of the role of ru- of the diseasestate produced by the presence of the bella virus in pregnancy. infectious agent is employed as the initial step for Today, I will review some of our studies on the this purpose as laboratory screening procedures are virologic and serologic phenomena demonstrable in not yet practical. The wide spectrum of clinical human products of conception after maternal ru- disease produced by pathogenic organisms and par. bella in the first trimcster. Already evidence indi' ticularly the occurrence of asymptomatic infections cates that certain features of this discase are com- present major problems in assessmentof the dis' mon to other congenitally acquired infections, most ruptive role of infections in pregnancy. notably those due to cytomegalovirus, toxoplasma Though the teratogenic potential of rubella virus gonclii and treponema pallidum. Thus, study of the was detected in the early 1940's by clinical means rubella viral-host relation may prove a prototype of and the role of this virus in pregnancy repeatedly chronic coirgenital infections of man. asscsscdby epidemiologic methods, understanding Certain of the results to be presented were re' of pathogenetic mechanism of congenital disease cently obtained in collaboration with Drs. Thomas awaited specific isolation technics. Weller and Franklin Ncva at thc Harvard School The reccnt introduction of cell culture technics of Public Health (Weller et al., 1964; Alford et al., for propagation of rubella virus reported by Weller 1964; Alford, 1965, 1966). and Neva and independently by Parkman, Buescher, During epidemics of rubella in Boston in 1963 and Artenstein in 1962, coupled with the occurence '64,'65, of a nationwide epidemic of rubella in 1963, TABLE 1* have provided both technics and materials neces- SUT'IN{ARYOF ATTEMPTS TO RECOVERRUBELLA sary for laboratory investigations of congenital ru- VIRUS FROM HUMAN PRODUCTSOF CONCEPTION bella infections, It is now recognized thaL chronic disseminated Percent Percent No. Positiue Negatiue infection of the product of conception may occur Total examined 68 46 54 as a result of maternal rubella early in pregnancy Placenta 51 47 53 that virus may persist in the fetus throughout and Fetal tissues 42 26 78 -lEirrun, Professorof Pediatrics,University of Alabama tf.o- So. Med. J. 59:745, 1966. Medical Center, Birmingham, Alabama.

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il 464 Alford Ala. J. Med.Sci.

TABLE 2* RELATION BETWEEN DURATION OF PREGNANCY AT TI}IT] OF ilA.r'ERNAI, RUREI,LA AND THE SUBSE,QUENT RECOVERY OF RUBEI-LA VIRUS FROIT THE PRODUCT OF CONCEPTION

Products ol Conception Studied It'umber flacentae Number fetuses Duration ol Number from pregnancy Number zoJtichtirns P ercent Number Nurnber Number Number (tuhs.) examined isolated positiue examined ltositiue examined positire

J 0-2 I (14) :) I J 0 3-4 t2 6 (50) 8 4 6 J

j) 5-6 I5 ll \/J' l0 8 8

7-8 8 4 (50) t) J 5 I 9-r0 r7 6 (3t; l3 t2 2 u-12 6 I (33) 6 9 t 0

I l3-16 3 I (33) J 0 +From Amer. .I. Dis. Child. 100:4ir6,1965.

'64, and 168 specimensrepresenting varioLrs portiolls tiva as rvell as urine obtaincd from these infants. of products of conception rvcre obtained from 68 Other investigators have demonstrated virus in women whose pregnancics rvere r,oluntarily termi- spinal fluid as Iate as 9 rnonths after delivery. nated during the first trimester becatrseof maternal Horvever, after birth, dcr.nonstrable viral excretion rubella. The results of virus isolations obtaine(l rates diminish rapicllv rvith aclvancing age of in- from examination of thesc nraterialsare summarize(l fant and maximal isolation rates are achieved by in Table l. The recoverl rate froln thc 68 worncn virologic examination pcrforn-rctl on material col- was 4670 with a placental isolation rate of 47L, lected during the first month of life. Virus was also closely approximating that of the total, ancl with a isolated from brain, lir,er, kidney and heart tissue fetal isolation rate of only 26lo. However, this latter obtained at autops) follo$'ine cleath of 3 of the in. figure is in accord rvith recent estimatcs of risk of fan ts. fetal dlmage reportcd frorn prospective epidernio- These data indicate that pcrsistent rubella infec- logic studies on the role of mbella virus in preg- tion of the conceptus is a variablc event after tna- nancy. Virus was recovercd from fetal brain, heart, ternal infection in the first trinrestcr.However, after kidney, stomach, skin-muscle-skeletaltissuc and, in infcction is establishcd, r'irtrs ntav persist through- one case, from fetal bloocl cells and was also iso- ont the first trinrester ilt both placental and numcr- lated from amniotic fluid. Thesc isolates rvere olt- ous fetal tissucsor apparcntlv selcctivelvin placen. tained from products of conception collcctecl as latc tal tissues.Selectire placctrral infcction most oftcn as 57 days after strbsidencc of the rash in tlrc follorvs maternal rnltella in thc second 8 wecks of mother. pregllancv. In sonre cases,cspecialll' those after rna- The relation between the cltrration of prcenatrcv ternal rubella in the first 8 u'ccks, r'irus mav con- at the time of maternal ntbclla and thc strbseouent tinrte to replicate throughout prcgtlancy in rnanv recoveryof rubella vinrs front thc conccptus is surrr- fetal tissucs,beine rccovcrablc fronr the neu'born at marized in Table 2. Thc total isolation rate in- birth and for sornc nronths tl'rcrcaftcr. Pcrsistcnt creased from a low of l-lcl; in the firsi 2 rvccks of conceptual infcctions fronr tnatcntal nrbclla in the pregnancy, reached a nraximurrt of 73lo in the irth first 8 rvecks of preer-rancvare apparently rlorc through the 6th week antl decreascrl thcrcafter to colnlnon and tencl to be associatccln'ith a nrorc 33fo. l'lacental rcco!'eries of virus cxcecded fctal recoveries in each age catcgorv thus accounting for TABLE 3* our high total isolation rate. tr{orevcr, nrost of thc RESUL-I'SOF RI.'BT-I,I-.\\F-L;TR,\I,IZING,,\N'fII}ODY fetal isolateswcrc associatedrvith rnatcrnal rubella I)t,.fER\IINAl'IONS OF FF-'fAI, ST-R.\, CORD, I,I*. II.{NT'S AND C]HII,I)RT-N'S SF]RI OR-f,,\INIiD }'Ot-. from the 3rd through the 8th wcck of prcgnano, LO\VING CONGENT-r.{L'I-RI\IES-l'[,R RLrBF.tLA ACQUIRED rN rvhereas selcctive placental infcctions wcrc col.llllloll THE FIRST OF l'REGNANCY. A'r-D IN CO.\IPARAI}LI, CON'TROL SERA rvith exposurcs after tlrc 8th rveck of pregl)altc\'. It is to be noted that pcrsistent fctal infection .lr'ith- II-16 uk. I day- 6-12 I -)" ollt concomitant placental infcctiotr l'ras not ltccrr Patients letus 6 rnos. mos. yrs,

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il 465 Vol. 3, No. 4, 1966 CONGENITALRUEELLA

initiated in utero sometime after the l6th week of malignant form of disease in the fetus and neonate gestation in spite of the presence of maternal anti- than- do those following maternal rubella in the sec- body. ond 8 weeks of pregnancy. Results similar to these maternal antibody disap- have been reported by Horstmann and Cooper' As is to be expected, seia of control casesduring the lat- These data also support the concept that pro' pears from the the first year of life. F{owever, in sera gressive maturation of the conceptlls during the ier part of of rubella syndrome persistencc of anti' f-irst trimestcr is an importaut factor in the rubella from cases demonstrated for Years with levels viral-host ^clation. The relative roles of the ma- body may be to those in maternal sera' In infants in turation of the placenta (as a determinent of the cornparablc with demonstrated viral excretion, the effectiveness of thc placental barrier) and matura' our series antibody dirninishes during tion of the fetus (in terrus of capacity to resist or initially dominant, 7G 6 months of life. Levels of antibody are overcorne infection with rr-rbella virus) remains to the first this period by primary anti' be established. maintained during 7M body procluction. Similar results have been reported Though less well publicized, eviclence for chronic by Bellanti. antigenii stimulation in congenital rubella infec- Thus, for the retrospective diagnosis of the ru' tions can be demonstrated both serologically and bella syndrome, virus isolation and determinations imrnunologically. Conseqttcntll',tcchnics for the de- of the ptrysicochemical nature of neutralizing anti tection of antibodY are eqtrallv or perhaps even body miy be valuable aids during the first 6 months more useful in retrospectivcdiagnosis of the rubella -fhereaftcr, of life. antibody persistence in the pre' syndrorne than those of virus isolation. school age child maY be of value. The resultsof determinations of rubclla neutraliz- The immttnoelectrophoretic analyses of immuno' ing antiboclies in fetal serutn, cord, infant's and globulins in serum from infants congenitally in- children's sera obtained after maternal rubclla ac- Iectcd rvith rubella virus and in control cord sera quired in the first trirnester of prcgnancy and in are of particular interest and have been separated iomparable control sera are summarized in Table 3' frorn the previous data for special treatment' A sum' Rubetta antibody was present in all fetal sera and mary of these determinations are presented in in most sera obtained from casesof the rubella syn' Table 4. drome in the first 5 years of life, as well as in most was documented in all fetal sera but sera derived from control casesin the first 6 months 7G globulin ^yM globulin r{'as dctected. of life. This antibody rvas absent from the sera in neither nor 7A the majority of control cases obtained from o ^yG globulin was demonstrable in all cord or early rnonths to 5 years of age. infant- sera from congenitally infected and control ^yM globulin rvas documentcd in In the first trimcster, levels of mbella neutraliz- infants. However scra from the infected casesbut in only ing antibody from both infected and control fetuses, six of eight cord sera; the prccipitin arcs cleiived from women with a prior history of rubella, trvo of eight control globulin in the sera of congenitally in' rn'ereless than, but parallel to the levels in simul- against 7M veloped more rapidll' ancl were taneously collected maternal sera.Detcrminations of feitect infants cle than that in the two positive control sera' certain of the physicochemical and immunochemi' denser quantitative analyses revealed marked cal properties of fetal antibody indicate that it re- Subsequent clevation of this globulin in sera frotn rubella in- sides in the 7G globulin fraction in sera from both infants with quantities var,ving from 40'200 infected and control embrl'os. From these and other fccted studies,fctal antibody dcrnonstrableduring the first trirnestcr in both normal and rubella infected etn- TABLE 4 bryos is assumeclto be maternal in origin' RESL;LTSOF I\IITUNOELECTROPHORE'IICANALYSIS tiF- EE-iel- sERA oBTAINED FRoNI EIIBRYoS FoL- In the cascs studied herc, chronic rubella infcc- iorfr,r-C FIRs-r rRI\IESTER I\IATE,RNAL RUBELLA FETUSES,AS wELL AS oF the conceptus aPpeared to exert little effect .clri-rnorl coNTRoL tion of Ciiir.n-exD r,cnt-Y INFANT sERA FRoNI PATIENTS on thc accumulation of 7G antibody in fctal sera coicE,iriALLY INFECTED wlrH RUBELLA vIRUS AND OF CONTROL CORD SERA during the last two trirnesters of pregnancy. Con- sequentlv, this antibody was the dominant rubella I mmun ogl obulins Present antibody in sera from both rubella syndrome and 7S^lt /9S"Y16a l/tt control cases at birth. Again thb rnaterial is be' Ty?es of Total No, Exam. No.* No.* No.* lieved to be maternal in origin, but recent studies SeraExomined can be formed by the human ll-16 Wk. Fetttscs indicating that 7G 700 in Rubella 7 fetus does not permit an unqualified statement Control i 700 the case of rubella infected fetus. Cord and Early Infant Rubella 8 8 6* 2*, Flowever, significant levels of "yM antibody were Control 8 820 demonstrable in cord sera from the rubella syn- *Precipitin more quickly and rvere more cord sera' The arcs developed drome cases,but not in the control abundanl than those iIr control sera. data on 'yM antibody suggests that after rubella **Two preciPitin arcs' Amer. Dis. Child. 110:457' 1965. infection of the conceptus antibody may actually be J.

-}.=

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il 466 Atford Ala. J. Med. Sci.

mgms./100 ml. sera. Furthermore, ^yA globulin was Retrospectivediagnosis by serologicmeans of congenitally detected in immunoelectrophoresis acquiredrubella infections.N. Eng. J. of Med. 270:1039- in two of eight r041. sera from infected casesbut was not demonstraied in the eight control cord sera studied. DR. ROBERT BIGLEY: (This work was done in It appears that the specific in utero antibody pro- collaboration with Drs. Robert D. Koler, Jose O. duction associaredwith congenital rubella infeciion Campos, & Peter Stenzel.) Patients with hereditary is accompanied by elevations of 7M and 7A globu- hemolytic anemia due to pyruvate kinase deficiency lins. detectable by simple immunoelectrophiretic have practically no pyruvate kinase in their rcd cells methods which are applicable in clinical diagnostic but ncimal levels of white cell enzyme. This appar- laboratories. ent diffcrence in genetic control is borne out by our In collaboration previously reported that lqukocyte and u'ith Dr. Jon Straumfjord, De- observations partmcnt of Clinical pathology, and Dr. George C0LUMN88 Liver Cassady,Neonatal Section, Department of ped^:trics here, immunoglobulins have been determined L.y irnmunoelectrophoresis performed in the Clinical PyruvoleKinose Laboratories on sera collected from ll0 infants ob- tained at or shortly after birth (Alforcl et al., in press). \.2\^

7M globulin was demonstrated in 4l and coexist- ant 7A globulin in 15 of these sera. Clinical and laboratory cvidence of perinatally acquired infec- tions were documented in 32 of the 4l infants with levels of .yM detectable by imrnunoelectrophoresis. These cascs includecl 17 congenital rubelia infec. s tions, 3 congenital syphilis infections and I congeni. \b tal cytomegalovirus infection, all of which have Leen f proven by laboratory and clinical means. Evidence s s of infection was documented in only I of the 69 infants with s levels of serum 7M below detection by immunoelectrophoresis. These results suggest that immunoelcctrophoretic 200 screening of cord Jera may ml EffluentVolume prove useful in the early detection of certain con. Fig. 1 gcnital infections and when coupled with specific isolation and serologic technics as well as longitudi- nal clinical studies may lead to a bettcr understand- ing of the overall role of infections in pregnancy. Shortly, such a program will be instituted here at -A.labama.In all infants with elevated immunoglo. Rbc b.ulins, attcmprs will be made to determine u Jp"_ cific etiology ro accounr for rhis abnorrnal finding. Hopefully, this approach may better define the clinical spectra of illnesses produced by organisms Wbc known to be associated with chronic in utero in- fections and unmask the role of other infectious agents in the production of like diseasesin rnan. Liver .@ BIBLIOGRAPHY

Alfg.idlC. A.,.J..,-F..A. Neva,and T. H. Welter. vrrologlc 1964. and serologic studies on human products of ion_ Liver ceptron atter marernal rubella. N. Eng. j. r275-r28r . of NIed. 2Zi: DtAE-.r2 5 Alj:111,_C.,+, I965. Studies anribody in congeniral ruDelra tntecttons.J... ,on I. _physicochemical and immun'ologic investigations of rubella i,."trutiiGg a;;ia.dt.-A;;;:j. Dis. Child. I t0:4bb-468. Liver Alford.,9.A.,Jr. ,|966.Congenital rubella, a review of rhe DIAT- yll9tqCr.,and serologic phenomena occurring afrer ma_ rernal rubeila in the first trimester. So. Medl bg:7ai- (Presented J., .003M 748. _ before the Sbttr- inn"u"al'me"ting of the- Southern Medical Association, Ho"iton, f.xas, td6b.; Alford, C. J. Schaefer, V. Straumfjord, ('.'.. ,A.,Jr., J. Jr., and Uassady. Correlative clinical and laboratory stridies on lntants born to mothers with high- risk of iniection dur- ing pregnancy. (in publication.) Weller, T. H., C. A. Alford, t Jr., and F. A. Neva. 1964. Fig. 2

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il Vol. 3, No. 4, 1966 DrscussloN 467

rf00 ADPr l0-' o 0.5 360

320 625

280

240 0.875

200 ! r.zs

r60

I

68 l0 12 x l0-! PEP Fig. 3

320r ADPx l0-a

280 '',s -7" ./ ?44 ,./L0.625

-/ A ^,? 200 o0.875 l_ V t.25 r60

120

80 ?;;"

o.? 0.4 0.6 0.8 1.0 t.? 1.4 1.6 1.8 2.0 I x l0-e PEP Fig. 4

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il I 468 DrscussroN Ala. J. Med.Sci.

red cell pyruvate kinases are quite different pro- gests that the red cell enzyme may be unstable, con- teins by several physicochemical criteria, including tinuous production of enzyme in nucleated liver kinetics of substrate binding (Koler et al., 1964; cells maintaining a level of activity sufficient to sus- Campos et a|.,1965). tain hepatic glycolysis. Weber (Weber et al., 1965) has pointed out thar DR. ELLIOT VESELL: I wonder if Dr. Bigley has pyruvate kinase is an important regulator of glyco- been successfulin converting one form of this pyru- lysis, since it catalyzesa one-way reaction and is in- vate kinase to another form. duced by insulin and suppressed or at least de. creased in states of gluconeogenesis. Tanaka and DR. BIGLEY: We have not attempted this. Some co-workers (1965) have shown that rat liver contains Czechoslovakian workers (1960) have dissociated rab- two forms of pyruvate kinase, only one of which is bit rnuscle pyruvate kinase and I believe reasso- the insulin-inducible form. We have studied human ciated it with restored activity. F{owever, they were liver and also find two forms of the enzyme. dealing with one isozyme. Perhaps you also are Figure I shows the DEAE chromatogram of whole referring to the fact that some Swiss workers (Wies- liver. A fraction of the pyruvate kinase is not bound mann and Tonz, 1966) have suggested that the Km to the DEAE, and another is eluted at about .08 differences between the red cell & white cell PK N{ KCL. disappeared at a lower pH. Figure 2, starch gel electrophoresis, pH 8.0, shows that red cell enzyme movcs toward the anode and DR. VESELL: Do vou have anv leads to the sub- white cell enzyme does not move. Liver contains two unit structure of PK isozymes? forms, one with the mobility of erythrocyte enzyme, and one with the mobility of leukocyte'enzyme. DR. BIGLEY: Some workers in Michigan (Stein. metz and Deal, 1966), working with rabbit muscle The slow-moving electrophoretic form precipi- enzyme, have good evidence that PK is a dimer; the tates at about 6olo (NH4)2504, the fast-moving apparent three forms in various tissues in humans form at about 35% (NH4)rSOo. Rabbit antibody in- as well as three forms in animals (Fellenberg et al., duced to red cell enzyme inactivates the red cell 1963) also suggest that is a dimer. enzyme and the fast-moving electrophoretic liver form; white cell enzyme and the slow-moving liver form are not inactivated. REFERENCES Figure 3 shows kinetics of substrate binding for Biochim.Biophys. Acta 44:604,1960. white cell PK and the slow-moving electrophoretic Campos,J. O., R. D. Koler, and R. H. Bigley.1965. Nature form from liver. There is independent binding of 208:194. the substrates, Fellenberg,R. von, R. Richterich, and H. Aebi. 1963. phosphoenol pyruvate & ADp; the EnzymologiaBiologica et Clinica 3:240. apparent Michaelis constant for PEP is about l0-a Koler, R. D., R. H. Bigley,R. T. Jones,P. Van Belling- molar. Figure 4 shows kinetics of red cell pK and hen, and P. Thompson.1964. Cold Spring Harbor Sym- posiumon Quant. Biol., NervYork XXIX:213. the fast-moving liver PK. The binding of one sub- Steinmetz,M. A., and IV. C. Deal. 1966. Biochemistry strate is dependent upon the concentration of the 5:I 399. second substrate, presumably due to competitive Tanaka, T., Y. Harana,H. Nforimuraand R. Mori. 1965. Comm. substrate binding, and the Michaelis constant for Biochem.Biophys. Res. 2I:55. PEP is Weber,G., R. L. Singhal,N. B. Stamm,and S. K. Strivas- in the order of l0-3 molar. tava. 1965.Fed. Proc. 24:745. The red cell enzyme and the fast-moving electro- Wiesmann,U., and O. Tonz. 1966.Nature 209:612. phoretic form from liver are not different so far as they have been studied; their shared immune inac- tivation & kinetics suggesr identity. The white cell DR. ELLIOT' VESELL: I think it is important to enzyme and the slow-moving electrophoretic form explore some of the implications, as have been done from liver are also similar. philosophically, of altering the environment of Peo- ple who have defective genes producting various Two uses might be made of these observations. inborn errors of metabolism. It has been argued First, qualitative variations this of important gly- that by altering the environment one is really in- colytic enzyme in tissues such as liver can perhaps creasing the gene pool of these seemingly useless be evaluated in more easily biopsied peripheral genes when they are in double dose, and this raises blood cells. Second, they may help to determine the opposite point that perhaps in single dose they whether patients with PK deficiency hemolytic may have some selective advantage. But be this as anemia have deficient enzyme production or mu- it may, I think it is important to point out, as has tant enzyme. By the criteria outlined here, we have been so often in the literature, that by treating the not found evidence that our patients have mutant environment one is not altering the cause of the dis- enzyme. None of our patients have gross abnormali. ease which lies at the primary genetic level. At this ty of systemic carbohydrate metabolism. Comparing point I think it is nice to make the connection tissue distribution of PK isozymes in rats and man, between what we heard this morning; that is we it is likely that the red cell and fast-moving liver know the codons that give the different amino acids, enzyme is the insulin-inducible isozyme. This sug- so that by the fingerprint technic we can detect

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il Vol. 3, No. 4, 1966 DrscussroN 469

point mutations producing abnormal proteins, and age, the serum phenylalanine did not go up what- once this is done, it doesn't lie too far beyond the soever, despite a perfectly normal diet of 300 milli realm of possibility that one can approach the grams per kilo body weight phenylalanine intake. disease at this primary level by altering the codons A second casewith an initial serum phenylalanine involved to produce the right or normal amino level of 45 milligrams percent was placed on the acids. I think this is eventually the goal toward diet and this child, taken off the diet, has run a which treatment should possibly aim, though it phenylalanine of betrveen 4 and 6 milligrams per- seems so very far away at this moment. cent for several months now. Two other caseswith initial phenylalanine levels of 22 milligrams percent DR. DAVID HSIA: I thought perhaps it might be u'ere placed on the diet and after three months of rvorthwhile to say a few words in follow-up of what age were taken off the diet. Their phenylalanine Dr. O'Brien has been talking about, about the fact levels went up very slowly but eventually reached that these inborn errors of metabolism are begin- the level of arouncl 20 milligrams percent and then ning to fall apart, and they certainly are falling sta,vedat that level but would not go higher. In apart at a very rapid rate. The ones that we felt contrast, the siblings of the knorvn casesof phenyl- were fairly sacred as individual entities now rapidly ketonuria were taken off the diet and within 48 are becoming many different entities with sirnilar hours they would have serutn phenylalanine levels genetic and enzymatic patterns. I think that the up to 30 or 40 rnilligrams percent. It appears paper on kinase is a good example of pyruyate without yet having gotten the liver biopsv data, the kinds approaches that need to be takcl in of rvhich n'e l'ill p;et, that tve are dealing rvith several order to be able to recognize in thc future u'hat diffcrent entities which are characterized by high really amounts to clifferences of codon alrel'ge - seruln phenvl;rlanine, sonre of l'hich probablv arc ments. Perhaps the only way that you can be cert.rin phen,vlketonuric, and some of rvhich are pcrhaps that you are dealing with a single genetic cntitv heterozt'gotes, and some of rvhotn are probably not is that the affected members of a given familv harc phenvlketonuric at all. In re-analyzing thcse pa- disease,and that the next family with the the same iieuts, in looking at thenr all over again on the maybe doesn't have the sauredisease brtt has disease basis of the initial preseniing criteria, it is not very closely related disease.I rvould like to' another possibleto decide in atrv giveu casein the beginning say a few words about phenrlketonttria however, as to whether thcse patients actually were different this is the area in which most informa- because from the ones rvho turned out to have classical tion seems to have been gathered. All of ,vou phenl'lketonuria. Therefore this has, I think' raised arc familiar with the fact that there has beet-r very serious questions in all our minds as to rvhere very widespread screening for phenylketonuria, antl do wc go fronr here itt terl-nsof the screening pro- a few months ago the data were presented suggest- gram. I think the philosophy that manv of us ing that the frequency of phenylketonuria \\'as are begirrning to adopt is that the screcnitlgProgram somewhere in the order of one in 10,0O0as against rvill pick up cascs of hperphenylalaninemia-lct's the original estimate of one in 25,000 or oir'-' in not call them phenvlketonuria, that these probably 40,000.There has been, during the last few ntoitths, shouki be treated, but that at the end of three rather careful revision and re-analysis on the 1-lart months of age or so the,v should be takcn off the of several diffcrent grollps across the countrv as diet for short periods of tirne, in order to reassure to what really constitutes phenylketonuria amot.tg oursclvcs that the seruln phenylalanine goes uP to those which were picked up by screening, and irt an acceptablv high lcvel, put back on the diet ancl contrast perhaps to those u'hich rvere pickcd up as perhaps repeated again at six months of age' and siblings of known cases. tLrsulnnlari/c oLlr ()\rll Just so on. I think as a result of this rve will probably experience, during the last tlr'o or threc vcars rvc be able to acquire more iuformation as to what have all together accumulater-i arottnci 17 cirse: of rcallv constitutes a patient rvith phenYlketonuria. newborn infants with the diagnosis of phertrlkc- Out of curiositv, becausc of this I decided to go tonuria, of which five were siblings of kttorvrr cascs. back to orlr state instittttions and this sunlmer we Among the remaining oues, tve hat'c recctrtlr' hatl strrveved for serttm phettrlalanine levels 2,000 known the opportunity of re-exanrinitrgthcm, anrl of these paticnts n'ith rneutal retardation in one of the state at least four out of thc remaining ll or 12 have institutions. In this stlrvev u'e fortncl that all except patterns of hyperphenylalaninemia rvhich are t)ot r"rineof them had perfcctlv normal serum phenyla' phenylketonuria. to summarize brieflv, the first Just lanine levels. Eight of the r-rineha

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.-J - 470 DISCUSSION Ala. J. Med. Sci. averagc was sorrrewherc around 33 rnilligrams per- to something we call phenylketonuria, a good num- cent. So I suspect that probably there is a real ber of these mav be double heterozygotes or mixed group of patients u'hom one can call phenylke- heterozygotesstates. Again, this is going to make it tonuric ir-r the classical sense, that there is a fairly more complicated, although we know already what significant number rvho have hyperphenylalani- the single gene is doing. ncmia without hypertyrosinemia during the new- born period, and sorne of these cannot at the mo- DR. WALTER NANCE: I'd lihe to raise the ques- ment be distinguished from the classical group. tion relatiue to the diagnosis and treatment ol We probably will have to approach this problem by ntetabolic errors to the panelists and others who dietarv treatment, taking it off the diet, and so on. haue spoken here, whether they are concerned n,bout And I think this same analogy u'ill undoubtedly the kinds of mothers that these treated, homozygotes apply to high tyrosine levels during the newborn u,ill mahe when they in turn become pregnant. We period, perhaps to the definitive diagnosis of maple already hnow that in the case of phenylhetonuria, syrup urine disease which seems to have several phenylhetonuric rnothers haue abnormal offspring different entities at this point, perhaps to several entirely independent of the different types of homocystinuria and so on and fact that those indi- so forth. I think therefore for someone who has uiduals, in other words, the offsprings are not in been interested in inborn errors and in specific en- themselaes homozygotes, and yet they are seuerely zyme defects, this is sort of a very difficult period ntentally defectiue. I thinh that if this is generally but I think one in rvhich a great deal of new knowl- the case,that there is another problem entirely that edge is being acquired by these technics. tue are dealing with and that one may simply be transferring to the next generation the problem of DR. CHARLTON M-{BRY: In response to Dr. what to do with these seaerely defectiue indiuiduals. Hsia's speculatiott-it,here do TLtego u,ith these I utould lihe to hear Dr. Hsia address himself to this screening tests,artrl in response to Dr. O'Brien's uery problem. obuious skirting of tlrc u,hole issue of screening for inborn errors ot' ntetabolism, I zuould like to point DR. HSIA: This is a very philosophical ques- out tltat tltere are at least I0 or I I if tt,ill, , ),ott tion but one which I feel at the present timc neurontetabolic disordersuhiclt can be treateclsome that as physicians we are primarily obligated in our way or other, and u,ltich can detected if either be society to do the best we can for any given indi bloocl ot' urine is obtained in tlte nurser\t. These vidual single patient, and I feel this very strongly. testscttrt be autontoted usirtg either continuous f lou, So that if ,vou, by an,v of thcse scrceninq ntethods, chernistry or using spectrographic anal of tltese ,*sis pick up a given patient with a diseasc which is luids, so tltat we hat,e tltis esserttiallyirt our ltands f treatable, or perhaps treatable, I think as a physi- rtow. Tlte questiott of autotrtation arises since u,e cian you are obligated to go ahead and treat this are talhing about tnillions of kids now euery ]-ear, patient. Perhaps by the time this patienr and ttobody is u,illirtg really €Jrows to talh at tlte ground up. I\{ethods can be incorporated to maintain leuel ctf actiuit\ of ltort, to get tltis done, and I this patient through pregnancy in a relatively tuould lihe to ltear sonie discussiortabout autonta- normal metabolic state to prevent thc next genera- tion of tltis. tion although they u'ill be heterozygotes,of having been damaged by being carried in utero.In the case DR. ARNO MOTULSKY: I n'ant to cornnrcltt not of phenvlketonuria, I think that most people would regarding autornation but rcgarding interprctation agrce that in a homozygous phenylketonuric moth- of sot't.tcof thesc cliseascsu'c havc hcard abotrt. If er, \'olr u'ould put her on a lorv phenylalanine diet there is consiclerablehcterogcncity, as rvas pointccl during pregnancy ivhich theoretically-I don't think out, $'e should rcalizc that rnanv of thesc paticnts it has ever bcen tried-rvill obviate this difficulty in wc rnight consider as homozvgotes carrving thc the future. I think that wc are not wise enough at cloublc closc of the same gene probably are not the moment to make thcse arbitrary decisions of hornozvgotcs,but thc situation is going to be morc saying that rve shor-rlclor shoulcl not treat because cornplicatcd in that sonrc arc going to be rnixed geneticallv rve arc adding to thc population poor hcterozygotes, in othcr u'ords, heterozvgotes for two protoplasm. I think some of you may remember that tliffcrcnt genes u,hich may interact in clifferent ways, in the days of Francis Galton and others in the late and again the hcrnoglobins can sct an example l9th century this was thc very popular kind of thing there, becausethcrc n'c havc populations that have that only the intelligent should have chilclren and sickling in hemoglobin C and sickling in thalas- the stupid people should not brecd, and I don't semia, and we havc cntities of sickling hemoglobin think there has been any great effort to indicatc C discasc and sicklir-rg thalassemia

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il Vol.3, No. 4, 1966 DTSCUSSTON 47r

'l'he should go on screening for other things. fact children that dietl and those tltat litted all had u'hat is we have sort of got ourselves stuck rvith screening appeared to be microcephaly by measu,rernentalong for phenylkctonuria-but the question is should we with other problems, it was thougltt that it might for instance develop methods, at probablv trcmcn- represent a chrontosontal aberration. In following dous cost which, despite atrtonration, rvill still in- up the family, we learned that a first cousin u,as volve a fair arnount of highlv skillcd technician a phenylhetonuric, institutionalized, and had a time; to use for screcning all of thc ncn'borns in serunt leael of 42 milligrams, and of course imme- America for amino acid clisorulersirr the Lrlood?I studied this and think this is a philosophical

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il 472 DrscussroN Ala. J. Med. Sci. the overall clinical observation, and I am enthusias- tarded and.she has children who were last heard of tic that someone has a mother on a diet. Just last in 1937, left in an orphanage, so we are trying to week Dr. Denison called me and he has found case get the details on that family and see what has nttrnber 29. This patient, however, is moderately re- happened to those children.

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::re PANEL 4: EPIDEMIOLOGY OF SELECTED GENETIC DISEASES Moderator: PrrnR Pnecocx,D.P.H., D.I.H., D.T.M. & H. Peter Peacock,D.P.H., D.I.H., D.T.M.&H.: Rheurnatic Feuer as a Genetic Disease:The Epidemiology. George Cassady,M.D.: Hereditary Renal Dysfurtction and Deafness. Ralph W. Gilmore, D.M.D.: Distribution of Dental Caries. Emanuel lVlargolis,M.D.: Sex-linkedAlbinism Associatr:dwith Deafnes.s.

RheumaficFever As A GeneticDisease: The Epidemiology

PeterB. Peocock,M.8., Ch.B.,D.P.H., D.l.H., D.T.M.&H..

hr 1840 llouillard described a predisposition to nratic fcvcr on Wilson's hypothesis(assurning a high rhcumatic fever rvhich was transmitted frorn thc penetrance) while in fact, only 9 pairs of the 3l in parents, often associatcd with a sanguine tempera- thc cornbined series showed this. A comparatively lnent, fair skin and blond hair, and sometimes lorv penetrance seemed possible. brought out by exposure to cold. Many other early Stevenson(1956) basing his arguments on his own workers including Snydcr (1907),Poynton (1920)and scries (443 families observcd over 4-5 ycars) and on Irvine-Joncs (1928), noticed that a high proportion a possiblc failure of \Vilson to allow for further of children with rheumatic fever gave a family his- cases occurring, as unaffected siblings grew older, tory of rheumatic fever. stated that the antosomal reccssivegene theory rc- Wilson (1937,1956) followcd ll2 rhcurnatic fami- mained unprovcn. lies (471 childrcn) for up to 20 years and for.rndthat Various groups havc attcrnpted to correlatc rhcu- a9;V.of the familics had parental rheumatism, ancl matic fevcr rvith rccl ccll blood groups; some find- in another 23lo sonte other close relative (grand- inE lcss O and morc .{ than was cxpectcd. The dif- parcnt, unclc, aunt, cousin) u,as affected. She found fcrenccs (sce Table 2) are howevcr obviously small. (1951) that irr 54 gcr"reticallysusceptiblc families, 40 It has bccn suggcstcd by Glynn, A. (1956, 1959) children out of l2l had rheurlatic fevef, compare

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:rc 474 Peacock Ala. J. Med. Sci.

TABLE I PERCENTAGE OF RELATIVES OF RHEU\{ATIC FEVER CASES AND CONTROLS WHO THEN{SELVES GAVE A HISTORY OF HA\/ING HAD RHEUNIATIC FEVER

Glasgow London Harriet Lane Home

Rh. Feaer Controls Rh. Feaer Controls Rh. Feaer Controls

Siblings J,I L, t .8-1-.5 I I .7-*.9 4.1-+I .2 15.5-+2.4 4.0-{-l.l Parents 10.0t1.5 5.6-r-I .6 I l.gtl.l 9.0-+2.0 27.7t3'7 6.2-f2'o Aunts, Uncles 9.1 3.8 Grandparents 18.2 2.3 Taken from Rcad, Frances, E. M., Ciocco, A., Taussig, Helen B. (1938) Amer. J. Hl'g. 27, 719 British Medical Research Council (1927) "Social Conditions and Acute Rheumatism" Special Report SeriesNo. l14, London.

TABLE 2 DIFFERENCES IN THE PERCENTAGES OF PATIENTS \VITH RHEU}IATIC FEVER AND CONTROLS BELONGING TO BLOOD GROUPS O AND A

E.tcessin Patients of Groult First 'Jlhere Number of Author Year Patients Controls OA Maseted Bristol 1940 200 6,780 +3.6 +0.6 Walsh Sydney 1956 259 I,123 -2.r +5.8 Addis Glasgor,r' \954 538 5,898 _1.4 +0.2 Clarke Liverpool 1960 263 16,057 -8.9 +4.9 Glynn Taplow 1960 609 15,046 -3.1 -0.9 Khatlab Cairo 1960 120 10,000 -9.1 +8.9 Trung Paris 1961 434 257 -0.4 -0.5 Buckwalter Iowa 1962 752 49,979 -4.8 +2.4 Taken from Dublin, T. D., Rernanke, A. D., Pitt, Elaine, N{asscll,B. F., Allen, F. H., Amezcua(1964) Brit. Nfed.J. 2:775.

TABLE 3 PERCENTAGESOF NON-SECRETORSAMONG RHEUMATIC FEVER CASESAND CONTROLS

First Number Examined Percentage non-secretors Author Year Rh, Feuer Controls Rh. Feter Controls Glynn I 959 533 669 28.9 22.9 Clarke I 960 263 851 27.8 24.3 Buckwalter I 962 752 49,979 25.8 23.0 Dublin 1964 (males) t74 430 23.6 25.6 (females) 429 t74 32.4 27.6

TABLE 4 SOURCE AND DISPOSAL OF SUSPECT RHEUI\IATIC FEVER CASES FOUND DURING THE 1957.1960SURVEY OF THE CITY OF GERil{ISTON (Population 139,407)

Locals Seen and Examined Source Died Not Locak Lef t Area Refusals Accepted Not Accepted Hospital records follow-up 6 70 97 2 88 l5 Official notification (non-hosp.) 9 l5 2 54(+27) 4 Unofficial notification r2 15(+10) 2 X-rays (44,872) I 3l ll General clinics (30,000 p.a.) 2(+49) I Tape recordings (6,025) 4 l8 t3 Schools (5,616) 6 Antenatal clinics (2,257) 24 95 Mun. employees (1,042) / l0 Work seekers (40,088) a Health visitors e(+3) 5 Scarlet fever (1,045) 6 General publicity 45 12 , 85 l2l 1 zrl 148 219 426

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.#NEEE€ Vol. 3, No. 4, 1956 RHEUMATICFEVER 475 of the rninor criteria on lvhich thc diagnosis of thesis that rhcurnatic fevcr is due to an autosomal rheumatic fever may be based. The irnportancc of recessive on the n'cll-documented assumption that this in surveys is apparent. lf, of the population are Potential suffcrcrs from In ury own experience in an initial study in Nfoose rheumatic fever. (Assuming the gele is not lethal the Jau', Canada (l9ir5), Il out of our first 47 patients in the procreative age, then l8lo would carry hacl a parcnt or sibling with a history of rheumatic recessiveand with an average family size of 5, then fevcr. This would fit nicclv I'ith \Vilson's h)'po- 30.25% of parents and siblings should evcntuallv develop rheumatic fever). TABLE 5 Subsequently, in the City of Germiston, South NUI{BERSOF RHET]}TATICFEVT-R PATIENTS WITH Africa, a careful survey was undertaken to try and oR wrf'HoUT A r;Alflrulutf;to"Y oF RHEr]]rATrC trace, as nearly as possible, every case of rheunaatic fevcr which had occurred. (SeeTable 4). Arnong thc EuroPean Non-EuroPean 207 patients placed on prophylactic medication and Among Il[ale Female trI ale F emale whosc history was investigatcd carefully r'r'e found Sibs, parcnts, childrcn l-1 :12 6 a family history of rheumatic fever as follows (see Arrnts, uncles, thc tirnc of grandparcnts 17 I I Tablc 5). The ages of our patients at Ijirst cotrsins ir 6 this study are given in Table 6. None of above 38 60 ittcidcncc Total I83 We demonstrated the average annual for first attacks of rheumatic fever to be 44 per 100,000 for Europeans and 15 pcr 100,000 for non- TABLE 6 -l-able in 7 AGES OF RHEU}IATIC FEVER PATIENTS IN Europeans. Using the distribution sivcn GL,R]\{ISfON I'ROPHYLAC'IIC PROGRAN{ and the equation

Il'* \i European Non-European ,ryN _- {' IUU Age in Years Ilfale Female trtale F emale i=_,, ll

<5 2 .l I 5-9 t5 I5 I for each race separately, rve fouucl that the actual IO-I4 30 2il 24 who had had at- l5-t9 l9 21 24 and expectcd number of paticnts o 20-24 3 1l J tacks of rheurnatic fcvcr atrtong rclativcs \vas as 9r,J- 528 25 given in Table 8. Our findings suggest strongly that while rheu- TABLE 7 matic fever may bc a familial diseasc it is not PERCENTAGE DISTRIBUTION OF ACCEPTED RHEU- ]\.TATIC FEVER CASES BY RACE AND AGE AT TIN{E hereditarilv detern'rincd. OF FIRST ATTACK REFERENCES

Age in Years European N on-European Bouillartl, I'. J. I ll-10. "Traite cliniquc du Rhutnatisme d Artictrlaire," I'aris. J. B. Baillierc. <58 t G. L. 1959. "Rhettmatic fcver cpidcmiology ir-9 36 ll Bywaters, E. 10-14 33 qo and prevention" (t-d. Cruickshank R., Glynn, A. A.) r5-t9 l0 20 Oxford. Black*'cll. 94. 20-29 5 165 Chung, C. S., F.. L. Pitt, T. D. Dr.rblin.196ir' Antcr J' Hum- 30-39 4 l0 Genct. l7:1152. 40-49 3 4 Diamond, [,. F. 1957.Pediatrics I9:90f]. l)itkorvskv, S. I)., l'-. Stevcnson, J. tI. Campbell. 1943. f,quation for expected numbcr agc "x" who hlrc had an J. Amer. IIcd. Assoc.l2l:991. attack of rhcumatic fever Dublin, T. I)., A. I). Bernanke, E. Pitt, I]. F. N{asscll,F. H. Allen, Amcztra. 1964.Brit. NIed. J. 2:775. u*: - il:- ! -li 100 Pi Gauld, R. 1.., l-. NI. Franccs,1910. .I. Clin. lnvcst. l9:393. i:-. Glynn, A. A., t,. E. Glvnn, E. J. Holborow. 1956. Laricct l'herc i is the annual incidcncc ratc for first attat:k of t.?:.o rheumatic fcver Ghnn, A. ;\., I-. E. Glynn, E. J. Holbororv. l{)l-r{f.Brit. Pi is the population at risk i vears l\Ied. J. 2:266. A1 is thc attar:k experiente at age i in the population Glvnn. I-. l-.. t.. J. Holborow. 1952.J. Path. Bact.6'1:775. <:otttcrned Glvnn, L. 1.., l-. J. Holborow. 1961. Arthr. and Rhettm. 4:203. Greerr,C. A. t9'12.Ann. Rhcrrm. Dis. 3:4. TABLE 8 Irvinc-.foncs,Ldith I. II. l92fl,Arch, Int. Mcd.42:78'1. Irvinc-.fones,Edith I. NI. 1933. Amer. Dis. Child. 1i: ACTT]AI, AND EXPECTEI) NUNIRF-R OF P.{TIENf'S J. I 184. WITH RHEU]\TATIC I'E,VER WHO HAD HAD Kaufman, O., E. Schecrer,1938. trIenschl. Vercb. rr. Konstit. A'TTACKS OF RHF]UTTA'TIC FEVER A}IONG RELATIVT,S 2:687. Peacock,P. N. B. 1955. Canad. J. Pub. Hcalth 46:486. Povnton, F.J., D.Paterson, J. C. Spencc. 1920. Lancct 2:1086. Relatiae PoPulation Relathtes Attached R. W., C. K. Kincaid. 1952. Amer. J. \Icd. S<:i' inuolued at risk Expected Actual Quinn, 223:487. Sibs, parents, Snydcr, J. R. 1907.J. Amer. N'Icd.Assoc. 48:490. children 871 12.6 (9 Stevenson,A. C., E. A. Cheescman. 1956. Ann. Human Aunts, Uncles, Gcnctics2l :139. Grandparents 1,652 9c| Wilson, NIay G., NI. D. Schl'eitzer. 1937. J. Clin. lnvest. First Cousins 834 9.2 u I 6:5irll. No rclatives involved r47.9 I ll'r Wilson, NIay G. (1940). "Rheumatic Fevcr." ir-cw York' Commonwealth Fund. 'zu w Wilson, Nfav G., NI. D. Schweitzer.1954. Cir

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s=re 476 Cassady Ala. J. Med.Sci.

HereditaryRenal Dysfunctionand Deafness

GeorgeCossody, M.D.*

I was initially honorcrl but also sonrcn,hat to collect a ctrnrtrl:rtivelv largc cnough scrit's to chagrined when first asked to participate in this drarv any significant conclusions. symposium-for someone now invesiigating thc .{t the \,erv tuln of the centurv (1902) a family physiology of the newly born infant to discuss a n,as describcd at Grr-v'sHospital by Dr. Guthric. disease that has yet to be reported in the neonatal This sarnc familv rras subsequcntly described threc period is at best a paradox! After careful consider- additional tirnes bv threc additional authors, a total ation, I decided thit the problem could best be of four reports bctu'ecn l!)02 ancl 1927 (Guthric, resolved by merely altering .,Heredi- my title frorn 1962;Kendall ancl Hartz, l9l2; Hurst, 1923; Alport, tary Rcnal Dysfunction ,,A ancl Deafness" to Multi_ 1927; Perkoff et al., l9ril). As a rcsult of ltis per- disciplinary Interdcpartmental Collaborative Study? sistencc,the fourth getrtlcnran to report this family, To many of you this u,ill be a very farniliar sort of Dr. .\lport, was able to obscn'e thc natural coursc title. of the diseasc,accuratclv clescribingthe proeressivc Morc than twcnty farnilies u,ith this svndronre severity of the renal rliscascitr t.ualcsand observing from Norrh Carolina, Virginia, Florida, Vrryf"",i for thc first tirnc that tlcafness\\'as a striking and and Indiana wcre investigated betrveen l9ir9 and predominant fcatrtrc of thc srtrclronre.In l9irl I'er- 1964.Genetic clctails of fii,e families in rvhich 2gl koff in Salt Lakc Citv sttrdierl onc laree family rvith memb^ers were pcrsonallv examinecl u,erc reported thc aid of the N{ornton tabernacle records. Hc was in l96l (Cohcn ct al., l96l). This nork rvas rlonc ablc to personallv exanritrcatttl stttclytnost mcttlbers with the co-operationancl lcadership of Dr. Maiuron of the fanrily anrl, as a result, his conclusionsrvcrc Cohen, a plant scncticist, ancl the conclusions rluite valirl ancl thc clinical finclings rvere atnazingly reachcd^were-supported bv rlork in drosophila anrl similar to those itt thc Guthric--{lport familv. maizc. C)ur clinical stuclies,ultimatclv reporting in This was thc statc of litcrature at thc timc wc J965 (Cassadyer al., l96J) thc findin.qs in 476 mem_ first became intercstcclin the svndrome. By 1961,at bcrs of 7 familics, rvcrc corxplcted rviih rhe hclp of thc time lve publishc

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q_---ff Vo[.3, No. 4, 1956 RENALDYSFUNCTION 477

that thc vast majority of instances of so-called be- spill some red cells, demonstrate proteinuria, and nigl hematuria in youltg children is represented by display some renal impairment during their preg' this disorder. Many years of follow-up, audiograms, nancy. At the condusion of the pregnancy, the func' and intensive family investigation will reveal in the tional renal status clears very rapidly and no uri- majority of these cases that the family is afflicted nary or functional abnormality may be detectable with hereditary renal disease dysfunction and deaf- between pregnancies. In one such patient massive ness. This has certainly been our experience, the proteinuria, and significantly reduced creatinine experience of the Guy's Hospital investigators and it clearance were demonstrated in the last trimester of has been the experience of all investigators to date. pregnancy but, within three weeks of delivery w€ There are actually thrce sets of presenting symp- were unable to determine any abnormality of renal function by routine clearance tests and renal biopsy toms in this disorder. The first form is most com- very mild mon in the young male but may occasionally be was essentially normal. The deafness is be seen in the young female. In this instance the renal until the 30's or 40's at which point it may clysfunction presents as hematuria, usually micro- picked up audiometrically and becomes progressive. scopic, but occasionally with concurrent URI's The common finding in thc urine is not hematuria grossly discolored urine may be passed. The initial but is pyuria. rnis-diagnosis is usually acute and/or chronic glom- What the meaning of this is, we don't know. Re- crulonephritis. If the work-up extends to the point cent studies at Yale suggest that chemical or me' of renal biopsy, glomerular changes are seen, inter- chanical acute or chronic minimal damage to preted variously as sub-acute, chronic, or mem- kidneys results in an increased susceptibility to in- branous glomerulonephritis. If, however, you look fection. In our experience and in most of the litera' at the clinical condition of these patients, the ab- ture however. infection is not common. The mcan- sence of edema, hypertension, preceding clinical or ing of all this is unclear to us at the moment. immunologic evidences of streptococcal infection, Before concluding our brief discussion of the and persistence of the abnormal urinary findings clinical findings, it might be best to note additional should suggest a striking contrast to the usual acute findings mentioned by some other authors. It has glomerulonephritis of childhood. Urea retention is been noted by some that eye defects and cataracts rarely if ever present at this stage in this form of are associated with the syndrome. In a few in- the disease. stances, this has been considered as a characteristic Another interesting featuie is that if a simple component of the syndrome. Most of the families audiometric scan is done in these young patients a have not been reported to have ocular defects and very characteristic bilateral nerve deafness becomes this is a most important fact to remember. In our cvident. This is important and I mention audio- patients with cataracts, the advanced age of lnem' 'high metric scanning because early the deafness is bers of the cataract group and the presence of tonc' and does not impair hearing of ordinary con. uremia in the few young members of the grouP vcrsation. It must be stressed that the characteristic lead us to suspect that any increase in cataracts or cochlear defect in these patients is picked up early lens anomalies above that found in the general only by audiometric examination. Rapid progres- population is entirely secondary to changes brought sion of this deafness occurs and, by the time they about by renal failure. are in their late teens, patients with this form of Our study provided solid data concerning the the disease begin to have urea retention and func- genetic mode of inheritance of the disorder. In ottr tional renal failure. They generally die with uremia families, if we looked only at the affected mother, and severe deafness in their late teens or early 20's. we had the very interesting finding that 73fo of hcr A second form of the disease is seen in older sons and 751" of her daughters were affected. This males. Perhaps the most common earlv symptom in certainly does not fit expected 50,-50ratios. If we these older males with the disorder is a clinically look at the affected father, we see that 84fo of his apparent deafness.They have an apparently benign, daughters will be affected with this disorder while intermittent, usually microscopic hematuria which only 43fo of his sons will have the disease. is generally unknown both to the patient and fre- Our interpretation of these facts is simply that, quently to their physician. It does not appear to in the female during oogenesis, the autosorne bear- compromise life expectancy and renal dcterioration ing the defective gene of this disorder generallv does not appear to be characteristic. In contrast to preferentially segregates with the primary oocyte the concurrent deterioration of hearing and renal rather than being discarded with the polar bodr'. 'young function in the male' form of the disease, Therefore, an excessof available affected "ova" are progression of deafness with minimal functional produced, with the result being an excess of af- 'old-male' renal impairment characterizes the form. fected males and females born to affected mothers. A third form of the disorder presenting as clin" The male, on the other hand, during spermato- ical chronic pyelonephritis is seen in most females genesis is unable to "get rid" of his good genetic with the disorder. This usually first appears during material but the preferential association of the de- the child-bearing age. It is often misinterpreted as fective gene with the X chromosome results in an pre-eclampsia or early toxenria. These patients mav cxcess of affected females but a deficiencv of af-

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reffE 478 Cassady Ala. J. Med. Sci. fected malcs frorn the father. Cassadr',(ieorge. K. Rrortn, If. Cohen, a-nd W. DeNlaria' 1965. Hcrcditarv renal dysfunction and deafness' Pedi- This disease is not, therefore, transrnittcd as a atrics 35:967-978. simple autosomal dominant but appears to repre- Cohen, N{. N{., G. Cassady,and B. I'. Hanna. l96l' A sent an example of non-random dysjunction in the genetic study of hereditary renal dysfunction with associ' Gen. l3:379' first meiotic division with preferential associarion ited nerve deafness.Amer. J. Hum. or congenital hereditary of that gene bearing the defective material with Guthrie, L. G. 1962. "Ideopathic" and family haematuria. Lancet l:1243. the X-chromosome. Hurst, A. F. I923. Hereditary familial congenital haemor- With this suggestion of preferential selection for rhagic nephritis. Guy's Hosp. Rep. 73:368. defective genes-on this rather anti-Darwinian note Kendall, G., and A. F. Hurst. l9l2' Hereditary familial Guy's Hosp. Rep' -I will close. congenital haemorrhagic nephritis. 66:I 37. REFER,ENCES Perkoff, G. T., F. E. Stephens, D. A. Dolowitz, and F. H. Alport, A. C. 1927.Hereditary familial congenitalhaemor- Tyler. 1951. A clinical study of hereditary interstitial rhagicncphritis. Brit. Med.J. l:504. pyelonephritis. Arch. Intern. NIed. 88:191.

Distributionof DenfalCaries

RolphW. Gilmore,D.M,D."

The considerablc data concerned with variations rrent.that ncither of the observed distriltutions was in the occurrence and extent of dental caries among Gaussian since the assumption of a normal distri- human populations have been reviewed by Volker bution is inherent to commonly used statistical pro- and Caldwell (1962) and by Davies (1965). It is cedures. The frequency distribution of smooth sur- clear that the mean decayed, missing, and filled face cavity counts could be approximated by a (DMF) teeth increases with age, and that females, negative binomial clistribution, which is one of the at early ages, since their teeth erupt sooner than class of so-called contagious distributions. Later, males, exhibit slightly greater values than males Gurland (1959), appl,ving the fully efficient maxi- for the permanent dentition. Although the number mum likelihood method to these same data, im- of children within a family appears to have no proved the fit of the negative binomial distribution effect on DMF level, the caries experience of sibs to counts of smooth surface cal'ities. The cavity is similar and reflects that of the parents, on the counts of those tooth surfaces exhibiting pits and average. Substantial differences in caries exist fur- fissures could be described as binomial distribution ther with respect to factors such as socio-economic in the case of children with mostly primary or level, nationality, geographic area, racial groups, permanent teeth. The mixed dentition period, when type of diet, and fluoride level of water supply. the primary teeth are being replaced by the per- manent Although there has been considerable activity in teeth, departed significantly from the collecting data on the occurrence and extent of binomial distributiorr. caries in association with various factors, relativelv Roche and colleagues (1966) have reported on little interest has been shown in the basic distribu- cariesdata about increments.They found that total tions of these data. Apparently little attention has surfacc counts for cleciduous tecth closely follow a been given to comparing the distribution of ob. negative binornial distribution. It rvould be of con- served data with expected distributions dcrived siderable interest to cxtend this type of study to rigorously from the theory of probability. pernranent teeth. The outstanding exception is the definitive work .{lthough the ncgative binomial distribution does of Grainger and. Reid (1954) who fitted various not imply infection, it does indicate that the occur- theoretical distributions to the frequency clistribu- rence of a single carious surface predisposes to fur- tions of data concerning the prevalence of dental ther occurrences within an individual. Grainger and caries. Since plots of total counts of carious surfaces Reid (1954) have interpreted the.underlying nega- suggestedsuperimposition of different distributions, tive binomial distribution of smooth surface caries to a dentally meaningful separation of the data was mean that environmental influences (as opposed to made into counts of carious smooth surfaces and factors inherent to the tooth) are of major impor- counts of surfaces exhibiting pits and fissures. They tance to smooth surface caries. This interpretation found that the frequency distribution of prevalence can be extended to caries increment data for de- Cata for smooth surfaces differed from that of pit ciduous teeth that also follow this distribution. In and fissure surfaces taken together. It seems perti- the case of pit and fissure caries the binomial dis- -liJpu.t-"nt tribution was interpreted as indicating that factors of oral Biology, University of Alabama im- Schoolof f)entistry,Birmingham, Alabama. inherent to the tooth were of relatively greater

/-

provided by the Maternal and Child Health Library, Georgetown University Vol. 3, No. 4, 1966 DENTALCARIES 479

portance than environment in determining the oc- tests to the value of a body of theory in the manage- currence of caries within an individual. Such an rnent of large amounts of data. It would scem interpretation appears compatible with the nature unrealistic to suppose that full development of of caries as disclosed by both clinical ancl laboratorv theory expresscd mathematically concerning dental studies. caries in populations could precede study of the Basic studies on the distributions of dental caries underlying distributions of caries. are important for scveral reasons.As more and more data are collected, data reduction becomes impor- REFERENCES tant. There is an advantage in being able to sum- marize Barkla, D. H., A. F. Roche, J. D. Jago, and J. S. N{aritz. many tables of data in a single sentence 1966. Possible sex differences in incidence of caries in specifying thc distriburion and the values of its human deciduous canines and molars. Arch. Oral. Biol. parameters, ll:201-208. for this uniquely describes a particular Davies, G. N. 1965. The significance of epidemiological set of data. It is also of both theoretical and prac- studies in relation to caries resistance,in Ciba Foundation tical Symposiums Caries Resistant Teeth, Little, Brown and import4nce in rhe sraristical analysis of data to Co., Boston. understand the underlying distributions of data Finn, S. B., and R. C. Caldwell. 1963. A comparison of the since these affect the caries experience of monozygotic twins, dizygotic twins interpretation of results and unrelated children. Arch. Oral Biol. 8:571-585. (Grainger, 1966). Experience indicates that sharpen- Grainger, R. N{., and D. B. W. Reid. 1954. Distriburion of ing of diagnostic methods frequently dental caries in children. J. Dent. Res. 33:613-623. catalyzes de- Grainger, R. M. C. 1966. Analysis of caries increments by finitive genetic studies. In this connection it is inter- age and tooth surface in fluoridated and non-fluoridated populations, esting that heredity, as presumed by electronic computer. Adv. in Fluorine from study of Res. and I)ent. Caries Prevention 4:53-66. human twins, influenced smooth surface caries sub- Gurland, J. 1959. Some applications of the negative bi- stantially, although no effects were nomial and other contagious distributions. Amer. J. evident for pit Public Health 49:1388-1399. and fissure surfaces (Finn and Caldwell, 1963). Cer- Volker, J. F., and R. C. Caldwell. 1962. The epidemiology tainly the rapid progress in population generics of dental caries in Clinical Pedodontic.s,S. R. Finn ed., ar- W. B. Saunders Co., Philadelphia.

Sex-linkedAlbinism Associated vrith Deafness

EmonuelMorgolis, M.D.*

This repon concerns a Jewish family originating perceptive deafness; the caloric test denoted a from North Africa. Among the nunterous member.s marked hypofunction of the vestibular apparatus. of the pedigree two instan-cesof first cousin and two The routine laboratory examination and x-ray instances of second cousin marriages were found. films revealed no abnormalities. The consanguinity in these caseshad no relation to In skin biopsy no increase in the abnormalities described. number of melano- cytes was apparent in the hyperpigmented patches There is no necessity to discuss individually the and no obvious decrease in number or abnormal affected members of the present family, since they distribution of melanocytes in the hypopigmentecl resemble each other in appearance. They are af- areas. The pigment found was argentaffinic not flicted with generalized albinism and have completc sudanophilic, did not contain iron, thus being most deafness from birth. probably melanin. The term "generalized albinism" proposed by No morphologic and/or numerical aberrations of Fitzpatrick (1960) (insread of "incomplere" or "im- chromosomes could be detccted in the karyogram of perfect" of the older classification) denotes ',an ab- one affected member and his mother, prepared from sence of melanin in the hair, skin and retinal pig- skin tissue culture. ment epithelium, ltut presenceof melanin in the iris The following facts of genetic interest have beer-r and occasionally the skin." established in the present pedigree: In all of the affected rneml_rersof our pedigree, The abnormalities first appeared in generation indeed, some pigmentation of the iris was found; III. All four boys (III-ll-17-22-23) were albinos and and in all of them pigmented spots could be seen deaf-mutes. One of them (III-22) died ar the age of on the skin, though not to the same degree and orr five of an intercurrent disease. All six female sib- the same part of the body. lings of these albino boys were reported. normal. Audiometric studies were performed on some of Two of rhem (III-16-24) died in childhood. The rest the members of the pedigree and showed completc married. Three of rhem (III-IZ-15-?5) produced a total of four affected (IV-31-3541-61) and four non- ]-oi-utlor"nt of Epidemiology, University of Alabama r\tedicalCenter, Birmingham, Alabama. affecred (IV-28-32-40-43)boys. All the girls bom in

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trff 480 Margolis Ala.J. Med.Sci.

this generation were normal. However, some of genotype and since derangement at any one of the them (IV-3G34-56-58)in the subsequent generarion intervening steps may lead to the same or a similar produced affected boys (V-69-74-106-107).Of all the phenotype, heterogeneity is not surprising (M.- deaf-mute albinos only two had offsprings. Indi- Kusick et al., l96ir). vidual III-17 married a normal Arab woman, who A unitarian hypothesis, namely, that we are deal- had, by a previous marriage, two normal children. ing with one syndrome and with one mode of in- From her second marriage with III-17 a normal girl heritance will find the variability encountered in (IV45) was born. Later this girl married a normal this association of albinism with deafness perplex- Arab man (IV-44) and gave birth to an albino deaf- ing. C)ne may try to put together the parts of the mute boy (V-78). The second affected member of puzzle by considering some entircly speculative and the pedigree (IV6l) who did nor avoid marriagc remote possibilities. and parenthood, produced two normal boys (V-IOS- One possibility is that the locus responsible for 109). It is noreworthy that, in spite of their defects this syndrome is in fact on the X chromosome and and difficulty in communication, all affected indi- that the mutant allele is usually expressed in fe- viduals were of normal intelligence and socially u,ell males (sex-linked dominant inhcritancc). In some adjusted. families, however, such as this reported in this In view of all available data, we are lead to con- paper, the X chromosome u'hich carries the mutant clude that the abnormalities are transmitted by one allele ma,v undergo inactivation, of the type postu- recessive,X-borne, sex-linked not sex-limited, gene. lated by Lyon, preferentially, perhaps because of The later distinction being based on the fact that some slight structural abnonnality, giving rise to neither of the descendantsof the affected IV-61 was an appearance of sex-linked recessive inheritance. a deaf-mute albino. Alternatively, the mutant allele may be normallv Numerous abnormalities have bccn associated responsiblefor autosomal dominant inheritance but with albinism. These include epilepsy, polydactyl,v, may have been translocated to one of the X chromo- short stature, and deafness. The incidence of these somes n'hich again may have undergone preferential abnormalities accompanying albinism is not known itractivation in carrier females in this family (Frazer, (Fitzpatrick, 1960). 1964). Our thorough study of the karyotypc The association of albinism with deafness seems of one of thc affected members of the pedigree and to have been first observed in dogs and cats and his mother concluctedat the "Institut de Progenese" was known to Darwin, who in the sixth edition of in Paris, trnder Lejeune, did not show variations in "The Origin of Species" mentioned also the fact thc structure, r.rur.nber,or arrangement of thc that more males are found among the deaf whitc chromosomes. cats. It has been found in some instances that thcr In 1942 Dorothy Wolf (1942) described rhree gen- associationof dcafnessrvith other defects is fortui- erations of deaf white cats and provided an exten- tous anrl cltre to independent segregation of two sive bibliography of the subject. Recently "Observa- genes,one causing clcafnessand thc second the othcr tions on the Histological Features, Development and anomalics. Thc corrsiderationof such a coincidental Pathogenesis of the Inner Ear of the Deaf White associationin our casc became obligatory when in Cat" was publishecl (Bosher, 1965, l966). Affectc

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*ryfEre Vol. 3, No. 4, 1955 ALBINISMAND DEAFNESS 481

ously to reproduce this syndromc again and again C) malignant tumors of melanocytes (rnalignant rvith precise exactitude. It is possible, though un- melanomas) do not respond to X-ray therapy and in likely, that such event will repcatedly occur 14 this respect resemble most nerve cell tumors; D) times in three generations. Crossing over would have abnormalities of pigmentation arc associated with it tendency to separate this linked characteristic, so abnormalities of the nervous system; E) two of thesc that therc would no longer be any particular asso- systems,the sympathetic and the melanocytic, havc ciation in a given individual. in common some relation to tyrosine metabolism: The one genc hypothesis seellts more plausiblc. the rnelanocytes form melanin, the chromaffin cells Multifaceted clinical syndromes, the result of a adrenal-like substances,from tyrosine, both procc'sscs single mutant gene, such as thc hcritable disorders passing the DOPA stage. of the conncctivc tissuc and somc rnetabolic heredi- Why is the tyrosine-tyrosinasc tnechanism suP- tary disease,are rvell knorvn. The pleiotropic action pressed in the neuro-ganglionic elements derived of a single abnorrnal gene is due to its control of from the neural crest, is an open question. It is in- some basic biological process. Thc diverse features teresting, anyway, that the cells that give rise to the seen in such complicated anomalies somctimes can sympathetic and melanocytic system, appear to leave be traced back to a simpler defect at earlier stages the crest earlier and are topographically separated of development (Frazer, lgirg). The problcm is to from the neural tube. The neuralganglion cells, ot-t

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€mre 482 Margolis Ala. J. Med. Sci.

-fhese facts fit u'ell any gcnetic hvpothesis, the Klein,D. 1947.Arch. Klauss-Shift-Vereb.Vorsch. 22:336. Langman,J. 1963."Sec. Int. Conf. Cong.trtalform." 247. ncural crcst hvpothcsis inclttded. Licata.R. H. et. al.. 1962.Anat. Rec.142:252. There is no doubt, however, that thc most telling McKusick,V. et. al., 1965.Nledicine 44:6. Patten,B. Nf. 1953.Human Embryology108. argument in favor of the neural ct'cst hypothesis Rawles,f{, 1948.Physiol. Revs. 28:383. would be provided if a phenocopy of the syndrome Strong& Elvin. 1964."Human Neuroanatomy"I3. could be induced in laboratory animals (c.g. cats) Tietz,W. 1963.Am. J. Hum. Genet.I5:259. Weston,J. A. 1963.Develop. Biology 6:279. while affecting the neural crest of the embryos at Wolf, D. 1942.J. Hered.33:2-it9. critical stage. Ziprkou'ski,L., and -{. Adam.1964. Arch. Derm. 89:151. The neural crest could be attacked by physical, Questiotts chemical, or better still, by immunologic means. A number of experirnents have been reported in DR. H.\\S ZEL,L\\'EGER: f)r. Cassady,wc havc rvhich animals injected with brain, kidney and heart followed trto faurilies with Alport's syndrome and extracts before and during pregnancy gave birth to we have made sirnilar observations.We found pye- newborn with defects of the brain, kidney, heart lonephritis in girls and chronic nephritis beginning respectively (Langman, 1963).Furthermo.", ihe pro- with hematuria in boys.We have not found the late duction of specific congenital malformations b,v beginning adult male type you described but we utilizing specific tissue antibodies has been at- have not had as many cases.How do you explain tempted, e.g., cardiac antigen prepared by process- this variation in phenotypic rnanifestationsin males ing homogenized heart tissue, injected into rabbits, and females if this should be the same gene? and experimental anti-heart serum thus obtained DR. CASSADY: We don't know. \Ve have made the (Licata, 1962). same ol)servations as ,vou ancl cannot explain them. In the case of the neural crest, it might be, as DR. ANDREW LORINZ: There is another heredi Ebert writes (personal James communication) that tary trait, the nail patella syndrorne,which has been "in view of the minute quantities of antigens avail- associated in some farnilies with renal disease. In able, something other than conventional techniques this, there is evidence that there is close linkage rvill be required . . for example, the sensitization with ABO blood group types. In the Alport syn- of splenic cells in cultures confronted with neural drome, have you had an opportunity to study the crest cells, followed by the administration of such linkage with blood group types? sensitized spleen cells to the neural crest of early DR. CASSADY: Preliminary linkage studies re- cmbryos in vivo." vealed no linkage between this syndrome and the It will be exciting to explore whether embrvos major blood group types, or the ability to taste treated with such supposed antineural crest agents will develop anomalies of some, or all systems orig- P.t'c' inating in the neural crest and more specifically DR. SMELO: Dr. Peacock,those of us in practice whether in this nray a phenocopy of the albinism are hard-pressed to discriminate between rheuma- rvith deafness svndrome could be obtaincd. toid arthritis a.nd rheumatic fever in the mild cases. I wonder what criteria 1'ou used in your epidemi- REFERENCES ologic study. DR. PEACOCK: \\'e were very careful about using K., and C. S. Hollpike. 1965. Proc. Roy. Soc. B. Dr. Jones' criteria. I teach my otvn students that you K., and C. S. Hollpike. 1966.J. Laryns. 80:222. can no more har,e a touch of rheumatic fever than G. B., and T. Madonia. 1964. O torl-ri nolaryng. a touch of pregnancy. Either you've got it or you haven't got it. This is from the point of view of Basis of Inherited insurance and life expectancy and all sorts of things, l:ll8-151. and when we talk about rheumatic fever, we are to Med. Genetics 163. quite definite that it was a clear-cut episode of one Medicine 88. of the three major criteria plus at least two minors, Oxford University not counting history.

provided by the Maternal and Child Health Library, Georgetown University PANEL 5: CLINICAL CYTOGENETICS \Ioclerator: DAvrDW. Sinrrn, M.D. Rolrert Summitt, II.D., and Robert L. Atnip, N{.D.: ChromosornalAberrations with Nor- mal Phenotype:Four Examples. David W. Smith, N'I.D.: lrlon-spbcificityof Indiuidual Malf ormations in Distinctiue Aneu- ploidy Syndromes. FrederickHecht, N{.D.; PhenotypicDif f erentiationof AutosomalChromosomal Abnormali- ties. Anomalies on the Gross,Radiologic, Autopsy, Cellular, and the Molecular Leuels in the D, (13-15)Trisomy Syndrome. Valerie A. Cowie, l\[.D., D.P.i\I.: ChromosomalFindings in a Population-BasedSample of Mongols. Cecil Jacobson,NI.D.: RecurrenceRisks in ChromosomalAbnormalities. (Manuscript not receiuedin tinte for bublication.)

! ChromosomalAberrations with NormalPhenofyPe'1 i ; FourExamples

RobertL. Summittond RobertL. Atnipz

I should like to make a few comments concerning chromosome variations in the normal population and present some of our recent findings in the in- vestigation of four families. Two of these families werc ascertained through a child who had or was #it d r$u suspected of having stigmata of one of the defined $tI d'ursae trisomy syndromes, while the other two were dis- covered when an aberrant chromosome was found it;, fru.ih diefrqqgffi in a normal adult control subject in a cytogenetic I2T3B study of children with mental retardation and mul- tiple congenital anomalies. fr* Jacobs and her colleagues (1964) have aptly classi' *$***af qF***.Pw fied chromosomal aberrations in the general popu- lation in two groups: The first includes actual h* ttdifhte fu e*ql suft ffi n # structural rearrangements and the second, altera- c(6'12+XX) tions which are not necessarily caused by structural rcarrangements. Our material, we believe, includes * three corresponding to her first group and one ex- emplifying the second. *tf*dlt# t ffi# #fi The first family was studied because of the birth D16 L7 18 of an infant with Dr trisomy due either to a Dr/D translocation or an isochromosome of the long arm T E r. t- of Dr (Figure l). In addition to the large metacen- ilf Irs Irp r1\ tric chromosome only four other D group chromo- r\: I somes were readily identifiable. The fifth was re- placed by a chromosome the long arms of which Fig. 1. Karyotype of infant with Dr trisomy due r fni, work wassupported by a grant from the Children's to DrlD translocation or isochromsosome long arm Bureau(Genetics Laboratory 404), USPHSGrant HD-O1020 D1 (large metacentric labelled "T"), four normal and USPHSPost-doctoral Fellowship lF2 HD-24, 776-01. chromosomes in the D group and a D chromosome 2 From the Departmentsof Pediatricsand Anatomy, Uni- with long satellited short arms with alternating posi- vcrsity of TennesseeNfedical Units, Memphis, Tennessee. tively and negatively heteropyknotic regions.

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snfffi 484 Summitt Ala. J. Med.Sci.

were similar to those of"the D group but which bore short arrns which were quite long and unusual in appearance with, in many cells, alternating regions of negative and positive heteropyknosis. The short qi T arms tcnded to remain in close proximity to each # #-s %: W: othcr and in optimal cells appeared to bear small * satcllitcs. While the large metacentric was present +1 :' {. # " $.'i only in cells from the proposita the structurally $*effi# altercd D was found in her phenotypically norrnal t2 3B father (Figure 2a) and older sister (Figure 2b). Such a structurally alterecl chromosome might have arisen by means of a translocation from another chromosorne to the D. However such an interpreta- ffi$*effi* tion does not seem likely becauseof the presence of ffiffiffiffiffiffit the altered D in and its transmission to normal 9i6.1! + X) inclividuals in whom no apparent deletion chromo- some which might have been the source of the $ translocated segment could be identified. Chandra and Hungerford (1963) suggested that a chromo- #r s& HX;;. *: some of similar appearance which they found in a D161718 normal father and daughter might have arisen from -1-,, a pericentric inversion. However such an explana- tion does not satisfactorily explain the obvious in- ffiw **$m * crease in the total lcngth of their altered chromo- some certainly could not account for the F GY* and increase in overall length of the altered chromosome in our family. On the other hand crossing ovcr in the inverted segment cotrld produce a large chromo'

Fis. 2. Karyotypes of father, (2a above) and.older sistei (2b belbw) of patient whose karyotype is de- picted in Figure 1, also revealing aberrant D chrom- osome with-elongated short arms. f #--, c"-# &' Fw$q fl*

.* Qq if. "sd3 ffiri$ ffi:: -*$ 1238 ;Htrktr$n$#;ffiffiffii c (6'12+X) $ tFq?u*pv r;+u{}*$,$iil{t;l$#$;.'a*: 'E+ C(6'1 2 *X X) Y,&*$e&$,, & ,.-ffi $s\&s*rotp;# *ffi ;is D 16 17 18 0161718 T i I ls" ffittr **&* I sngh t***** I F GY FG F

Fig. 3. Karyotypes of infant (3a above) and father (3b iight), both containing morphologically dissimi- lar 16's, one larger than normal with a prominent region of secondary constriction in the long arm adjacent to the centromere.

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.:re Vol. 3, No. 4, 1966 CHROMOSOMALABERRATION 485 some but the unusual morphologic characteristics, sible influcucc that such a structurally altered including the satellites, of the chromosome itr ques- chrornosome rnight havc in producing a meiotic tion would seem to preclude a pericentric inversion crror leading to translocation or isochromosome for- with recombination in the inverted segment. The mation such as was found in the proposita. As de la aberrant chromosome in ortr family is somewhat Chapelle and others (1963) have pointed out thc similar to two cases reported by the L,dinburgh association in familics of such chromosomes with group (1964) brrt appears to involve longer short other chromosorne aberrations in carriers or their arrns. Their rcport suggested that such aberrant relatives and with the occurrence of developmental chromosomes might result not from actual duplica- abnormalities or repeated abortions is an intriguing tion or addition of chromatin but could represent possibility whilc on the other hand most investiga- inherited variations in thc behavior of heterochrom- tions of normal individuals are undertaken because atic regions such as the short arms of thc acrocen- thev arc relatives of a developmentally abnormal trics or othcr regions of sccondary constriction. patient. Nevertheless this speculation caused us Such an explanation rvould r)ot secrn likely in our sone reservations in optimistically counselling the family in view of the increascin lcngth of thc short family concerned. arm of the D chromosorne.Pelhaps bccattsc of its Our second family was discovered when chrotrto- characteristic appcarancc ancl thc prescncc of satcl- somal analysis was performed on a newborn infant lites a duplication or evcn higher order of replica- tlclivcred bv a 42 ,vear-olcl cliabetic mother. Thc tion of the short arm seemsmore likely. infant was suspected of being a monsol lty the rc- It is interesting to speculate concerning the pos- ferring physician who informed the farnily accor

t % {$ # ,ffi $.i l q% ffi ffi* ffiflh L# $h$hEi $'J#j ffh 2 3 B -Srf u*qttt s *b ##q.et t' [} tI te ;f tr ;A*se*hhtue ill{}f}j**fillrutd&n}t} c(6-12+X)

** '* w *t * k sh.* !:: d ,,E #; #-#b ffi#*ffi ft ffi $b#ft * *h D l6 t7 18

T I :8 I 34#{rusr #*#r ffi i5p I G Y -,i_

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*ffE 486 Summitt Ala. J. Med. Sci. ryr #$ p$p$tr$rr}{}i $$ *ffi iii * 1 23BLz3B ff$$ffitr&e?fi$ffg$$; r*$F}t;i$'',$&e$$$fr;, C(6-12+XX) tft*dt{ts gn elt n $fl;{lr1 trEs *in aintJ$ D167718 t6 T7 18 !tn*r ss**,* i* T FGi gt I *fr*r I l5! I;* { r G Y l

Fig. 6. Karyotype of mother of propositus whose Fig. 4. Karyotype revealing satellited chromo- karyotype is- depicted in Figure 5, a-lsorevealing dis- some 17. similar 2's, the more metacentric on the left.

ingly. We saw the infant and felt that he was not a mongol. Chromosomal analysis was performed to support our clinical impression. The karyotype was normal except for a prominent size difference be- tween the homologues of pair 16 (Figure 3a), one #w ?r being consistently larger with longer short arms and prominence of the region of the secondary constric- lffi$htion. This chromosome was also found (Figure 3b) H$ in cells from the phenotypically *s normal & father. $ It is l23B sinrilar to two casesrcported by Jacobs et al. (1g64), -* ** and conforms to most of the four criteria set forth U--t|$ lt,, g, by Jacobs for inclusion in hcr Group II (chromo- sorlres u'ith altered appearance not necessarily due *,d$SS&o'**{tf*}*,&$$ to structural rearrangement), namely l) that the C(6-12+X) variant chromosome be identifiable in every cell of good quality, 2) that it be prcsumptively present in all tissues as indicated by its presence in fibroblast *k&&*** H$ S; {* cultures, 3) that it be familial and 4) rhat thc aber- D 16 17 1B rations be confined to chromosomes which possess a secondar-vconstriction and that they involve the T area of thc sccondar,vconstriction. Thc likelihood H,-H** **d** * that this aberration is thc result of an inherite

--J

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---il Vol. 3, No. 4, 1966 CHROMOSOMAL ABERRATION 487 authors (R.L.A.) in a blind controlled cytogcnetic study of children with mcntal retardation arid mul- ?l tiple congcnital anomalies. These were the only *& ; .1 two abnormalities discovered in a group of 50 nor- nral adult controls-a small sample yet revealing &$ $$$ $tr $&$ 4fo of. structural rearrangcments in comparison to the 05fo reported by Court Rrown (1965). First (Figure 4) a satcllited chromosome of thc E gf; group, probably l7-rvith apparent loss of a portion T! r$ nnt* of its short arm-was found in an apparently nor- mal 33 year-old nrale. None of the acrocentrics r'# seerned to possessshort arms more Prominent than r g usual to suggcst reciprocal translocation, but such , s,# *tr a small incrcase in mass might be difficult to dis- {1q, tisl #-\ W.if cern. The satellitcd 17 n'as not found in three nor- rnal, healthy offspring of the propositus. His parents F*ul---l , H have not yet been studied. Jacobs (1964) reported a case involving a tclocentric D and a satcllited E 5r ;# which she felt was tlue to translocation of the short FU %* # arm of the D to thc short arm of the E leaving a telocentric D. \\'hilc thc karvotype of her propositus :t t# $ffi $&,rf included both thc teloccntric D and the satellited E, that of one of his offspring revealed a telocentric D but a normal E rvhilc that of another included a #*J satellited E but no telocentric D, both unbalanced {g combinations possiblc from the balanced transloca' *j: ftlJ ,fr l&& tion. Both offspring were phenotypically normal. L23B Perhaps the chromosome which donated the satel- lited short arm to the l7 is present in one or more partial of prop-ositus of the offspring of the propositus in our family but Fie. ?. Composite karyotype ."a'i"oitte. providirig additional evidence of dis- cannot be detected in material from peripheral si*it"i-i;i,-wittr ttre riore metacentric on the right' blood leukocytes. those zygotes without recombination involving thc aberration which we never- Last is a rather subtle inverted segment would produce observable off- be bona fide-it has been con- theless believe to spring, all of which should be phenotypically two other observers. The firmed independently by normal. 16 year-old male propositus is a healthy, intelligent This series of fifty normal adult controls is one of pair were structurally in whom the members f2 of five with which the authors are acquainted at consistently more dissirnilar, one (Figure 5) being the University of Tennessee and the University of While one chromosome metacentric than the other. Wisconsin. The first series of fifty, studied in 1963' shorter than the was not consistently longer or revealed no abnormalities while one abnormality ratios were other in total length the apparent arm was found in each of two of three subsequent series: chromosome consistently different. This variant .{n XO/XX mosaic (Patau 1966) and an XO/XY of might represent a translocation the reciprocal rnosaicin a fertile male (Daly 1966). Thus investi- a deletion which cannot. be seen in the microscope, sation of 250 individuals selected at random on or a duplication, but is more interestingly inter- *trom we have data two are aneuploidy mosaics and preted as a pericentric inversion. It was also found two are abnormal u'ith structural rearrangements (Figure 6) in the mother of the propositus and in rvhile none was found with an alteratiorl conform- five of her siblings so far investigated. All two of irrg to Jacob's GrouP II. are normal and fertile. Figure 7 is a composite of REFERENCES the A and B groups of chromosomes from the propositus and his mother made to provide further Chandra, H. S. and D. A. Hungerford. l9tilt. An abcrrant female and hcr fathcr, both support the consistency of the altered arm ratio. i..totom" (13-15) in a human apparently normal. Cytogenetics 2:34. Against the explanation that this chromosome is ae ia Ctapelle, A., P. Aula and E. Kivalo. 1963' Enlarge.d probablv the result of pericentric inversion is the lack of both short ar-m or satellite region-a heritable trait unassociated with developmental disorde r. Cytogenetics miscarriages and abnormal offspring. Meiotic re- 2:129. 1965' combination involving the inverted segment would Court Brown,W. M., P. A. Jacobsand \f' Rrirnton. Chromosome studies on randomly chosen men and lromen. produce two unbalanced chromosomes, one consist- Lancet 2:561. ing mostly of the long arms of the homologous 2's, Daly, R. 1966. Personal communication. 'Iacobs, P. A., M. Brunton and W. \1. Court Brorvn, 1964. the other made up primarily of their short arms. Cytogenetic studies in leukocl'tes on the general popula- It is possible that such a duplication/deletion zygote tion: subjects of ages 65 ycars and over. -{nn. Httm. Genet. 27:.353. would not survive to implantation, so that only Patau, K. A. 1966. Personal commttnication.

provided by the Maternal and Chitd Health Library, Georgetown University 488 Smith Ala. J. Med.Sci.

Non-specificityof lndividualMalformations in DisfinctiveAneuploidy Syndromes

DovidW. Smith,M.D.,

With thc cxception of sorne of the sex chromo- tiple malfonnation rvhich are not due to a chronro- some aneuploidies, thc known viable aneuploidy somal abnormality. It usually requires the detection states interfere with morphogenesis; giving rise to of othcr less common anomalies to allow for a spc- patterns of multiple malformation. Given a paticnt cific clinical diagnosis. Such defects are listed in with multiple structural defects, the clinician must Table 2 for certain of the aneuploidy syndromes. encleavor to arrive at a diagnosis of a specific Yet each of thcse features may also be found in etiological entity. In this quest it is crucial to appre- other syndronles of multiple defect and no single ciate that most individual structural defects are non- rnalformation can yet be said with assurance to bc specific and it is only the total pattern of anomalies pathognomonic of a given etiological entity. Therc. which may have etiological specificity. For example fore the clinical diagnosis in multiple malforma- there are 7 different abnormalities which may be tion casesrelies on a consideration of the total pat- found as frequent to occasional features in 4 of the tern of altered morphogenesis with a general aneuploidy syndromes (Table l) and these same appreciation for thc non-specificity of individual defects are also frequcnt featurcs in patterns of mul-

TABLE I NON.SP}.CIFICANO\TALIE,S IN FOUR ANT,UPI,OIDYSYNDRO\IES

Doun's I8 Trisomy I ) Trisomy XO Syndrome Syrtdrome Syndrome Syndrome (iross \[ental Deficiencv + + + +/- Short Statrlrc + + +/- + Inncr Epicanthic Folds + +/- ? +/- Ilalformed Auricles I I'art.icular I + + + Ircat rr res Nlicrognathia +/- + + + .Simian Creases -T/ - 'friradii + + +/- I)istal Palmar Axial -i- + +/-

= usual fincling + - {,'- occasionalfinding ? = apparently unusual

TABLE 2 R,,\RI] I)I,-I;I.,C'I'S\\'T{ICH HAVE (;REAI'L,R DIAGNOSTIC SPE,(]IFI(;I'I'\'FOR ,,\ I'-\R'I'IC]L]I,,\R ANf,UPI,OIDY SYNDROIIE, BU-I' ARE NOT P,^\THOGNO\IONI(]

Down's 18 Trisomy I ) Trisotny xo Syndrome Syndrome Synrlrome Syndrone H r potonia (.llenchcd hand \lid-facial & forebrain ()onadal d1'sgenesis defect pward l slant to palpcbral I-orv arch digital dermal Posterior scalp dcfccts Oongenital lympht'tlt'rna fissu rcs pattern

Short sternrrm \Veb neck

, Il.'pr.,rr-rcnt of Pcrliatrir s, Iinivcrsitl' of Washington, \(':ltt lc, Washington.

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PhenotypicDifferentiation of Autosomal ChromosomalAbnormalities. Anomalies on the Gross,Radiologic, Autopsy, Cellular, and MolecularLevels in the Dl (tg-1 5) trisomy Syndrome

Frederick Hecht, M.D.1

Paticnts with cliffering chrontosomal abnornrali- with dcvelopnrcntal rctardatiotr and phvsical atlotn- ties rnay appear to shorv striking phenotypic over- alies. Patients with the D' trisomy syndrome, for lap with one another, a situation reminiscent of cxample, may present rvith a rather characteristic that observed with a small group of small chromo- pattern of major malformations. Fig. 2A shows a somal deletions known as "nainutes" in -I)rosophita patient with the Dr trisomy syndrome with cleft rnelanogaster. More than 20 of these small deletions lip-palate and polydactyly, in whom microphthal- variously involvine the X chromosome and the 3 mia was also present. The original casc (Pdtau, ct autosomal chromosomes were found to affect thc al., 1960) with D' trisomy had this triad of major gross plrenotype of Drosophila in an apparently anomalies-eye malformation, clift lip-palate, and identical fashion (Schultz, 1929). polydactyly-and 13 of the next 14 cases described Do different karyotypes in man always lead to (Conen et al., 1962; Ellis et al., 196l; Lubs, Koenig clifferent phenotypes? Thc thesis of this paper is and Branclt, 1961;Northcutt, 1962;Smith ct al 1953; 'l-otvnes that they do, if they restrlt in significantly different Smith, Patau and Therman l96l; et al 1962) sums of genetically active material. with the Dr trisomy syndrome had at lcast 2 of thcsc The problem of overlapping phcnorypes can bc findings. However, patients with less cxtreme ph\'- consideiecl in tcrrns of a model of overlapping sical anomalies are being recognizccl to have attto- squares. Each square in Fig. I represcnts the phcno- somal chromosomal abberations. We havc stttdied a type of an individual with a different chromosomal severely rctarded Dr trisomic paticnt rvho tlocs t-tot abnormality. If, as in Fig. lA, one examines 3 have cleft lip or poll'dact,vly (Fig. 2I]). Hc has a phenotypic aspects of each individual and finds I cleft in thc tip of his fifth digit, rvhich is presum- similar aspect and 2 dissimilar aspects,then onc has ably an abortivc form of polyclactylv (Fig. 2C). Re- relativelv little assurance that the 2 phenotypes arc peated chromosomal studies of pcriphcral bloocl significantly different from one anothcr. However, lymphocytes have shown an additional D chromo- if, as in Fig. lB, one multiplies rhe number of some and no eviclencefor a trormal population of aspectsexamined by 10, even though the percentage cells. of phenotvpic overlap remains the samc, onc fcels Minor physical anon.ralicsrr-ray be of considerablc rnuch better assurerlthat one is dcalinE with 2 dit- diagnostic help because of thcir cornmonness itt fcrent phenotypes paticnts lvith a given chrornosornal aberration and Patients with chromosonral abnormalities shoul

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rc 490 Hecht Ala. J. Med. Sci.

H

N \ e&,. :,wL " 1.e i *dr '.rB

- ;*4.8::!':-gj *-f

{iS. Z. The clinical phenotype in patients with the Dr trisomy syndrome. A: Patient with cleft lip-palate, polydactyly, and microphthalmia. B:E: Another patient u'ith the same chromosomal abnormality showing fewer facial malformatibns (B). but characteristic-minor anomalies including abortive polydactyly manifest as a cleft in the tip of the fifth digit (C), deep V-shaped piionidal depression (D), and soft tissue web across the axilla (E).

defects arc an cxanrple of snch a u.rinor lnalforma- Anomaliesat Autopsy tion in the f)r trisonrv syndrome. f-hese scalp dc- CarcfLrl autops)' studies fects usuall_v arc rccognized by the obstetrician, are sitnilarlv valuable in patients l'ith established sometimes with a fccling of dismay at the thought or suspccteclchromosornal aberrations. that hc may have abraded the baby's scalp rvith his forceps. Other cornparable minor malformations in In the Dr trisomy, for cxarnple, arrhinencephaly the Dr trisomy syndrome include a deep \'-shapecl l'ith absenceof the olfactorv bulbs and tracts has pilonidal dcprcssion (Fig. 2D) and a tight band of bccn a colnmon finding at autopsy. Other gross soft tissuc across thc anterior aspect of the axilla, anomalies detected through post-mortem examina- which prevents the patient from raising his arrns up tion in the Dr trisomv svndromc include intestinal closc to his hcad (Fig. 2E). rnalrotation, accessor)'spleens, and bicornuate uterus (N{ottet and Jensen, 1965).Nficroscopic exarnination Anomalieson RadiologicStudy of post-mortem Dr trisomic tissue has disclosed other anomalies, such as srnall renal and adrenal Radiologic examination of patients $'ith chrouro- cvsts (N{ottet and Jcnscn, I 965), rr'hich \\'cre Lllt- somal abnormalities permits the detcction of acldi- suspecLedclinicallv. tional features, which may not be clinicallrv appar- ent. For example, in tl're Dr trisomy syndrome, therc CellularAnomalies are frequent anontaliesof rib nnmber and morphol- ogy. Thc pclvis appears to be rnisshapen rvith lorv The microscopic anornaliesdetectcd in aneuploid acetabular angles, but with normal iliac anglcs individuals at autopsy generally reflect aberrant (Hecht, Loop, Graham, 1965). By contrast, in tri- clevelopment on the tissue level. Examination of somy lB the pelvis tcnds to be small with normal single cells may disclose further abnormalities in angles, and in trisomy ll (Down's syndrome) both these patients. For cxample, Dr-trisomic patients acetabular and iliac angles tend to be lorv. It is have been found to have polymorphonuclear neu- anticipated thar furrhcr radiologic studies will per- trophils with an increased number of nuclear pro- mit the construction of a radiologic phenotypc jections (Fig. 3) (Huehns, et al., 1964) (Walzer, et which will be relatively specific ro each rype of a1,..1966).One patient with Dr rrisomy has been re- chromosomal abnormality. ported to have a normal number of nuclear projec-

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TABLE I NORNIAL HEXIOGLOBINS AND THEIR AI}NORITAI, ITY IN THE D1 TRISONTY SYNDROME

C lasses Nam.e Polybebtide Abnormality of ol chain in D1 trisoml' hemoglobin Itemoglobin comPosition syndrome Embryonic Hb Corver I €-1 Hb Goucr 2 d,:€t l)crsistcnt to birth Percentogeof Neutrophils Fetal HbF e,:Iz l)crsistent in infancv wifh Oneor more Adult HbA dt?z Projection s Hb A2 oz6s l)claved rise after birth

ing electron microscopy, chrornosomal abnolmalities nray affect othcr organelles, such as mitochondria and ribosomes, in wal's which arc detectablc b,v electron rnicroscopy.

3-4 5 doys- 3-6 6 mos- do's .Yrs MolecularAnomalies ili'rtr,'Ti,i Sttrd,v of the chromosornally abrrormal indivitl- FiS. 3. Percentage of neutrophils with 1 or more nuclear projection as a funetion of age in D, trisomic ual'sphcnot,ype 0n the rnolecularlevel will, it it and in normal (non-D1-trisomic)children. The num- hoped, provide clues as to thc nrcchanismsof tera- ber of proje-ctions-drops with age, suggesting that Lne rncreasednumber of nuclear projections in the togenesis. D'. trisomy may be due to a delav in cellutai mitui- In the DL trisomy sevcral rnolecular ation. (Based on data from Watier et at. 1g66). abnorrnali- ties have been found. Thosc involvine hemoglobin are of special interest becausc of their rclation to tions in ncutrophils (Fine, et al., l965), but staris- the nornal ontogeny of this protcin. It is norv tical anall'sisof the data discloseda significant in- known that normal inclir,icluals posscss at least .:r creasein neutrophilic projections (Hccht and Scott, hemoglobins in thc coursc of clcvelopment (Tablc 1965). Prcliminary evidcnce suggeststhat there is l). The embryonic hemoglobin, Gowcr l, appears also an increase in nuclear projections in eosin- to predominate in early human lifc (Hecht, ct al., ophils in this syndrome (Lurzner and Hechr, l966). t966). Gower I and another crnbryonic henroelobin, The occurrcnce of morphologic abnonnalities Gower 2, are nomrally no longcr detectable aftcr involving a particular type of cell may possibly pro- the third month of gestation (Huchns, Dancc, vidc insieht into the genetic control over the archi- Reaver, Keil, Hecht and \{otulsky 1964; Huchns, lccture of that ccll. For cxample, the fact that thc f)ance, Beaver, Hecht and Motrrlsky, 1964). Fetal Dr trisorny ancl a numbcr of other chromosomal hernoglobin (Hb F) is thc prctlorninant fraction irr aberratiorrsaffcct thc rnorphology of thc neutrophil, late embryonic ancl fctal life,

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ff= 492 Hecht Ala. J. Med. Sci.

'l'r'is':tn:<' On tlrc rnolt'crrlarlcvel thcse abnourraliticsnray Ili-li nt:t orclrhalic.Ann. Paediat. l()9:198- 205. be consitlcrt:tl as clclays in normal clcvelopment (lonen, I'. l-., K. (). I'lrillips and L. S. IIautner. l9(i2. trIul- (\Valzcr,et al., 1965,ancl [-ce, ct al., 1966).If htrman tiple clevelopmcntal anomalies ancl trisomv of a l3-1ir group <:hromosome ('D') sl ndrome , Canad. II. A. J. lay arc supposecl to bc plaving Fine, R. N., I1. Y. F. Woo \Vang, and C. W. Hcath, Ir. allcero. 1965. Nuclcar projections of nerrtrophils in thc l3- l5 trisomy svrrdrome. I)ediatrics 35:712-714. Summaryand Conclusions Hecht, F. Unpublishcd data. 'fhc Hccht, I'., J. lV. Loop, and C. B. Graham. I96ir. It is prc

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#trnre Vol. 3, No. 4, 1966 CHROMOSOMALFINDINGS OF MONGOLS 493

ChromosomalFindings in a Population-Based Sampleof Mongols

In the Mcdical Rescarch Council I'sychiatric gols found trisorny-G in 213, D/G translocatiott itr ? Genetics Rcsearch f.rnit we har,c teceutly investi- (0.9 per cent), G/G translocation in 3 (1.3 per ccnt), gated a population-based samplc of nottgols. It is a combination of D/D translocatiou and trisomv-G well known that gross organic dcfects such as con- in onc casc rvhich takcn together with six cascsrvith genital heart malforurations are of ten associated n'.

rfl

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il 494 Cowie Ala. J. Med. Sci. the baby was very lethargic and unresponsive. days before tcrnt, shc recovcred quickly ancl from 'fhis child died at 2t/2 months. She had been in birth onwards rvas a strong hcalthy baby, feeding hospital since the age of 3 weeks with dyspnoea and well and exceptionally free from respiratory infec- cardiac failure. Postmortem she was found to have tions considering that shc was a mongol. She an enlarged heart with extensive atrial and ventricu- showed most of thc usual clinical features of Ial defects, the septurn being almost absent and tnotrgolism, and hacl sir-rgle transversc palmar with a single atrioventricular valve. The ventricle creaseson both hands and a single interphalangeal had a very thick wall. The ductus arteriosus was creasc on thc right little finger. F{cr heart was patent but narrow. There \vas a confluent broncho- apparently normal on clinical examination, and pneumonia in the whole of the lolver lobe of thc there was no evidence of gross congenital mal- right lung. formations, although her clitoris was abnorrnally Turning now to thc patient with the D/G trans- large. location, the mother was 28 and the father 38 at She was exccptional in that she was thc least the time of his birth. The parenrs $'ere Irish. They hypotonic baby in thc series and her neurological both had apparently normal karyotypes. They had responseswere nearcr to the standard norms avail- five phenotypically normal older children. The able than those of any other mongol studicd in the patient was born I I days before term. FIe was a series. wcak babv from the beginning, and had bluc Unforttrnatcly wc were unablc to follow up this asphyxia at birth. At birth the midwife remarked baby beyond the agc of 26 wecks as thc parcnts on his mongoloid appcarancc, and on clinical exam- \4rere unwilling to co-operate. She has, however, ination he was found to have many of the features becn seen recently at the age of 2r/2 years by her associated with mongolism including slanting pal- paediatrician who reports that her good health pebral fissures; bilateral epicanthic folds; strabis- and good physical developrnent continue and that rnus; Brushfield's spots; a small nose with a de- her psychological attainnents are very ncarly that pressed bridge and forward-facing nostrils; an of a normal child of the samc age, though no "inverted-V" mouth; a short, broad neck; prominent psychometric tests have bcen carried out (Dt. and abnormally shaped ears with rolled tops, an Stroud, persor-ralcornmunication, 1966). abnormally horizontally-placcd though double pal- nrar crcase in the left hand; and mottling of the Discussion skin and a poor periphcral circulation although the The D,G and thc G/G translocation mongols hcart appeared to be normal. He suffered from in this series wele both puny babies who dicd repeated attacks of bronchitis. He rvas a markedll' earlt. The er,idence is not sufficient, however, to hypotonic baby and poor in his ncurological re. postulatc a causal connection between their karyo- sponses. types and the severitv of their biological handicaps. -{t the age of 7 nronths hc was found dcad irr Other mongols in thc series who were trisonric his cot. Postmortem, bronchopneumonia and focal \vere equall,v handicapped or r{orse. nccrosis of the liver werc found. The heart was On the other hand, the babv with trisomy-G found to be normal. and a balanccd BiC or R/X translocation stood Thc third child, with a Ri C or B/X translocarion out f rom thc rvhole series as ttnusually vigorous in addition to trisoml'-G, u,as in urarked contrast and healthl', ancl with neurological responscswhich to these two babies who appearecl to bc in a rnore nearlv approached the nort'ual standarclsthan position of great biological disadvantage and who those of anv othcr babv in the group. It is opcn failed to survive longer than a ferv neeks. This to question n'hcther thc balanced translocation in third child was born to .{nglo-Jewish parcnts, 3! this case has cor.rfcrrecl a biological advantagc. tla,vs before terrn. The rnother was g8 ancl the -{lternatively, as blootl culturcs only were done, fathcr 39 at thc patient's birth. There rvere ttvo the possibilitv of tnosaicism canlrot be mled ottt. oklcr surviving sibs, phenotypically normal but not REFERENCE cxanrincd cytogenetically, and the mothcr hacl trvo rrriscarriages,cach at six rvccks. The mothcr ap- Richards, B. \\'.,..\. Sterr-art, P. E. Svlvester, and V. Jasic- patients d peared to be a balancecl carricr for a B/C or B/k wicz. 1965. Cltogcnic survcl' of 22ir diagnose clinicalh as mongols. J. Ilcnt. Def. Rcs. 9:245-259. translocation. Thcre wcrc 7 chromosor-uesin group A, thc additional chrontosor.nercscmbled a chrorno- Questiorts: sorne No. 2. There was one chromosome rnissing DR NfIHAI-Y BAR'I'.{I-OS: Instcad of asking ques- in cach of thc groups B and CX. Chronlosomes tions from the panel I n'oulcl likc to supplement the of thc D group appeared normal, but thcre was precccling disctrssion with some additional data an additional chromosomc resembling a chromo- originating from our laboratorics. 'Ihe sornc No. 16. The mongol baby appeared to havc first slide shorvs human mctaphasc chrorno- thc sarnekaryotype, but in addition had trisomy-G. somes 'l'hc from peripheral blood stained by an am- birth was complicated by placenta praevia, moniacal silver proceclure. This stain, previouslv an

provided by the Maternal and Child Health Library, Georgetown University Vol. 3, No. 4, 1955 CHROMOSOMALFINDINGS OF MONGOLS 495 solnes. \Vith this procedure iu Drosophila seg- occasionally shorv slight cliffct'enccs rvhich are rncnts of chromosomes stainetl to cliffcrcnt degrees usually confined to one region. This observation, (Black and Anslcy, 1964), this observation if further substantiated, woulcl suggcst the possi- prornptecl us to apply the same techniquc on human bility of "Lyonization" on the autosomes. material. Aftcr some modification of thc original linally, the last slide illustrates three clistinct procedtrre we obtained similar rcsttlts on chromo- t1'pesof staining patterns obscrvcd among the large sornes of pcriphcral bloocl cells divicling in Phyto- acroccntic chromosomes. This oltservation raises the l-remaggltrtinin-stimulatedmcdium. This slide shows hope for distinguishing metnbers of the Dl3-lir a metaphase where different rcgions of the chromo- gl'ollp frorn each other. sonlesshow variations in thcir staining intensitics. I would like to entphasize that as far as the An initial attcmpt was macle to dcfine the stainitlg staining patterns are conccrlrecl otlr rcsttlts are characteristics of indivicltral chromosonles. The re- prclirninary. N,Iuch nrore experiencc is ncecled with sult is shown on thc following slides.On the second this technique to work out the definite staining slide we have an Al chromosomc with. a cliagram pattern for all members of the hltmatr complement rer.'caling the prescncc of sonie l8 or 19 segments. in one and in different tissues.It is hope(I, how- On the A2 chromosome, u'hich is shown on thc cver, that this approach cventtrally may become thircl slicle, 17 reeions nray be iclcntified based on useful in detecting minor strtlctural altcrrations of rlifferent staining plopertics. the chromosomesand thus will extctttl the clinical I woulcl likc to point out that thesc patterns application of cytogenetics. appcar to be fairly consistenl frotn urctaphase to REFERENCE rnetaphasc in thc same material and from the same Illack, l{. }t., and H. R. Ansle-v. l9ti4. Histonc staining pcrson. N{cmbers

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rn- PANEL 6: INDICATIONS FOR CHRON{OSO}I[, .,\NALYSIS

lloclerator: Sene C. Frxlnv, NI.D.

Eric Ensel, \I.D.: Main Clinical Indications lo C/rromosoul( Studic.s. Lillian Lockhart, N,I.D.: Sex Chromatin Stttdir:.s. .fohn F. .[ackson, NI.D.: Drtcction of trfosaicisnt. Hans Zellr'veger,trI.D.; Indicatiorts for Chrontosrtrnal Anal1,si.sirt hlongolism. Riclrarcl A. Iinch, N,I.D.: Sontc Indications for tlte Analysis of Mt:iotic Chrornosome.s. Sara C. Finler', lI.D.: Clrromosornc Studie,sitt Sltorttaneou,s Abortir.trt.s-

Main ClinicalIndications To ChromosomeStudiesl

Eric Engel,M.D.*

The

I I I. I'orcnl.s -With Familial Conccntration of fSupportcd by a grant (Glt ll6li>) from the Unitcd .statcs Anomalies I'ublic Health Service and a grant from the Children's -With Rureau (Project 423), United Srares I)eparrmenr of Health. Repeatetl Abortion or Still- Education and Wclfare. birth aDepartment of Nledicine, Vanderbilt Univcrsitv School -\Vith an Affected Child 'I'cnn. of Meclicinc, Nashvillc,

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{iq. .1.- P_ycnodysostosis. Note peculiar -facies, open fontanelles and increased bone density. The finding of a-d.eleted G group chromosome suggests phenotypic expression of a rare recessive trait locafed in the relion of hemizygosity corresponding to the deletion.

an indiviclual of eithcr sex u'ithout known parental tl'rc chrouratir.r negativc srrbjccts selcctcd for consanguinity. In the case of a chrornosomal dele- oligospermia or azoospernria (NtcIlrce et al, 1966). tion a full expression of a rare recessivetrait could Several of thcse caseswith tcsticular chromosomal be accounted for by actual hemizygosity rather anomalies did not rliscloscanv somatic chromosome than homallelism for the nrlrtant gene (Elmore et variation, a fact rr'lrich strcsscsthc importancc of al, 1966; deGrouchy et al, 1966). This rvas rhc such an approach. rnechanism that wc ir-rvoked to explain a case (fig- (5) The group of paticnts r.r'itlrnrcntal rctar.rla- rrre l) in whom scvere exprcssion of a very rarc tion or criminal bchavior shou's a relativelv high autosomal recessive,affccting the bones (so-callerl incirlcncc of clrronrosornal anornalics particularll. pycnodysostosis) coincided with short arm and sex chronrosor)rcanollralirs. Thc incitlerrcc

{-E-

provided by the Maternal and Child Health Library, Georgetown University 498 Engel Ala. J. Med. Sci.

-f hypothetical schcnre horvcvcr. herc are 3 main clinical groups of such patients as revealed by an anal_vsisof the cases reportcd in the literaturc (Baloclimoset al, 1966) (table 2). w In thc first erotrp, u'e have patients rvith tall- ^ll f;tr .tlf,i lless, retar(1atiorr,aggrcssivcness and an apparentlv il normal scrrral dcvclopncnt. Thcsc cases rnay bc . fullv clisonric for an intact Y chromosorne. [!ISftfr Irrlnl To thc seconrl group bclons cascs n'ith hypo- olllArll*i6 rtr rtt lt.r, gonarlism ol' even intclsexuality rvith oru'ithottt talhrcss anrl rncntal retardation. In linc rvith the clinical hr poeonaclism onc nright suppose that therc is :ul undetectcd structul'al clcfcct of the tluplicatcrl Y chromosoure rvhich perhaps also itr- terfelccl u'ith thc sccotrtlnreiotic clisjunctional pro- 'I-hat ccss. occasional cltrplication of a deficient Y chronrosomc lna,v <-rccuris inclicatcd in a recent rcport of t\\'o brothcrs (Baloclimos ct al, 1966), both hrpoeonadic, onlv otre of u'hom l'as XYY n-hilc the othcr was (16) XY. An alternative explanation for group II patients l'ould imply an XO XYY mosaicism with thc XO stemline only vestigial or undetcctccl. In the third group arc thc rare casesu'ith various autosomal synch'omcs, Sturge \\'eber's, mandibulo- facial cl1'sostosis,\Iarfan's syndrome-n'ith or rvith- Fig. 2. (4?) XYY male pseudohermaphrodite. out hr pogonadism. In thesc cases an autosomal rnutation and a Y cluplication rnust coexist. is also my bclief that all the XYY and XXYY l'ho (6) Genotvpic stuclicsparticularly from the nrar- were or rvill be put to death for crimes rvhich rorv cells ma,v have a clircct diagnostic and prog- thcy were perhaps genetically endowed to commit nostic significancc in certain blood proliferative will once rise up, fincl their judges, and plague disorclers. Thus in males rvith chronic myeloicl their celestial lives with all kinds of nondisjunc- lcukemia antl the Ph1 chromosome.the concornitant tional mischiefl loss of another G group mcmber by the leukemic In contrast to the above phenovtpe of tall, ag- cells (Engel et al, 1965) mav signify a milder and gressive male criminals the next figure shorvs an rnore prolongecl cvolution, a possibility again XYY patient without detectable mosaicism as it stresserl bv a recent report by deGrouchy (de- was established from both blood and marrow prep- (iouchv et al, 1966). In other cascsof this condi- arations (Engel anrl Franks). This little girl is tion a clonal evoltrtion involving acquisition of 29 months olcl, 88 cn"r.tall, shows moderate mental supernLlmcrarv chromosomes and not rarely the retardation and was seen for some seizures. The tloubling of the Philadclphia (Court Bro'rvn ancl clitoris is enlarged and there are trvo massesin the Tough, 1963; deGrouchv ct al, 1965; Engel antl labia majores. Apparently this is a case of male- \[cKec, 1966) chrornosome Inav occur at the time pseudohermaphroditism or rather of strpermale of acute blastic crisis. pseudohermaphroclitism! Trving to make scnse of (7) Parents shotrld be studic(l rvhcncvcr they the YY syndrome is not easy. This is our orvn corne to the attcntion through TABLE 2 a) repeated abortion or stillbirth b) a familial concentration of various anomalies Groult I Group 2 Groult ) c) n'hen ther- have prodtrcerl an affcctccl incli- Cases llitlt: Cases With: Cases Witlt: r idual -l-allness -Hypogonadism -Various likc bricflv statc nature or Svndromes I u'oulcl to thc of some -Retardation -I n tcrsexuality -Sturge Weber observations in this largc group. While most -Aggressiveness -Franceschetti stuclies do not disclose any cletectablc variation -"Eu -gonadisrn" -l{arfan there rvill be cascswith (With or Without (With or Without l) interchanges or translocations Tallness and Sexual Retardation) Deficiency 2) r,arious tvpes of aneuploiclv 3) isolated changes of one chromosornc. Full Disomy Nullosomy- Autosomal (Intact Y Disomy l\Iutation The problems which arise frorn the presence of Double Dose) (Defective Y Wirh Y a translocation in one parent has several aspects. Double Dose) Disomy In major group there is actual transmission of Or: a Undetected such a translocation to the offspring in a balanced xo/xYY or unbalanced form (Hamerton et al, 196l) de- Mosaicism pending on thc various types (adjacent or alter-

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ffiffifre Vol. 3, No. 4, 1966 CLINICALINDICATIONS 499

nate) of meiotic segrcgation aclticvcd by thc gametesand their viability. Repeated abortion may follow since the genetically unbalanced offspring of a structural hcterozygoternay die during intrau- terine developrncnt (Schmid, 1962). On the other hand there are carriers of certain balanccd trans- locations whosc dcfective offspring do not inherit the translocation but ir-r lvhonl missegregation of another type occurs. For instance, rcgular aurl straight trisomic rnongols lr,ere born to parents, scveral of whom had a D/D translocation (Hamer- ton ct al, 1963; Yunis et al, 1964) and one cI rvhom at least had a D/G translocation (N{oor- head et al, 196l). Asiclc frorn thc translocation carriers some par- Fig. 3. Facial similarities between modern portrait and chromosomally defective girl ! ents may bc phenotypically normal yet exhibit one of the several types of aneuploidy such as changc supposedlv much morc benigtr or heretofore l) mosaicism with a diploid nonnal and an unnoticed. The phenomenon has been described in aneuploid ccll linc, the latter apparently rcspon- l)rosophila where it rvas found that chromosome siblc for sccondary meiotic nondisjunction (Blank changes, for instance heterozygosity for an auto- et al, 1962; Smith et al, 1962; Ferrier, 1964).Alons sornal inversion, increased the amount of primary thcse lines gonadal mosaicism appcars more than nondisjunction of the X and other chromosomes likely as suggestcd by the consistency of certain (Sturtcvant et al, 1942). Deciphering the human abnorn.ral rneiotic figures for,rncl in the testis of genome in a search for all thc structural rearratrge- solne individuals with normal sornatic chromo- rnents liable to similar cffects is painstaking. In the somes (Mcllrec et al, 1966). casc of positivc findings the facts have almost al- 2) Therc may be hyperploidy for the scx chromo- rvays bccn uncovered retrospectivcly although they somcs, for instance either XYY (Hauscka et al , can still apply for counselling and fnture familv 1962 or XXX ,Sparkeset al, 1966),a t,vpc of anomalr planning. The key to a somc$'hat more prospective sometimcs rcsponsible for manifold aneuploirlics approach coulcl be founcl in systematic chromosomc in the offspring. studies of a whole population perhaps helpcd by 3) One may observe an isolated and apparentlr computers or in a morc exact understanding of thc rninor l'ariation of one chrornosome. This raises factors leacling to thc constitution of faulty gametcs an intcrcsting qucstion since such a variation, or z)'gotes. I cloubt if cither rvill cver be achicvecl while it could bc part of the so called normal but one can hope to progress along thcsc lincs. "poly,rnorphism" (Court Brorvn et al, 1965) might Incleed broaclly stated, viral factors, radiation ef- also rcprcscnt a definite anomaly interfering .rvith fects, autoirnmune mechanisms and others havc chromosonral behavior. This perplcxins problern, lleen thought to intcrferc in sonlc \{'av rvith gamcto- for cxarnplc, is reflectcd bv thc family clescribctl gencsisand earlv clevelopment, by Sharv (1962) where a mothcr heterozyous for a Our prcrious observations,with Forbcs, of a link (i short arrn clclctiotr had 3 of hcr ir childrcn with llcnreen cliabetesmellittrs (Forbes ancl Engcl, 1963), a G trisomy and f)own's Syndrome. certain autoilnrnune disordcrs (Engel and Forbcs, 4) The study can yield art unustral proportiotr l!)63) ancl thc production of a child rvith Turncr's of anetrploirl or variant solnatic cclls (Dekaban Svndromc in ccrtain families (Engel an

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sffre 500 Enset Ala. J. Med. Sci.

BIBLIOGRAPHY Poupinet, C. Salmon, II. Lamv. 1964. Chromosomes marquerlrs familiaux et aneuploidic. Role possiblc de llalorlirnos,l\f. (1.,H. Lisco,I. Onvin, \V. llerrill, and.T. F. I'interaction chromosomiquc.Ann Genet. 7:76. I)ingman. 1966.XYY karyotype in a cascof familial hypo- Hamerton, J. L., \/. A. Cowie, F. Giannelli, S. M. Rriggs, gorradism.J. Clin Endocrinol. 26:443. P. E. I)olani. 1961. Differential transmissionof Down's Illank, C. E., 8,. Gamnrell, M. D. Caser',\[. Ford. 1962. S1'nclromc(\Iongolism) through male and fcmale translo- llosaicisrn in a rnothcr Iith a rnonoscl child. Brit. NIed. (ati()n carriers. l.ancct. 2:956. J.2:37u. Hamerton, J. L., F. Giannclli, C. O. Carter. 1963.A family (iascv, I\I. D., L. .I. Seeall, D. R. K. Street, C. E. Blank. shorving transmission of a D/D reciprocal translocation | 966. Sex chromosome abnormalities in nvo state hos- anti a casc of regular 2l trisomic Down's Syntlromc. pitals for patients requiring special sccurity. Nature Cr togcnctics.2:194. (I-ondon). 209:641. Hauschka, T. S., J. E. Hasson, II. N. Goldstein, G. F. Koelf, man rvith progcnv (lourt Ilrorvn W. NI., I. N{. Tough. 1963. Cvrogeneric .'\. A. Sandbcrg. l9(i2. An XYY inclicating familial tcndcncy to non-disjunction. Am. J. stuclies in chronic mleloid leukemia. Advanccs Canccr (lcnctics. Res.7:351. Human l4:22. 'ftrrpin. (lourt Lc jerrnc,J., \I. Gautier, R. 1959.Etude des chrom- Brown W. NI., P. A. Jacobs, )I. Brunton. 1965. ()somcs (lhromosome somati(lues de netrf cnfants mongolicns. C. R. stuclics on randomly chosen men and wom- .{r'ati. Sr:i. (Paris). 248 l t'21. cn. Lancct.2:561. jerrne, l\{. Boes- I-t: .f., f . I aforrrcade, R. Rerger, .I. -I-roisVialatte, l)ekaban, A. S., Nf. A. Bender. G. E. Economos. 1963. rr'illlalrl, P. Scringc, R. Turlrin. 1963. cas dc tlele- Chromosome studics in mongoloids and thcir families. tion particlle des bras courts (l'un chromosome5. C. R. Cl togcnctics. 2:6| . Acacl.Sci. (I'aris).257:3098. l'idrviruls,J. H., D. G. Harnden, A. H. Cameron, V. 1\I. Lejeunc, J., R. tsergcr,J. l,afotrrcadc,N[. C. Rethore. 196(i. (lrossc, O. H. Wolffe. 1960. A ncn' trisomic svndrome. I-a deletion particllc drt bras long du chromosome l8' l,anrct.l:787. Intlividualisation ri'un nottvel ctat morbidc. Ann (icnct. l-lmore, S. lI., W. E. Nance, B. .1. ]IcGee, 11. Engcl-de \l:32. .rvith \lontmollin, E. Engel. 1966. Pvcnodl'sosrosis a Lcjeunc, .1.,R. Rcrgcr, \I. O. Rethore, L. Archambault, H. familial clrromosomeanomaly. Amcr. .f. \Ied. 40:273. .Jerome, S. T'hieff ry, J. Aicarde, N{. Broyer, J. Lafour- l.ngel, E., C. I'. Hastings, R. E. Ilcrrill, B. S. IIcFarland, cade, J. (lrtrveillcr, J. Turpin. 1964. Ilonosomie partielle W. E. Nance. 1966. Apparent cri-du-chat and "arrtimon- porrr un pctit acrocentrique. C. R. Acad. Sci. (Paris). golism" in one patient. Lancet. l:1130. 25!):4I 87. lingel, E., R. Franks. Unpublished. l-ejetrne, .1. 1963. Atttosomal disorders. Pediatrics. 32:326. I'-ngcl, E., I). E. .fenkins, R. E. Tipton, B. J. IlcGee, ilI. \Iillrec, tI. E., \V. H. Price, W. N{. Court Brown, W. S. Engel-dc I{ontmollin. 1965. Phl-positive chronic myelo- l-ulloch, J. E. Nervsam, N. trIaclean. 1966. Chromosome genous leukemia with absenceof another G Chromosome stuclieson testicular cells from 50 subfertile men. Lancet. in a malc New Engl. J. Nfed. 273:738. 2 :ti9. Engel, l)., L. C. trIcKee, 1966. Double Ph1 chromosomesin \laclean, N., D. G. Harnden, W. M. Court Brown' J. Bond' leukemia. Lancet. 2:337. I). I. \Iantle. 1964. Sex chromosome abnormalities in F-ngel,E., Nf. E,ngel-deNlontmollin, A. Christie. 1964. Iful- nc',"b.r.n babies. Lancet. l:286. tiple chromosome alterations in a young $'oman with a \loorhead, P. S., W. J. Ilellman, C. Wenar. 1961.A famil- trisomic mongoloid child. Abstracr 373, American Societv ial chromosome translocation associatedwith speech and Hrrman Genctics,Boulder, Colorado. mental retardation. Am. J. Hum. Genetics,l3:32' Engel, E., A. P. Forbes. 1963. Furrher observarions on rhe I'atau. K., D. W. Smith, E. Therman, S. L. Inhorn, H. P. association of Hashimoto's th_vroiditis and thyroid anti- Wagner. 1960. \Iultiple congenital anomaly caused by alr bodies with Turners Syndrome. American Thi'roid Asso- extra chromosome.Lancet. I:790. ciation in Harper Hospital Bull. 2l:83. l)rice, \V. H., J..{. Strong, P. B. Whatmore, W. F. l-ngel, E., A. P. Forbes. 1965.Cvrogenetic and clinical find- \lcClemont. 1966.Criminal patients with XYY sex chrom- ings.in 48 patients with congenitalh' defective or absenr osome complcment. Lancet. l:565. ovaries. Medicine. 44 135. Reisman, L. E., S. Kasahara,C. Y. Chtrng, A. Darnell, B. Fcrrier, S. 1964. Enfant mongolien-parent mosaique. Hall. 1966. Anti-mongolism. Studies in an infant with a _.These 2957, F,d. l{edecine et Hygiene, Geneve. partial monosomy of the 2l chromosome. Lancet. l:394. Fialkorv, P. J. 1966. Autoimmuniti and chromosomalaber- St:hachenmann,G., IV. Schmid, I{. Fraccaro, A. Mannini, rations. Am. J. Hum. Genetics.l8:g3. L. Tiepolo, G. P. Perona, E. Sartori. 1965. Chromosomes Fo-rbes,A. P., E. Engel. 1963. The high incidence of dia- in coloboma and anal atrcsia. Lancet. 2:290. betes'mellitus in 4l patients with gonadal dy,sgenesisand Schmid, W. 1962. A familial chromosome abnormality asso- their close rclatives. Nletabolism. 12:428. ciated with repeated abortions. Cytogenetics' l:199. Grouchy, J. de, ?. Royer, Ch. Salmon, l\I. Lamy. 1964. Shau', \I. 1962. Familial mongolism. Cytogenetics.l:141. Deletion partielle des bras longs du chromosome t'9. path. Sm;th, D. W., E. tr{. Therman, K. Patau, S' L. Inhorn. Biol. I2:579. 1962. lfosaicism in mother of two mongoloids. Amer. J. Grouchy, J. de, C. Salmon, D. Salmon, p. \faroteaux. 1966. Dis. Child. 104:534. Deletion du bras court d'un chromosome l3-lb, hyper-' Sparkes,'G R. S., I. C. Veomett, S. W. Wri$rt., 1966. Trisomv telorisme 9-! phenotvpe haptoglobine HpO dans une rr'ithorrt Don'n's Syndrome. Lancet. l:270. meme famille. Ann. Genet. 9:80. Strrrtcvant,A. H. in T. H. l\Iorgan and A. H. Sturtevant. Grorrch_v,_J.de, C. de Nava, G. Bilski-pasquier, J. Boisse. 1942 trtaintenance of a Drosophila stnck center, in connec- 1966. Chromosome Phl -et _perte de petiti acroCentriques tion with investigations on the constitution of the germi- dans une leucemie myeloide chroniqire-9:73. a evolution pro- nal material in relation to heredity. Carnegie Institute Iongeechez un homme. Ann. Genet. Year Book.43:164. Grouchy, J. de, C. de Nava, G. Bilski-pasquier.196b. Dupli- Vallotton, I{. B., A. P. Forbes. 1966. Antibodies to cvto- cation d'un Ph1 et suggestion cl'une evolution clonalc olasm of ova. Lancet. 2:264' dans une leucemie myeloide chronique en transforma- Yrinis, J. .T.,It. Alter, E. B. Hook, M. Mayer. 1964. Famil- tion aigue. Notrr'. Rev. FrancaiseHimatol. 5:69. ial D/D translocation. Report of a pedigree and DNA Grorrchy, J. de, S. Thieffry, NI. Arthuis, J. Gerbeaux, S. rcplication analvsis.New Engl. J. Med. 271:1133.

Sex ChromatinStudies

Lillion Lockhort,M.D.*

Since Barr's report in 1949, of the sexual dimor- phism in ganglion cells of the cat and later reports of similar findings in the human, the use of the *D"pu.t-"nt of Pediatrics, University of Texas Medical Rranch, Galveston, Texas. study of the sex chromatin mass in interphase nu-

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-i: -i=,.,.sDE€ Vol. 3, No. 4, 1966 SEXCHROMATIN STUDIES 501

clei has spread greatlv. For sonre time it was be- the rnenstrual cycle rvith the lorvest incitlencc at lieved that thc nrassreprcsentcd all or part of both menstruation, howcvcr, Brainerd could not verifv X chromosomcs in the fcmale. Ohno, however, in this. Jacobs is quoted as finding administered estro- 1959, dcmorlstl'atcd that the Barr body represented gens to cause a decreased incidence. The lorveretl 'I-hc a single X. rccognition of the mass is made positivity in the first two days of life proposecl bv possitrle b,v the clifferential staining reaction by Dr. Smith has been consideredto possibly be associ- basic dy'esof thc chromatin of one of the X's of ated with the high level of estrogen in this agc fcmalc cells n'hen it is more condensed and hetero- group. chromatic than the other chromatin. Various stains. Shetty and Sharma recently prcscnted a trenlen' such as thionin, hcrnatoxylin, aceto-orcein, Fuelgen, dous study on 200 healthy children controls and 67 are the more cornmonly used. The range of normal females prior to, during and after prednisone ther- values of positivity have varied depending on the apy. There was a statistically significant dccrease itr tissue studied, the staining technique, and the the incidence of sex chromatin by four - Provided by the Maternal and Child Health Library, GeorgetownUniversity

b 502 Lockhart Ala. J. Med. Sci.

range for sex chromatin counts. The possibility that in interphasc nuclei? These questions are yet un- such patients may have an isochromosome or de- answerablc. \\re would, however, like to recommend leted X had to be considered, however, they did not its more universal use as a screening study in fer- have abnormal X chromosomes on karyotyping. I tility clinics, in mental hospitals and in all develop- am not sure that we could recognize a small Barr mentally retarded patients. It is a necessarydiag- body with our stain, as has been found in such cases, nostic tool in casesof ambiguous gcnitalia. In fact, becauseof the great variability in size that we see even in minor abnornralities of the genitalia, such normally. as cryptorchidism and non palpable testes, it is a In reviewing ollr cases, it was obvious that the rnust. most common sex chromosome anomaly-XxY- When one sccs typical Barr bodies in a pheno- was lacking in our referrals, probably because prior tvpic malc, chromosomal analysis is indicated for to puberty the clinical manifestationsmay not be so verification. When a negative result is obtained in a obvious as in paticnts with greater than two X's. phenott'pic female, one lnust consider that it may bc We are norv screcning all children r.r'ith develop- a false negative resulting from various metabolic mental anomalies on the pediatric service with a sex influences. If after repeating such a stuclv along chromatin study in hopcs of finding such patients. n'ith normal controls, discrcpancv still exists, Many questions remain concerning the sex chromosomal analysis is again nceclcd to vcrify thc chromatin-Why does its incidence vary in different diagnosis.It rvill continue to be a uscful screening tissues?Why does it vary from tintc to time in a test becauseof the relativclv snrall amount of tirnc single inclividual? \Vhy rloesit vary in differing age atrd expense rcquirecl in cornparisolt to performing groups? Why is it most often peripherally located a chronrosonralanalysis.

Detectionof Mosaicism

John F. lockson,M.D.*

'I-hc dctection of rnosaicisrtris an iurportant prob- as n'cll as in providing cridcnce cventr,ralll'cstablish- lcm in the practical application of c1'togenetics, ing expectcd variation of chromosorne colrnts rvithin particularly in regard to familial transmission of the nonnal population. Since culture techniquesand trisomic syndrornes.Both parents of anl' trisonric criteria for accepting or rejecting individual cells child ideally should have chromosome analvsis at for counting van fronr one laboratory to another, the tirne that the child is studied. Such information probabilities should ideall_vbc establishcd by cach is of irnmediate valuc fol gcnctic counselling.Accur- laboratorv group to tcst the sisnificancc of its orvn ate delineation of mosaic patterns is ultimatclv int. data. In collaboration u'ith Doctor P. E. Puller', portant in thc ovcrall correlation of clinical findings cornputer printecl tables of probaltilitics have lteen with cytogenetic studies. devised as an airl to the clctcction of tnosaicisrn The detection of rnosaicisi-r.rprcscltts a particular (Jacksonancl I'trllo', 1966). problern in that cven in nonnal people variations Table I is a photoeraph of a contputcrprintctl in acttral chromosomc counts are to be cxpected tablc giving convcntionalinclusivc probabilities.P.\ duc to lnany factors. Artifact probabl,v accounts for lcprcsents the proirortion of nrodal cclls, l'B thc some portion of thc ncgatively skcrvcd distribution proportion of hy'pornodal cells and PC the propor- around thc modal number becauseof chromosomc tion of hypermodal cells, excludins poll'ploicls, loss frorn brokcn cells. True variation in chromo- basecl on large numbcrs of chronrosollte coultts in some count from mitotic crrors in somatic tissues normal individuals. .{s an cxamplc utilizing this resulting in chromosonle llulnbcrs othcr than thc particular segment of the tablc, onc can scc that 2l rnoclalnumber also occurs. This is particularly evi- rnodal cells out of 27 shotrld include all but one il) clcnt rvith advancing age (Boyd, et al., 1966). Occa- a thousand analyscs.Thus, if less than 2l cells arc sional individuals havc true mosaic chromosomc rnodal, there is only onc chancc in a thousand that constitutions, lvith a significant proportion of their this particular anall'sis is rvithin the nonnal range. tissues being composeclof trvo or more stemlines of Horvevcr, cumulative probability tables do not takc cells with differcnt chromosome numbers. full advantage of the fact that thc clistribution is Knowing thc probability for finding aneuploidv negatively skewcd about the rnodc. shoulcl be of grcat help in the detection of mosaics, Table 2 shorvs a portion of a computer printed probability table listing probabilitics for specific cell counts. If in a total of 40 cells counted, 36 are -.O".,rrl"n, of Prevcntir c \ledicinc, Laboratorr- for modal, 4 hypomodal, and 0 hypemrodal, the prob- \ledical Genctics, [inivcrsit.v of ]fississippi ]Icdical Oentcr, Ja

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TABLE I

27 CELLS i{l IH PA = 0.9468t144t Pts = 0.OJ t6L34t pC = 0.Ol950Zl 2l IU 2l M0l-tALr PR0B : O.2290963 26 T0 27 MODALT PROB = O.17607g2 25 I0 27 M0DALT pR0B = O.gZ9 ll0l 24 I0 27 M0L)ALr PROB = 0.947t922 23 t0 2l M0DALT pR0B = 0.9g7ZOZ5 22 T0 27 M0ttALr pR0B = 0.99T4lSO ZL I0 2l M0DALT pR0B = 0.9995g79 = 2e CELLS wl Ix PA 0.9q68844, Pts = 0.03 )61)4, pC = 0.01 }5OZI 28 I0 28 M00ALr PROB = 0.2169277 2'l I0 28 MODALr pROB = 0.5576491 26 I0 2A flODALr PROB = O.Bl56T02 2, I0 28 MODALr PROB = 0.941 1096 24 I0 28 UODALT PROB = 0.9850879 23 T0 28 MODAL, ?RDB = 0.9969294 22 fO 2E MODALT PROB = 0.9994751 29 CELLS l{lIfl PA = 0.9468844, PB = 0.03t6L34, PC = O.0l950ZI 29 I0 29 MODALT pR0B = O.Z0i4O55 28 t0 29 MODALT pR0B = 0.5)95505 27 T0 29 M0tiALr PROB = 0.80L9652 26 t0 29 FODALT PROB = 0.9344q69 25 T0 29 MODALT PROB = 0.9gZ75ZO 24 T0 29 1.,!0DALr pR0B = 0.9963004 2) I0 29 ttODALr PROB = 0.99 91404 30 CELLS l'tlTH PA = 0.9468844r PB = 0.0J361.34r PC = 0.0l95OZl l0 I0 l0 MOuAL, pR0B = 0.1944953 29 I0 30 MOOALr PROB = O.5ZLgO?z 28 T0 t0 MODALT PROB = 0.Zgg0269 27 T0 30 MODALT pR0g = 0.927+099 26 IO 30 MODALT pR0B = 0.9g01962 25 r0 30 r,t00ALr PR0B = 0. gg55BOg 24 T0 30 H0UALr PR0B = 0.9991790 chrornosome analysis. However, if with 36 modal that these cells wcre rnost likely XO in view of the cells therc are 0 hypomodal and 4 hypermodal, rhe negative sex chromatin pattern. In our laboratory, probability falls ro .0003, indicating in numerical the finding of this cell count distribution whcre 3 fashion that hypermodal cell counts are a stronger cells out of 60 were hypermodal was .002, so that wc indication of mosaicism than are hypomodal counts. concluded this was probably an XO/Xy mosaic. If a borderline probability is obtained, additional Subsequent clinical findings support this conclusion cell counts can be performed, and the correspond- in that laparotomy revcaled bilatcral testes, rathcr ing tables consultcd until a satisfactory number of than the streak ovaries cxpected in thc usual XC) cells has been counted. Once one clemonstrates sta. Turner's S,vndrome. tistically significant aneuploidy, then karyotype In addition to this typc of application, onc fre- analysis of the aneuploid stem-line is necessarv to quently faces the problem of gcnetic counselling for confirm consistencyof karyotype. parents of a trisomic child. We recently investigated This approach has helped us recently in the in- a family in which there were three mongoloid chil- vestigation of an intersex problem in an infant with dren. One had expired, and the two available sibs ambiguous genitalia. This infant had a chromatin each had the usual trisomy 21. Their clinically negative sex chromatin pattern, and on chromosome normal mother was found on analysis of peripherai analysis had 45 chromosomes in 52 cells, and 46 blood leukocytes to have approximately r/s of her chromosomes in 3 cells. Karyotype analysis of the cells with 47 chromosomcs, 2l trisomic on karyotvpe cells rvith 46 chromosomes revealed all three to be analysis. apparently XY cells. The cells H'ith 45 chromosomes Similar cytogenetic findings in parents of nrongols had orrly 4 chromosomes of rhe 2l-22, y group, so have been reported by sevcral investigators (Rlank,

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TABLE 2

PA 0.93630 P8 0.04970 PC 0.01390 40 AENORHALNOT CANCER

FREQUENCY PROBASI LTY A40 B O C 0 t. 0. 07 I 879 ----T-39--A39 I 0 -T--l'C I 40. 0.042684 rI 40. 0.152617 ----F-38--E-A38 I 0 --rC 2 790. 0.0 12356 I r I >60. 0.098362 A38 S 2 C 0 780. 0.157971 Att B 0 c 3 9880. o.oo2324 ----T-7A!7 B -zI C 2 29640. o.o24924 B C---T- 29640. 0. 089 437 B 3 C 0 9880. 0. I 062l4 A-Ttg0 E4 436 I I C 3 365560. 0.004564

136 I 3 C I 365560. 0.058342 0.052151 --I_55-435 I -r-0 -CC -4-5 - -32-900-40.658008. 0.000034 E- T:1006Tr 435 B 2 C 3 6580080. 0.004360 435 B I C Z 6580080. 0.015590 ---T-Tt-B435 B 4 C -Or ---651008.1290040. o.o27 872 5 C 0:oT993r ---r*14-134 - I 0 -E-5-C 6 ------73030280.38l8 380. 0. 000003 E--l 0.000063 A34 I 2 C 4 5757 5700 - 0.000566 ct al., 1962;Snrith, ct al., 1962;\Vcinstein and War- casesrcpol'tcd bringins thc total to morc than 20 kany, 1963,Verresen, ct al., 1964) in which there has (Jackson, 1966; Soukor.rp and lVarkany, 1966; Nfur- been a reasonable percentage of cells that were tri- phy and Reisman, 1966).Others have noted an even somic for chromosorne 21. There was no problem in higher incidence of ancuploidy than in our cases deciding rvhether or not these individuals were (Murphy and Rcisrnan, 1966). Further studies arc rnosaics. Horvcver, Ridler (1965) has reported one needed, espcciallv with regard to comparison of rnosaic mongol in which all of thc analyzed cells chromosome analvsis frorn tissue involved in con- from blood had 46 chromosomcs, and almost all of genital abnorrnalitv to some suitable control nra- the cells from skin had 47 chromosomes and were terial. trisomic for chromosome 21. Therefore, one musl REFERENCES still individualize in rcgard ro how many tissues Blank, C. E., 1.. (]ernmell,lI. D. Casey,lI. Lord. 1962. should be cxaminccl in thc investigation o[ cach Mosaicisur in:r r.rrothcr u'ith a rnongol child. Brit. \Icd. J. family. 2:3 78. Itoyd,J. T.. \V. \[. Corrrt-Rrorvn,.f.Vennart, G. E. Wood- The localization of trisonric cclls to a particular cock. l lXi(i. (lhromosomes Studies on IVomen I'ormerlv l.mplovctl as l.uminous-dial Painters. Brit. Nfed. l:377. tissue or tissucs points up thc J. possibility of the de_ Gropp,4., A. Jusscn, F. Odunjo. 1964. Near-Triploicl tection of localized rnosaicisrn. Since cleft palate is Chromosomc Constittrtion in Epithelial-cell Ctrlturcs of one of the most Palatal lf rrcosa I;r'om a Case of Clcft Palate. Lancct I : frequent finclings in the Df trisomy I t67. syndrome, we havc studied cleft palate tissuc seeking .fackson, .T. I'. l9(;(;. Chromosomes in Cleft-Palatc Tissucs. localized mosaicism as a cause of isolated clcft Lancct l:llir6. pai- F., and I'. l-. Ptrllcy. 1966. Detection of Clrrom- ate. \Ve Jackson, J. have now studietl a total of 9 individuals osom:rl \[osaicism bv Computer llethods. J. NIcd. Genct. by chromosomc analysis of tissue culture

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lndications For ChromosomalAnalYsis In Mongolism"

HonsZellweger, M.D.f

Most cytog':trcticlaboratories arc not staffed and NORMAL ccluipped to karyotype every case of mongolistn and paramongolisrn, although such informatiort would Formesfrusles bc desirablc in ordcr to know morc about thc inci' dence of thc variotts chrotnosotnal complements found in tnongolistn. The clinician, thcrefore, has MAR. to select those cases rvhere a chromosomal analysis is definitely indicatcd. This presentation is lirnited to a discussionof thc three tnain indications listed in Table l. Phvsicians and/or parents of a mongoloid child rnay ask for a chrontosomal analysis of a case which

l. CasesWhere the Diagnosis Nlongolisnt is Doubt' fuI: "Mongolisur is an all or none condition, au indi- vidual either is or is not mongoloid." Such state' ruents are found occasionally in texts. They do r"rotwithstand critical analysis any longer, although they contain a grain of truth, which becomcs evident when rnongolism is compared with other constitu' tional anomalies, such as, for instance Marfan's syndrome. A fully developed case of Marfan's syn' drome is charactcrized by a nurnber of signs and symptoms: macrosomia, dolichostenomelia, scoliosis, cctopia lentis, cardiovascular manifestations, muscu- Fie. l. Marfan's syndrome-"normal" and its tormes frustes teiains ovei to the state. The number of lar hypotonia, etc. Besides casespresenting all thesc liieJ--ot- rormes frustes increases the more their signs and s,vmptoms one finds nulnerous caseswith Jvmptomaiology approaches the state of "normalcy.'' a few or only one of these manifestations. Such cases, also callcd formcs frustes, represcnt inter- formc fruste.* Even morc oftcn casesaLc ctrcottnterctl bcnveen rnediary stagcs between clear-cut Marfan's syndrome prescntine diff iculties in distinguishing ;'fortoes "norntal state". and "normalcy" (scefigure l). Therc is a cotrtinuum frustcs" and tlre so-called '['he frustes incrcases thc of transitional statcs. Often it is almost irnpossiblc numbcr of casesof forn'res "clearcut rnore the symptomatology approachcs "normalcy". to decide rvhcthcr a case is a Marfan" or a 'fhe situation is different in mongolisnt as shorln TABLE 1 in figure 2. The small circlc on thc lcft side of figure 2 indicates the casesof rnongolism showing a A CHRO}IOSO\{AI- ANALYSIS IS INDICATED IN: varying number of mongoloid stigmata. Thc larger l. Cases where thc diagnosis mongolism is doubtf ul. "normals". 2. Mongoloids born of mothers below the agc of 30 years circle on the right side represents thc and both parents of such mongoloids. Sorne "normals" show a few mongoloid stigmata as 3. Mongoloids who have mongoloid relatives' well, rvhich rarely call for diagnostic considcrations . *Supported by United States Public Health Service Grants f , i, quite possiblethat only some of the so-callccl formcs f rustes are due to the mutant gene of lf arfan's #5" POI NBo5489-02 and f3 ROI HDOI822-0651. fDepartment of Pediatrics, Univcrsity Hospitals, Univer- syndromc. Othcr cases may belong to othcr nosological sity of Iorva, Iorva City, Iog'a. enti ties.

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wflre 506 Zellweger Ala. J. Med. Sci.

Mongoloid (skin cultures) have been reported (Ridler, ct al., I965; Zellweger, ct al., 1966). Forty-six normal chro- nlosomes arc fotrnd occasionally in paramoneoloicls. A normal chromosomal complement is compatible rvith the diagnosis paramongolism and hence does not help in dclineating paramongolism from thc "normal" state. Mosaicism with a normal cell clonc and a 2l tri- somic clonc can be found in rnongols, in phcno- typically "normal" individuals, ancl in paramongo- loids. In fact mosaicisrn is rclatively rnore frequcnt among thc latter, i.e. arnong cascsn,hcrc the rliag- nosis mongolism may bc in cloubt. Scvcral casesof I)own's syndromc .vith chrornosomal mosaicism anrl normal intelligence have becn reported. This investi- gator is not convinced that casesof mongolism rvith Koryotype normal intclligcnce (Lq. 100) exisr. He rvoukl bc inclined to consider such cascs as paramongoloid. Hc rcalizes that in an cxceptional casewith chromo- somal mosaicism sharp dclineation betn'cen l-non- ..Fig. ?: Mongolism, paramongolism, and mongoloid strgmatization leading over to the .,normal',-state golism and paramongolism may be cxtremclv diffi- (see text). Paramongolism and mongoloid stigmati- cult or even impossible. zation as transitional states between-mongolisin and the "normal" state. Chromosomal comfii-ements of Difficulties inherc to mosaicisnr not onlv rvith the various states. rcspect to clinical diaenosis. At times the recogr-ri- tion of mosaicism itsclf may be clifficLrlt. Thc cases to nrle out ntongolisnt. N{ongoloicl stigmata occrlr studied in our laboratory and prescnted in table 2 in various othcr conditions not listed in figure 2. illustrate the problem. The last four cascslisted in The diagnosis r.nonsolism is made prima vista in thc this table show a chromosomal tnosaicisrnwith lg tnajority of cascs and presents difficulties in vcn. 'lhcre to 53lo euploid and the rest 2l trisomic cells. The rare instances. are onlv a few casesrvith clin. diagnosis mosaic mongolism of these casesis beyond ical manifestations, l,hich fall betu,een rnongolisnr doubt. They account for 2.lo/o of the casesstucliecl and "norntalcy." These cascsarc indicatc

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disappcars and a mosaic er"ubrl'o rvith atr euploid TABLE 3 clone and a 2l trisomic clone develops. Non-dis- RISK OF HAVING MONGOLOIDCHILDREN junction during mitosis is probably not as frequent as non-disjunction during meiosis I of ovogonia, Younger Older Motlters Motlters where the prolonged duration of svnapsis (dictyo- \. Risk of tene) favors its occurrcnce.** having (-)NL l.o\\r HIGH 2) Chromosomal mosaicisrn can originate fronr mongoloid loss of a chromosome 2l during an early cleavage child trisomic zygote,whcrebr B, Risk of division of an aneuploid 2l having a one daughter cell remains trisomic, while the other SECONI) HIGH LOW cell would 46 normal chromosomcs. Furthcr mongoloid display child mitoses would lead to two clones with the respec- tive chromosomal complements. This mechanism may be rnore frequent than that described in thc cncountcred in about 9Og5To of all cases,is age previous paragraph. Chromosornal analysis of spon- dependent. Its incidence increases with increasing taneously abortctl material sugscsts that about two maternal age. Chromosomal analysis of these cases out of thrce 2l trisomic zygotes are spontaneouslr' shows a regular trisomy 21. B) There is an age inde- abortcd. One could conjecture from this observa- pendent type of mongolism found only in a minor- tion that nature has a tendenc-v to rid itself of ity of caseswhich has a considerablc rccurrence risk. aneuploid zygotes. One wonders, thereforc, rvhether This form can be subdivided into thrce sub-groups: a sirnilar regulatory mechanism could be operative N{aternal t^'isomy 21, parental mosaicism, ancl fa- for parts of an anctrploid zygotc, i.e. for individual milial intcrchange or translocation. aneuploid cclls. In other n'ords, cortld it be that Mongoloid girls rarely become prcgnant, be it nature attempts to eliminate thc supernrrrnerarv that thcir fertility is decreased, be it that thcy are chromosome at least in some cells in order to rc- lcss sought after as mating partners. Not more than cstablishcuploidy? Such a hvpothesis could best cx- a dozen mongoloid girls, with all togethcr 16 chil- plain thc frequent occurrence of nrosaicism consist- dren, ha'r'e been reported to have rcproduccd. Ovo- ing of a iar.qc2l trisomic anrl a srnall crrploid clonc. gonia of mongoloid girls are most probably trisomic Such explanation, however, rernains at prcsentlr for the number 2l chromosome. Meiosis of a 2l highly conjcctural. trisomic ovogonium may lead to a 2l disomic ovum (24 chrornosomcs)or an euploid ovum rvith onlv one (23 Thc risk of a 2l 2. Chrontosontal Analysis of Xlongoloids I)orrt of 2l chromosornc chromosomcs). a trisornic zygote is irO Mothers Belou tlte Age of )0 l'ear.s attd of Par-

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TABLE 4 TABLE RF-Ct.RRI-NCII RISK OF MONGOLISM rN FAMTLIES 5 WITH PARENTAL INTERCHANGE PROI}AI}ILI'I'Y oII F-A}{II,IAL TRISOMY 2I IF ONE HETEROZYGOSITY MONGOT,OID OCCURS IN 600 I,IVE BIRTHS THE PROBABILITY OF TWO }IONGOLOIDS IS l'arenlal Interchange RerLlrrence H eterozygosittt Ri.shof , T r ansloca t iitn C arri er ) ,ll ongolism In Aggregates Probability of ti>/21 and 2t/22 of 2 Mongoloids Interchange 50 Individuals Less than 0.01 \lother Carrier A b,'rr t 20 f./n 100 0.02 Father Carricr Lcss than 20 "'r 200 0.06 2l /21 Intcrchange (or 2l Isochromosome) \Iother or Father loo./,, and secon(l cousins, aLlnts and uncles included) amounts to 100 or rnore people one would expect that two mongoloids occur in about 27. of the kin- lrorn of morhers over 30 years of age had a DIG 'l dreds. his probability figure appearecl ro or GIG interchange respectively. However, inter- be sur- prisingly high. \\te clecided, therefore, to check these change mongolism can occur as a mutation, in which f ieures in a population other than the selccted carre it remains a sporadic manifestation. In fact group studiecl in our laboratorv. On two Field only 101" of the G/G and b\/o of the D/G inrer_ Clinics of the State Scn'ices for change mongoloids have a parent with interchange Crippled Children in Southern Iowa l.[9 parents of non-mongoloid hr terozygosity.In the latter situation the recurrenie children coming to the clinic for various pediatric risk of monsolism is considerable, as shown in table 4. and ortlropedic ailments were questioned for mon- soloid relatives. Six instancesof a mongoloirl relative were found: Once it was the probancl's sibline, 3. Familial Monsolism, in particular Familiat -fri- three somy 2l times a first cousin of the sibling, and twice a sib- 'f ling of a parent of the proband. In other words risomy 2l can be found in several siblings or may about 4 .oioof thc patients had a rnongoloid relativc. occur in a closc or distant relative of -orr- This then rvould indicate that about four percent soloid proband. " Investigations of familial mon- of the rvider families or kindreds in this area havc golism in the State of Iowa yielded l0 sibships with :r monsoloid relative, independent of thc ailrnent ,y?. (8 times) respectively three (twice) mongoloid of the proband rvho brought the family to our siblings. Moreover, mongolism in a first ciusin, attcntion. This could inclucle families where rhe unclc, or aunt. of a mongoloid proband was founcl proband, himself, is mongoloid. If we substitute, in 14 instances, while a second cousin of the pro- therefore, the proband consulting the clinics by a band or a first cousin of a parent of the probancl mongoloid proband, the incidence of two mongo- was mongoloid in Z0 instances. A more distint rela- loids in a kindred is of the order of rnagnitude tionship between two mongoloids was found in three which was predicted by the statistical computation. instances. Some of the mongoloid cousins, uncles, \Ve can conclude, therefore, from these consiclera- aunts, etc., were maternal relatives, others were pa- tions that the occurrenceof twcr casesof trisomy 2l ternal relatives of the proband. A familial incidence in a rvider kindred may be merely coincidental in of trisomy 2l was found in 3g instances, which a fair number of instances. Random association, amounts to about 2O/, of all mongoloids studied in however, does not cxplain all cases of familial tri- our laboratory. This figure is by no rneans repre- somy 2l as shown in figure 3, which shows four sentarive for the incidence of familial trisomv 2l families, each one with ntore than one case of tri- in the mongoloid population of our area since we somy 21. In farnily l) (KU) a firsr cousin of the karyotyped a selection of cases. Mostly familial cases proband's father is 2l trisomic. This could be, as and sporadic cases born of young mothers were rnentioneclabove, a coincidental associationof two studied while sporadic mongoloids born of older casesof trisomy 2I in this kindred. In family Z) mothers were not analyzed. (C$/H) four patients with mongolism were ob- The occurrence sf morc than one case of trisomv served. Two of them u,'erekarl'otyped ancl revealed 2l among second cousins or more distant relativcs a trisomy 21. The mongoloid II l0 is relared ro III may be coincidental. Such a contention becomesob- 8 and IV 10, being an uncle of III 8 and a grear vious if one keeps in mind that the over_all inci- uncle of IV 10. Likewise III 20 is relared to III g dence of mongolism is high, namely about I in 600 (first cousins) and to IV l0 (first cousin of the livebirths. We asked our paul biostatistician, Dr. mother of IV l0). Ffowever,III 20 and II l0 are not Leaverton, the following question: ,,What is the related to each other. The occurrence of one of them probability of two mongoloids occurring in aggre- in this family must be coincidenral. This observa- gates of 50, l0O, and people, 200 respecrively, iI"the tion calls for a careful pedigree analysis in every over-all incidence of mongolism is assumed to be instance of familial mongolism. Notwithstanding, I in 600 livebirths?" The answer is listed in table 5. the occurrence of three of the four mongoloids in If one assumes that a medium size kindred (first family 2 shows a sraristically significant aggregation

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c q) E o- E o(-) |lr E C\I E > o .tl E o C) E e, o a- t- b l- |J - E C! @ E o L- -?a- = o a- C E3 st .att G!V D{'(tt N .n 1r- c G' >.; aa- (! >\ c) ,tI =(tt E C tg, o (o^ rtl E -g o EE EL E o 3 Fcr E t- 9;- GO _c. E c)< x L) I Ecv .cts=' ll-t f (JrE o -- '=.Ct-l; = 3: ct) (l)-c-c (t' -?? E E3= o.) .9q')c, -99 -9 q)bE (q E EeeE '6 'o99aslsl o o-- (7) c 9ao o bEE E Eooglc') I I c eCC o =oo t v z. >=z at ooo TI rO t_t BTE HH H H H

Fig. 3. Examples of familial trisomy 21 (see text).

of cases.The occurrence of four casesof trisomy 2l families and kindreds with a significatrt tct.ttlcttcl in kindred 3) (SI) and of six mongoloids (three of to familial trisomy 21. Possible causes of farnilial them proven to be trisomy 2l) in the kindred 4) is triscmv 2l have been presented elscwhcre ((,rrrtis' certainly more than coincidental. The high inci' et aI., 1964). Whether or not familial trisontv 2l is dence of trisomy 2l in these two kindreds and in age dependent or not age depenclent is not ktrou'tr family 2 must have another reason than random at pl'escnt. More material has to be collected llcfot't' association. We conclude, therefore, that there exist this qucstion can be answercd. A c:rreftrl historv

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+-:=rc 510 Zellweger Ala. J. Med.Sci.

and chromosomal analysis is, however, mandatory Ferrier, S. 1964. Enfant mongolien-parent mosaique, th6se, Gen6ve. for all such observations. Hall, B. 1962. Dorvn's syndrome (mongolism) with normal chromosomes. Lancct ?:1026. Summary \IcKusick, V. A. I966. Heritable disorders of connecrive tissue, 3rd cdit. \Iosby, St. Louis, Mo. Ideally all cases of mongolism should be karyo- Peterson, C. D. and L. Luzzatti. 1965. The role of chromo- typed; however, technical considerations compel us some translocation in the recurrence risk of Down's syn- drome. I'ediat. 35:463. to presently limit the indication for chromosomal Polani, l'. E., 1966. Nfongolism.J. F. Kennedy Award Prc- analysis to cases where the diagnosis mongolism is sentation, Boston. Polani, P. E., J. L. Hamerton, F. Giannelli and C. O. in doubt, to mongoloids born of younger mothers Cartr:r. 1965. C1'togeneticsof Down's syndrome (mongo- and to instances of familial mongolism. lism). II Frequency of interchange trisomy in patients born at maternal age of less than 30 years. Cytogenetics 4:I 86. REFERENCES Ridler, \f. A. C., A. Shapiro, J. D. A. Delhany and G. F. Abbo, G., H. Zellwegerand R. Cuany. 1966.Sateilite asso- Smith. I965. A mosaic mongol with normal leukocyte ciation (SA) in familial mosaicism.Will appearrn Helv. chromosomcs.Brit. J. Psych. I I l:183. Paed. Acta. Sergovich,F. R., G. H. Valentine, D. H. Carr and H. C. Carter, C. O. and K. A. Evans. 1961. Risk of parents r.;ho Soltan. 1964. Nlongolism (Down's syndrome) with atypical had one child with Down's syndrome (mongolism) having clinical and cytogcneticfeatures. J. Ped. 65:197. another child similarly affected. Lancet 2:785. Zellweger, H. 1965. \'fongolismus-Down's syndrome. Erg. Cowie, V. A. and J. Kahn. 1965. A mongol child rvithout Inn. NIed. Kinderh. 22:268. trisomy G. Lancet 2:58-59. Zellweger, H., G. Abbo, N. K. Nielsen and K. Wallwork. Curtis, H., J. Tilley and C. Crowley. 1964. The elimination 1966. \Iosaic mongolism rvith normal chromosomal com- of chromosomal aberrations in liver cells bv cell division. plement in the rvhite blood cells.Will appear in Human- Radiat. Res. 22:730. scnetik.4:323.

SomeIndications for the Analysisof Meiofic Chromosomes

RichordA. Finch,M.D}t

The attalvsisof meiotic chromosomesproniscs to cdge of a specific chromosomal abnormality has be most useful in the investigation of the causcsof preccdccl the obscrvations regarding infertilit,v. sterility, serni-infertility, and spontaneous abortions In thc past fe\v vears there has been an increased in selectcd families. It has been known for lnan\' intcrcst in utilizing chromosonte analysis in the in- years that chromosomal aberrations in other organ- vcstigatior-rof the etiolog-r'of infertility per .sc.It isms can be corrclatcd rvith a decrease in the nunt- has bccn anticipatecl that a significant numbcr of ber of offspring as rvell as n'ith the production of pcrsons thus ir.rr,cstigatcdrr'ill have chromosomal abnormal offspring. Since improved methocls of abnorrnalitics that \rere previouslv unsuspccted. chrornosome anah'sis have enabled us to recogllizc \Iost of thcsc abnonnalities u'ill be detectcd bv con- structural changcs in human chronrosomes,rnost of vclltional nrethods of analvsis of mitotic chromo- thc cytogeneticists'attention has becn directed to- somcs. Horvclcr, as Schrnicl pointed out in 1962, wards such abnorrnalities associatcd lvith congcn- ncgativc cvtoloeical rcsults do bv no means rule out itally malformcd children. It has been through thcir a translocation or invcrsion. Btiijk and Kjcsslcr in manifestations, namcll', abnormal offsprine, that 196-1,clisclrssing the rolc of rneiotic chromosomc the majority of human chromosomal al;errations analvsis in infcrtility studies reemphasizeclthe fact havc becn detectetl- that "translocations of cqually sizcclparts and In the coursc oF investigating families in t'hich invcrsions. $'hich are noI paracentric, may escapc one or morc rnenlbers is a translocatiotr hctcr-ozr'- o'toloeical rletcction" when rcliance is placed whollv gote, ir has bcen noted that some of the carriers of on analr.'sisof mitotic chrornosornes.It is possiblc, a trallslocation chromosome have beerr chilcllcss or Itou'cvcr, to (letect thcsc traltslocations ancl inver- thcir marriages have resulted in spontaneous abor- sions bv a careful studr, of thc chromosome complc- 'f tions intermittcnt rvith normal prcgnancies. he llrcnt in rnciotic cells. llature of the chrornosomal abnormalitv has bccn I l'ould like to summarize norv the rcsults fronr readily evident from analysis of chromosomes in trvo scrics in rvhich meiotic studies have been car- rnetaphasc of mitosis: indecd in thcse cases knolvl- riecl otrt in the course of the work-up of males attending infertilitv clinics. *Present address: Department of Nledicine, University of Kjessler in 1965 reported initial results of chronr- North Carolina School of Nledicine, Chapel Hill, N. C. iSupported as a post-doctoral laboratory assistant in the osome studies in 130 childless males. At the time of Ovtogenetics Laboratorl', University of Alabama Medical this report onlv the results of mitotic analyseswerc (ienter, Birmingham, Alabama, bv Children's Rureau Project 1r3. availablc, brrt of the total, l2 patients displavctl

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cytological abnormal karotvpes, ilrclucling 5 with sex chromo- with the rcsults of histological and some mosaicism and onc with a balanced D/D studies. translocation. Trivalcnt formation was seen in the Analysis of meiotic chromosomcs is also indicated rneiotic preparations from the patient with the in the study of certain congenital malformation translocation. syndromes, especially those in which the parents of Mcllree et al in 1966 reported their results of children with trisomy-deletion synclromes are found meiotic stutlies in fifty subfertile males. The men to have normal mitotic karyotypes' rvere selected for meiotic analyses on the basis of Trujillo et al (1966) recently reportecl their stucl- having no gross physical deformity, and because ies on a child with multiple congenital anomalies the,vwere chromatin negative and had sPerm counts who had abnormally long arms on a chromosome of less than 20 million per ml. In two of the cases, of the B group. The appearance of the chromosome multivalents were seen at diakinesis suggesting the suggests that it arose from a reciprocal transloca' presence of a reciprocal translocation. It is interest- tion in one of the parents, but both parental mitotic ing to note that in neither of these cases was a karyotypes were norrnal. Other very similar cases translocation s€en in the mitotic preparations. In \{ere reported by Gagnon et al in 1963 and Bray another patient no structural abnormality was seen, ancl Josephine in 1964. Trujillo presented in dia- but thcre was one pair of small acrocentric chromo- gramatic form the various t,vpes of Sanretes that sornes that showcd only terminal chiasmata or ex- rnight be produced by a carrier of a small and un- isted as univalents. Although these findings may be detected translocation, as a result of crossing over preserlt in some apparently normal cells, in this in meiosis.In the course of the meiotic process,de- case they rvere associated with degeneration of the pending upon the extent of crossingover, a certain bivalcnts at diakinesis and a deficiency of cclls in percentage of gametes rvill be produced u'hich, if second meiotic metaphase. they are able to effect fcrtilization, will result in At least l0/o ot.the males in this small scries werc zygotes with gross structural chromosomal aberra- found to have chromosome abnormalities and in tions. In cases such as this, analysis of parcntal trvo instances the abnormality was not detectable in mitotic karyotypes gives no clue as to the origin of the mitotic karyotypes. the translocation. Meiotic analyses would then be At this time, a great many questions rcmain un- helpful in determining which parent carries the answered regarding the mechanisms by which cer- translocation ancl perhaps in identifying the chrom' tain abnormalities result in poor reproductive caPa- osomes involved. This information would be useful bilities. Dr. Zellweger in a rccent paper noted that not only from the standpoint of categorizing varia- of a total of 49 offspring of parents with balanced tions of similar chromosomal syndromes, bttt also in D/D translocations, only two children had the par- genetic counselling where familial translocations tial trisomy D syndrome. Hamerion in 196l found are involved. from analysis of the pedigrec of a D/G transloca- In adclition, fairly accurate assessmclltof the risk tion that male carriers producecl only carrier prog- of producing abnormal offsprir-rg may be made on eny and only the offspring of carrier females re- the basis of meiotic analyses in persons shown to ceived the unbalanced complement and were af- have chromosomally normal and abnot-mal stem fected with Dorvn's syndrome. It is now known that cell lines. In these mosaics it would be important to males also can transmit the unbalanced state. How- determine the existence and extent of gertn cell in- ever, from thesc and other observations it does seem volvement. As important as this information might that gametcs rvith unbalanced translocations may be be it will be of only limited value in genetic coun- at a selective disadvantage. Thc selection is clearly selling until such time as it is feasible to detect not absolute, since, although some translocation mosaics before they have producecl abnormal off- heterozygotes are infertile, others produce normal spring. as well as carrier and affected offspring. Whether In general meiotic studies will be most helpful in the selection against these garnetes is a function of cases of congenital malformation syndromes in the quantity of exchanged genetic material; the size which there are structural rather than numerical or nature of the chromosomes involved; or other chromosome abnormalities and in rvhich the paren' factots, including a widespread effect on the meiotic tal mitotic karyotypes are normal. process as a whole is not clear. More observations I would now like to discuss briefly some of the on normal and abnormal meiosis are needed before research that is being done with meiotic chromo- the exact role of chromosome abnormalities in thc somes. We have been considering, prirnarily, the etiology of sterility and semi-infertility can be dc- study of meiotic chromosomes in tcsticular cells. termined. ['rom these initial studies, however, it is The great majority of meiotic studies rvill undoullt- evident that meiotic analysis will be useful in select- edly, be carried out in male subjects. Although the ed cases. In order that a structural chromosome proceclures necessaryto obtain material for analvsis abnormality might be related causally with infer- are relatively innocuous, thcy are of strch a natllrc tility in a given instance it should be demonstrated that norrnal tissue rvill be difficult to obtain. The that there is an associatedabnormality in the num- clinical investigations in problerns of sterility and ber or function of the resulting gametes. Kjessler, known chromosomal syndromes will, therefore, pro- in his study, has attempted to'do this by correlating vide the bulk of new information regarding the sperm counts and sperm morphology and motility morpholow and behavior of chronlosomes in mei-

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il 512 Finch Ala. J. Med. Sci. osis. N{uch can l;c lcarncd regarding thc rlynamics ceive more widespread applications. N,Ieiotic studies of chromosome bchavior and the transmission of also provide a means by which the "partial trisomv- abnormal chromosomes by studying the various deletion" syndromes may be investigated when rneiotic stages in testicular cells. There are, how- parental mitotic karyotypes are apparently normal. ever, certain advantages in studying the chromo- It is acknowledged, of course, that even when somes of human ova. nreiotic abnormalities are demonstrated, the present Edwards in 1965 rcported his initial observations state of our knowledge of meiosis in humans, allows of the nraturation of human oocytes in vitro. He us to only theorize concerning the mechanisms by was able to observc all stages of meiosis through rvhich certain abnormalities manifest themselves second metaphase in some 133 living oocytes in cul- clinically-hence rhe need for additional meiotic ture. The availability of such methods that allow studies in normal and abnormal subjects. the observation of chromosomes in the active pro- cess of meiosis will be most useful-especially in REFER,ENCES studying the phenomenon of non-disjunction and Ilttdk, -I. A. and B. Kjessler.1964. Nleiosis in the human the effect of various chemical and perhaps viral male.Cytogenetics, 3:143-147. Bray, P. F. and SisterA. partial agents on the mciotic process. Meiotic Josephine.1964. auro- studies in somal trisomy and translocation. Report on an infant oocytes might also provide information regarding with multiple congeniral anomalies.J.A.M.A., l8i:98-101. the exact mechanisms by which structural chromo- f,dwards, R. S. 1965. Nlaturation in vitro of human oocytes. Lanccr 2:926-929. some abnormalities result in gametes with decreased Gagnon, J., L. Archambault, E. Laberge aifo w. Katyk- fertilization potential. Longtin. 1963. Trisomie partielle l8 par insertion ou translocation.4/18 L'Union l\{ed. Canada, 92:31l-319. At thc present time the re are no criteria by Hamerton, J. L., V. A. Cowie, F. Gianneli, S. Ir{. Briggs and which to identify the meiotic chromosomes. Specific P. E. Polani. 1961. Differential transmissionof Down's svndrome (mongolism) through male and female carriers. identification is not so critical in the infertilitv Lanccr, 2:956-958. studies but some tneans of correlaing meiotic anrl Kjessler, B. 1965. Karyoty,pesof 130 childless men. Lancet, mitotic observations 2:493-494. is necessary in studies of thc Kjessler, B. 1964. Nleiosis in a man with a D/D transloca- congenital malformation syndromes. tion and cl.nical sterility. Lancer, l:1421-1423. Nlcllree, 8., In sumrnary then, meiotic M. W. H. Price, W. N{. Court-Brown, lV. S. analyses have beer-r Tulloch, J. E. Nerlsan and N. N{aclean.1966. Chromo- shown to be of value in selected casesof male in- some studies on tcsticular cells from 50 subfertile men. fertility. Of the chromosomal Lancct.2:69-71. aberrations that might Schmid, W. 1962. A familial chromrsome abnormaliry asso- be detected by these methods, translocations are the ciated with repcated ab.rrtions.Cvtoeenetics, l:l99-209. most obvious and easily recognized Trujillo, J. l\1., R. S. Zeller, R. A. Plessalaand B. List- but it is con- Young. I966. Translvation heterozvgosisin man. Amer. ceivable that inversions and insertions may be asso- .lour. Hum. Gen., l8:215-22i. ciated with infertilitv as meiotic investigations Zcllwegcr, H. | 966. Fam'l;al lhromosrmal ahcrrarions P;rrt re- I. Ann. Paediat.,206'317.332.

ChromosomeStudiesIn Abortions*

Soro C. Finley,M.D.t

'l'hc ln otrr rliscussionof the indications for chror.no- frcqrrencl of chrornosomal abnormality in somal analysis,I worrld like to comment briefly on the fetrrs is highest in early pregnancy and Polani the need for morc information regarding the inci- (1966)in a recent review stared that nearly a third dence and types of chromosomal aberrations in of early spontaneous human abortions carry obvious abortuses. Chromosome studies have now been donc chromosome abnormalities to rvhich their failure to in a nurnbcr of laboratories on a total of approxi- rlevelop might be attributed, whereas among later mately 800 spontaneous abortions and in the data spontalteous abortions occurring after the third reported to rlatc, there is much variation in thc rnonth, thc proportion of chrornosomal anomalics

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:lrc Vol. 3, No. 4, 1966 IN SPONTANEOUSABORTIONS 513

-I-risottrv REFERENCES been rcportc(l in frtus(i l6' ho$'evcr, seems to bc thc nro<[ c{rl]lnt()tl }- grotrp trisomy in Polani. Paul E. 1966.Chromosome Anomalies and Abor- Neurol.8:67-70. abortuscs though thi.. <,f corlrsc. has not becn the tions.Develop. Med. Child. casc in livcbortts. (/UESTIONS: Another conllr)()l) firrrling irr atltlitittn to the vari- DR. DAVID SMITH: In the Wisconsin-Iowa ous trisonrics has bct'tt tltc Prcserlce tlf 45 chromo- family in which there are two brothers, each of comes with a tnissitrg cltrottrosonre in the C plus X rvhorn had children with Down's syndrome, as I group. N'{attv of thcsc karronpes have been intet'- recall, both of those brothers had hypercholestero- prctecl as XC) thousli tto rlistittction can be made lemia. It would be interesting to know if parents of l)etween C group tnott

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t- e=dEffii€ 514 DrscussroN Ala. J. Med.Sci.

TABLE I TRISONIY 2I TIOSAICS ASCERTAINED BY PRODUCTION OF OTFSPRING IVITH 2I TRISON{Y

Grand- Case Maternal I,Q. Number .Ser '4ge* D ermatoglyplrics Estimate Author I r 39 \Iongoloid type of dcr- 82 Smith et al. (1962). mal ridge pattern (N{aternal age and I.Q. data givcn in rePort of case 3) I F 40 l'almar and digital ridge 60 Blank et al. (1962) patterns caused au- thors to suspect diag- nosis of mongolism. 39 Reported as normal Cornpleted lOth grade \Veinstein and edrrration at age 16. Warkany (1963) 34 Radial loop on (R) 4th Normal Verresen ct al. (1964) digit, otherwise nor- mal. Hallucal pattern normal. Strm of palmer atd 1-horrght to bc normal. Fcrrier (1964) angles - ?9'. Ulnar loops on all digits ex- cepting radial loop on 3rd (R). Total finger ridse count - 140. Digital loop on 3rd interspace each hand. Hallucal patterns showed a large looP ()30 ridges) bilat- erally. *i.c. The agc of rlre rnorher of the 2l rrisomy mosaic at the time of the latter's birth.

tlividuals. I would direct your attention to the fact giving rise to mongol offspring, collectcd in Balti- that the grand maternal ages, that is, the age of the more, we could find no such cffect. mother at the time the mosaic was born, are all We analyzed the data in a variety of ways (Wclch very high. I would also direct your attention to the and Sigler, 1966).fig. 2 shows the number of affect- fact that these were all mothers: there was no mo- ed probands associated with differing grandmater- saicism in a father giving rise to an affected indi- nal ages in the affected an(l control groups. There vidual. The dermatoglyphics show variability, as is no evident increase in the nunbers of those with rloes thc I.Q., and this, of course, is not surprising. older grandmothers among tl"re affccted group. A Wondering what proportion of the affected off- more sensitive test-the paired "t." test-also did spring of young mothers would show such a grand- not shorv any statistically significant difference bc- maternal age effect, we collected a series of mon- tween the granclmaternal ages of the trvo groups. gols, the offspring of young morhers. In the course of collecting the data, r{'e came across another little known study, carricd out by Grcenberg in 1963, in CRAIOIIATERl\rAtAGE D]SJRIBUTIONFOR MO,THERS <30 YEARSA'I PRCBANDS81R]H which he looked at the grandmother's age, for rea- sons other than the ones I have mentioned here, 8 pertaining to rnongol offspring of young mothers, '7 and at the granclmaternal age of a control group 6 ('fable 2). He found a considerable increasc in the llumber of older grandmothers, 35 and over, in the 5 affccted groups, the chi-square test shon'ing a clif-

fercnce significant at the half percent level. Imaginc 3 our surprise llhcn in our group of voung mothel-s 2 :I I TABLE 2 rl GRAND}IATERNAL AGE: MOTHERS LESSTHAN 30 YEARSAT TIME OF PROBAND'SDEATH (Daraof Greenberg,1963)

Grandmother's Age trfongol Control at Mothefs Birth Probands Prohands Under 35 20 166 35 and Over I8 5l Total 38 2n 20 2s 30 X:=9.52;p(0.005. t Fig. 2. Grandmaternal ase at mothers birth

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==#flFre Vol. 3, No. 4, 1956 DrscussroN 515

TABLE 3 GRAND}IATERNAL AGE: MOTHERS LESSTHAN 30 YEARSAT BIRTH OF PROBAND

English Data (Essex) U.S. Data (Balthnore) (Greenberg, 1gfi) (Welch Ct Sigler' 1966) A B A' B' Grandmothefs Age Mongol Control hlongol Control at Mother's Birtlt Probands Probancls Probuntls Probands Under 35 20 166 54 56 35 and over l8 5l 97

Total 38 ,n ,13 ,rJ

A'B X2 = 9.52,p ( 0.00ir Il:I]' X: = 4.56,p ( 0.05

were puzzled as to why this might be, having, We Vonotron in IncrCence ol Dowris 9yrdrome as we thought, two reasonable studies with oPPosite with lncreosrng Molerrcl &e results. We now believe that the reason for this dif- ( doto of Cor ter ond Enre 1961) in ference is that we were looking at an essentially a different population. Table 3 shows our own re- 8 sults laid out in the same manner as those of & Greenberg, and I direct your attention to the con- E Y sig- D trol groups of both series (B and B'). They are ,'c nificantly clifferent in the numbers of older grancl- E rnothers, the difference being significant bel otttl I o the five percent level. These two populations are o therefore not comParable, and $'e think that this is -o o the reason why our own study did not confirm thc -i grandmaternal age effect. This difference in the number of older granclmothers is consistent rvith the difference in the maternal age distribution dur- Fig. 4. Variation in incidence of Down's Syn- ing the period of collection of both studies (Fig. 3). drome with increasing maternal age. the same If a large proportion of mosaics shon' mothers in the general Baltit-trorepopulation, thus matcrnal age as increaseclincidence with increasing accounting for our results. This does not alter our Don'n's st'n- has been demonstrated in the tvpical conclusion that grandrnatcrnal agc data as tt'ell as '1)' this tlrome cases(Carter and Evans, 196l) (Fig. rnaternal dermatoglvphics rvortld be useful in cle- of tnosaic rvould result in a relatively small number ciding n'hich mothers shoulcl be cramined for po- tcntial mosaicisnr.It cloessuggcst, I think, that the MATERNALAGE DISTRIBUTCIN proportion of rnosaicsr aries consitlcrablv fronr onc ENGLANDAND WALES 1953-57 population to another. BALTIMORE(WHITE BIRTHS)1946{2 - REFERENCES

Blank, C. f., E. Genrmell, \I. D. Cascr. \f . I-ord. 196!. \Iosaicism in a mother rvith a mongol child. Brit. \Iect. J, ii:378-380. Carter, C. O., K. -'\. Evans. 1961. Risk of parents *lto have had one child with Dort'n's svndronre (mongolism) having another child similarlv affected. Lancet. ii: 785-787. Chaudhuri, A., K. C. Chaudhuri. 1965. Chromosome mo- saicism in an Indian child rr-ith Dorr'n's synclrome. J. \Ied. Genet. 2:l3l-135. Fcrrier, S. 196.1. Enfant mongolien-parent mosaique. Etrrde de detrr families. (-l-hesis for the Degree of Doctor of \tcdicine. Thesis \o. 3957. Ulrirersity of Geneva.) J. g€nit. hum. l3:315-33(j. fttv IJ Grccnberg, R. C. 1963. Tn'o factors influencing the births :tr of mongols to voungcr mothers. \Ied. Officcr 109:62-61. F l'enrosc, L. S. 196+. Dermatoglvphics in mosaic mongolism 91s2- ancl allicd conditions. ln Genetics Today (Procecdings of rhe XI International Congress of Gcnetics, The Hagttc, F -I-hc 210 Netherlands. Sept. 1963). Pergamon Press, Ne'rv York. Ll-t Smith. D. \V., E. J[. Therman, K. A. Pateau, S. C. Inhorn' l{)62. \tosaicism in mothcr of nvo mongoloids. Amer' J. tr I)is. l0-1:534. lr, ( Child. o- Vcrrescn, H., H. Van Den Rerghe, S. Creemers. 1964. \Iosaic trisomv in a phenonpically normal mother of a mongol. Lance t. i: 526-52i. \\'einstein, E. D., \1'arkany. I963. \f aternal mosaicism 1q J, 20 25 30 35 10 45 and Down's sy'ndrome (mongolism). J. Pediat.63:599- AGE 603. MATERNAL \1'clch, .I. P., .{. T. Sigler. 1966' The influence of grand- maternal agc on the occurrence of Down's syndromc ant! Fig. 3. Maternal age distribution its r-clcvancc to nraternal chromosomal mosaicism. Prcsent-

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fl 516 DISCUSSION Ala. J. Med.Sci.

cd at SecotrJInuitation.al Conference on Human Behau- think therc may often be cells which play an im- ior, Genetics.(Louisville, 29 April-2 N{ay 1966.)To be published.Academic Press, New York. portant role and are difficult to find. What we find at any point in time may not necessarily corrclate precisely with thc patient's phenotype. I)R. WAYNE FINLEY: Correlation is not 1'et Possible between the number of physicai stigmata, DR. HANS ZELLWEGER: Thc fcn'cells that we intclligence, dcvelopment, and the associatedkaryo- count from patients cannot be represcntative for typc findings in patients with Down's syndromc. the total distribution of the two cloncs in the total Such a wide variation in clinical findings is present body. \Ve have recently founcl a case like that rc- in patients rcportcd in cach of the cytogenetic pat- ported by Ridler in which cclls from the blood terns found in Down's syndrome that no distinct werc nornal and cells from the skin wcre 2l- group such as 2l (G) -trisomy,2l(G)ll3-15(D) trans- trisomic. Hamilton has recently mcntioncd a mon- location, zl(G)l2l-22(C) translocarion,or any of thc goloid boy with an I.Q. of 100 and two trisomic rnosaic patterns can lte dcfined by phvsical exami- cells ancl about 40 normal cclls. If \{'e go that far, natiotr. then u,e dilute the term mosaicism; therefore, u'c In our scrics of 82 mongoloicl children of young should ask where is the limit between what lve mothers wc have found four with normal/21-trisomv rvould call mosaicism ancl $'hat rve could not call rlrosaic karyot1'pes. There are 26 documented cases mosaicism. reportcd and rvith Dr. John case fivc Jackson's DR. RALPH PLATOU: Dr. Lockhart, u'oultl mothers with normal/21-trisomy mosaic karyotypes vou givc us cxpcricncc or conviction on tJrc have given birth to 2l-trisomic infants. In the 30 )our variations in in casesthe I.Q. has varied from r.'erv lo$' to tl-rat of a the sex chromatin thc newborn? normal ranse and the phenotvpe has been norrnal DR. LILLIAN LOCKHART: \Vc have really harl in somc of thcm. Of 390 patients rvith Dou,n's syn- verv littlc experience in that arca. In about ll'r cascs tlromc sttrdicd by Dunsdon er al (1960),6-7lo had studied in the first two clays of life, all of thcrrr I.Q.'s above 45 while the four normal/2l-trisomic have bcen in the ran€ie for older chilclrcn. This is patients that rve studied had I.Q.'s of 50 or above a very small group of patients. rvhich n'as in the upper rangc for mongoloid chil- DR. YIELDING: I would likc to makc one morc tlren. I woulcl like for Dr. C. Rosecransto com- J. commcnt on Down',ssyndromc. It seemst{, me that ment on the psychological evalnation of these 4 it is a little difficult to know cxactly rvhat to expcct rnosaic rnongoloid patients. in terms of enzvme level becauseit is goirrg to cle- I)R. C. J. ROSECRANS: Psychologicalevaluations pend on thc naturc of thc cnzyme; whether or not of non-institutionalized mongoloid children are it is a rcgulatccl or an unregulated cnzyrne. It may scarce.\Vunsch (195i) in Rhode Island reported an bc useftrl to consider microorganisms as models. It LQ. median of 38 rvith a high of 61 in 17 mongo- has bccn sho$'n quite clcarlv using independent loitl childrcn living at home. None of our fotrr tcchniclncs that in microorganisms somc enzymes paticnts had cxceptionally high I.Q.'s; rhev do har.c rnav bc rlccreascd and somc rnay be increased, if scores in the top lSlo of the patients studied bv r'orr incrcasethe amount of gcnctic material. Andcr- \Vtrnsch. Thcse findings would tend to confirm that son ancl C)gg shorvcd quite clearly that grorving L. rnosaic mongoloicl patients havc a higher intellcc- coli-in thc prcscncc of camphor. u'ill double their Irral tlevelopment than 2l-trisontic patients. I)N{ content, presumably thcr-r thcir genctic loarl Is thcrc a dcfinite rclationship bctvr'centhe rati

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i#ffiare ClosingRemarks

Joseph F. Volker, D.D.S., Ph.D.1

It is my pleasure to close what I hope has been a pleasant ancl profitable International Seminar on Medical Genetics. Although the demands of my office have not permittetl the pleasures of continuous attendance, I have been u'ith 1'ou enough to be impressetl with the great progress that is being made in your specialty ancl the tremendous benefits that may become available to a substantial segment of the population in the years immecliate- ly ahead. As an amateur historian I have hacl a deep ancl continuing interest in Thomas .|effer- son. Although he is most remembered for being President of the Unitecl States, it is note- \A/orthy that he was one of the leading scientists of his time ancl the author of what was probably America's first comprehensive scientific monograph, No/es on Virginia. In the compendium there are many very cogent obs-'n'ations. C)f special interest to this sroup would be his descriptions of albinism ancl vitiligo anrl speculations concernins their eti- ology. He was, of course, a great agriculturist ancl recorrlecl in rletail the results of crossing black and white N'Ierino sheep. Being a mathematician, he attemptecl to predict the re- sults of these matings arithmetically. The limitations of such :rn al)l)roach are self-evitlent, but it shoulcl be rememberecl that this lvas the onlv nrethocl available to him in the pre- Ntendelian era. In 1916 I hacl the unusual pleasure of visitins the sarclen ol Gregor \Iendel in llrno, Czechoslovakia. The original monastic setting has been maintainecl, ancl one cannot hellr but be impressed with the very simple tools that u'ere available to the nran, who, in retro- spect, was one of the greatest scholars of the moclern era. Ry an unusual coinciclence, I am cul'rently invoh'erl in the preparation of a monograph relating to sonle of Pasteur's earlvl,vork on inlectious clisease. Llnrler these circumstances it has been natural to compare these scientific siants. Strangelv enough, they share the same birth year, 1822, ancl both lived until the latter part of the nineteenth century. Whereas Pasteur's pioneering efforts receivecl immecliate recognition ancl had immediate application to the solution of problems of human clisease, thosc of \lenclel lay clormant Ior manv years and only toclay is their full sisnificance being appreciated. In retrospect, it seems extraorclinar)' that each shoulcl have fosterecl a new science basic to moclern medical practice-Pasteur, bacteriolog)', ancl lfenclel, senetics. Of even sreater significance is the reaiization that lvhereas the discoveries of the former have been respon- sible for the primary advances in health sciences clurine the first half of the twentieth cen- tury, the observations of the latter may simil rrlr :rffect the next fiftv vears. I would like to thank all of the essayistsr,vho have sharecl their wisclom with us ancl the faculty ancl friencls who have made all of this possible. \Ve are cleliehtecl that you coulcl be with us. You have renen'ecl our hopes and bolsterecl our aspirations that this fJniversity will ultimately make a moclest contribution to the culture of our times ancl to this area. And with your indulgence, I n'ould like to close rvith a note that gives us conficlence rhar our efforts will be worthwhile. I used it in a presentation to the facuity this spring, ob- serving that poets have an almost supern:ltural abilitv to express in a minimum of worcls

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--* -il 518 VOLKER Ala. J. Med. Sci. the innermost thoughts of man. .|ohn Masefielclclid at the installation of the Chancellor of the University of Sheffield almost twenty years ago, when he said: "There are few earthly things more splendid than a University. In thesedays of broken frontiers and collapsing values, when the dams are down and the floods are making misery, when every future looks somewhat grim and every ancient foothold has become some- thing of a quagmire, wherever a University stands, it stands and shines; wherever it exists,the free minds of men, urged on to full and fair enquiry, may still bring wisdom into human affairs. "There are few earthly things more beautiful than a University. It is a place where those who hate ignorance may strive to know, where those who perceive truth may strive to make others see; where seekersand learners alike, banded together in the search for knowledge, will honour thought in all its finer ways,will welcome thinkers in distressor in exile, will upholcl ever the dignity of thought ancl learning and will exact standards in ,thesethings. "There are few things more enduring than a lJniversity. Religions may split into sect or heresy;dynasties may perish or be supplantecl,but for century after century the Uni- versity will continue, and the stream of life will passthrough it, and the thinker and the seekerwill be bound together in the undying causeof bringing thought into the world. "To be a member of one of thesegreat Societieslnust ever be a glacl clistinction."

Thank you.

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