Clinical Cytogenetics/Clinical Genetics A45 (0169) 13.1 (0170) 1.89

In situ hybridization shows direct evidence of skewed X inactivation in wo patients with 22q13.3 deletions have similar facies ard monozygotic twin females discordant for Duchenne muscular develcpental patterns. L. Ekie baum3, J. Singa dystrophy. S. M. Zneimer. N. R. Schneider and C. S. Richards . Btlt ,232 I. Tshima2 '3, _Cjinjj, Departments of Medical Jniversity of Texas Southwestern Medical Center at Dallas, and Genetics1 and Paediatrics2, University of Toronto, and GeneScreen, Dallas. Division of Clinical Genetics3. Hospital for Sick Ciildren, A novel combination of conventional and molecular cytogenetic Thronto, Canada techniques was used to examine the discordant expression of an X- inomy distal to 22q13 .3 due to a deletion is rarely linked recessive disorder in a set of monozygotic (MZ) twin females. described. We have recently seen two diildren with similar Both twins carry on their maternal X chromosome an approximately de novo del(22) (ql3.3) whose facial a oearare and 300 kb deletion within the dystrophin gene which is responsible for the neurodevelcpent-al progress are very similar. manifestation of Duchenne muscular dystrophy (DMD) in one MZ Both patients are only mildly dysmorphic. They both have twin (Richards CS et al. AJHG 1990;46:672). A unique 506 bp cDNA long narrow faces with prmninent protruling ears, prnen generated from exon 48 within this gene deletion was hybridized in situ chins, a flattened midface on profile, deep nasal labial to the twins' metaphase chromosomes, a probe which would grooves, a sneswhat praninent nose, frequent tongue presumably hybridize only to the normal X chromosome and not to the thrusting, and an increased carrying angle of their elbow. X chromosome carrying the gene deletion. Chromosomes were Slight terminal hypoplasia of the phalanges is also present identified by reverse-banding and by the addition of BrdUrd in culture in both children. Their height, weight, and head to distinguish early and late replicating X chromosomes that circumference are within normal limits. Both patients had correspond to active and inactive X chromosomes, respectively. This sacral dimples. strategy provided a direct means of determining the relationship Both patients were hypotonic in infancy, had brisk lower patterns and manifestation of an X-linked extremity reflexes, and learned to walk in the second half between X inactivation of the third year of life. Broad based gaits persisted at disease. age 5. Both children have expressive language delay which Hybridization experiments showed predominant inactivation of the is more severe than their global delay. Cognitive and fine normal X chromosome in the twin affected with DMD, which accounts motor skills are moderately delayed in both children. for this twin's manifesting the disease. Unexpectedly, the unaffected Patient A had additional features of a primary persistent twin also showed skewed, predominant inactivation of the X hyperplastic vitreous and Coats disease, as well as chromosome carrying the deletion within the dystrophin gene, superscapilar dimples. accounting for this twin's normal phenotype. Her skewed X Patient B had an umbilical hernia in infancy. inactivation pattern is not the random pattern predicted by the Lyon We conclude that del (22) (q13.3) causes a recognizable hypothesis that is seen in most carriers of X-linked recessive disorders. dysmorphic and developuental syndrome which is distinct from Further, the unaffected twin's X inactivation pattern is not only skewed, and more mild than rost cases of ring (22). but a mirror image of the pattern of her affected twin sister. is study The ring (22) syndrcme is more severe presumably because is the first to show direct evidence at the chromosome level of skewed in and opposite X inactivation of cytogenetically normal X chromosomes, addition to the deletion of 22q, cells with varying resulting in the manifestation of an X-linked recessive disorder in one numbers of rings can be present. of MZ twin females. These results support the suggestion that MZ twinning and skewed X inactivation may be associated events.

Clinical Genetics (0171) 1.90 (0172)

The relationship between lymphocyte cell surface markers and Syndrome of osteopetrosis associated with cerebro-oculo-facio- serotonin in autistic probands. R.K. Abramson, S. Self, P. skeletal changes: natural history in a patient with prolonged Genco, N. Smith, A. Pendleton, J.Valentine, H.H. Wright, M. survival. Cuccaro, and D.Powell. Univ. of S. Carolina Sch. Med., l.J. Anderson. S.R. Rosengren & R.M. Greenstein. Dept. of Columbia, SC and Med. Univ. of SC, Charleston, SC. Pediatrics, Univ. of Connecticut School of Medicine, Farmington. Familial hyperserotoninemia has been described recently Osteopetrosis is a rare metabolic bone disorder characterized by in about 30-40% of families with an autistic proband. Abnorm- a generalized increase in skeletal density and abnormalities of al immune function, a consistent finding in autistic probands, includes reduced lymphocyte proliferative response to mito- bone modeling. Inherited forms include the autosomal dominant gens, decreased E-rosette forming lymphocytes, and an altered benign type and the autosomal recessive congenita type. In both T /T8ratio. Some probands have absent rubella hemagglutina- forms, osteopetrosis and its complications are the sole tion inhibition antibody responses to rubella vaccine and abnormalities. In 1987, Lerman-Sagie et al. reported a new significantly reduced herpes simplex virus antibodies. syndrome of osteopetrosis associated with cerebro-oculo-facio- In the present study, whole blood serotonins(5HT) were skeletal changes in two male siblings who died by age 4 months. completed on 11 autistic probands. Three probands had 5HT We report a 23 month old girl who was similarly affected during values . 2SD above the mean, 3 had 5HT valuesA 1SD above the infancy but who has exhibited a prolonged clinical course. mean and 5 had normal mean values. Immune function studies She was born at 36 weeks to nonconsanguinous parents after a included B4, T., T4 T8, T11, the T4/T8 ratio, Mo-2, TAC, I3, pregnancy complicated by an elevated MSAFP and decreased and a series og lymphocyte activation markers. The mean fetal movement. Amniocentesis was normal. At birth she was percentage of T cell markers was significantly depressed for noted to have microcephaly, micrognathia, large, low set ears, T [X - 57+10.7, t-3.8, df-55, p-0.0002], T4[X-36t8, t-3.8, bulbous nose, multiple flexion contractures and rocker bottom feet. dr-55, p-0.002], T1, [X-76±7.7, t-3.2, df-55, p-0.001], and I3 Skeletal x-rays showed diffuse osteopetrosis. Laboratory studies [X-16±5, t-2.4, df-55, p-0.0l]. The absolute number of T cell performed at age 4 months showed calcium, phosphorus, markers was normal. and hematocrit within normal limits. At age 5 months, Correlational analysis of T and B cell subsets versus electrolytes 5HT level was completed using Pearson's correlation coefficent. she demonstrated intractable seizures and was diagnosed as Significant negative correlations were observed between 5HT having Lennox-Gastaut syndrome and cortical blindness. and the mean percentage of T3 [r - -0.62, p - 0.039] and T4 Normocytic anemia, hepatosplenomegaly and hypocalcemia [r - -0.61, p - 0.042]. The TAC marker was absent in 20% of became apparent at age 21 months. Renal ultrasound showed probands, had a value of 1 for 60% of probands, and a value of bilateral renal calculi and skeletal x-rays demonstrated marked 2 for 20%. The TAC marker was absent in probands with high osteopenia. Parathyroid hormone levels were normal. 5HT and had a value of 2 in probands with normal 5HT. Our patient is unique in that neither of the previously reported These preliminary data document a relationship between siblings exhibited metabolic abnormalities, anemia, seizure 5HT and abnormal immune function in certain autistic probands. disorder or osteopenia. This patient's course may represent the This might explain the presence of abnormal immune function in natural progression of this disorder in children who survive beyond certain probands and not others. It also emphasizes the infancy. importance of serotonin as a marker in autism. A46 Clinical Genetics (0173) 1.91 (0174) 1.207

A caxpauerized approach to X-linked Prental retardation Toluene Embryopathy Syndrome. G.L. Arnold(l) and Univ. of Colorado Health (XIZ). L. Wilkens-Haug(2). J.F. Arena and H.A. ilubs. University of Miami School of Sciences Center, Denver, CO(1) and Denver General Medicine, The Mailman Center for Child Development, Miami, FL. Hospital, Denver CO(2). Data fron the 34 syrdranes which are currently emerging fian the category of "non-specific XIM'R" were used to develop Inhalation of toluene based solvents in glue and this systen. Clinical and pedigree data, photographs of spray paint continues to be a popular form of norrmal and abnormal family members, literature references, and recreational substance abuse. Toluene vapor summaries fran both the McKusick catalogue and Birth Defects sniffing can induce reversible renal tubular E~nylopedia are entered into a Macintosh II computer system acidosis (RTA). Toluene abuse during pregnancy can through a flatbed scanner, keyboard, or modem. The primary result in significant perinatal morbidity for both advantages of the system include minimal learning time, mother and neonate. A few reports in the excellent quality of pbotographs, easy interaction between literature of prenatal toluene exposure suggest descriptive words and photographs, rapid comparison of both teratogenicity leading to dysmorphic features whole faces and specific features fran many individuals, and similar to the Fetal Alcohol Syndrome (FAS), growth direct linking of pedigree position to photographs. Informa- retardation, and microcephaly that may constitute a tion within families and between disorders can also easily be toluene embryopathy syndrome. cxs[ared. 10 women with 30 pregnancies involving chronic After the clinical information was entered, the database glue and paint sniffing were identified by history was utilized to search for syrdromes with ccOmon traits such as or drug screen. Of the 21 toluene exposed short stature or large testes. The results of these searches pregnancies which came to delivery, 3 resulted in were used to develop a series of simple branching diagrams such perinatal demise. Preterm labor occurred in 86% as shown below. These are designed to aid in reaching an and 57% delivered before 37 weeks. Newborn follow- appropriate diagnosis or differential diagnosis. This approach up was available on all 18 viable infants, and cold be expanded to provide a valuable diagnostic and teaching pediatric follow-up on 15/18. Intrauterine growth aid for many types of inherited or dysmorphic disorders. retardation was noted on 55%. 13 children were followed at one year of age or later. All were in foster care. Growth retardation and microcephaly were seen in 61.5%. Developmental delay occurred in 71.4% of children of mothers with a greater than 5 year history of abuse. Developmental delay occurred in 16.6% when the maternal history of abuse was less than 5 years. Some children showed similar facial dysmorphology and physical features similar to FAS. We plan to further discuss the physical and developmental findings on the children and the entity of fetal toluene embryopathy syndrome.

(01-75) 14.7 (0176)

Diagnostic value of ophthalmologic findings in myotonic CHOLELITHIASIS IN TWO INFANTS WITH DOWN SYNDROME. dystrophy: comparison with risks calculated by haplotype D. J. Aughton. P. Gibson. and A. Cacciarelli. analysis of closely linked RFLPs. T. Ashizawa, J.F. Departments of Pediatrics, Surgery, and Radiology, Hejtmancik, J. Liu, M.B. Perryman, H.F. Epstein and D.D. William Beaumont Hospital, Royal Oak, Michigan. Koch. Baylor College of Medicine, Houston. Cholelithiasis is often thought of as being Myotonic dystrophy (DM) is the most common adult form rare in children; however, this disorder is now of muscular dystrophy. Although one of the earliest known to occur not uncommonly in the pediatric age manifestations of DM is iridescent cortical lens opacities, group. Despite this, and despite the fact that their diagnostic specificity has been recently questioned. Down syndrome is a common and readily recognized To determine diagnostic value of lens opacities in DM, we condition, we are aware of only one report in the examined 98 at risk members of 9 DM kindreds. The risk for medical literature of cholelithiasis in a patient DM in each member of the families was calculated by with Down syndrome, an 18-year-old woman who also haplotype analysis of RFLPs using ApoC2 and CKMM, each had Hirschsprung disease [Ann Gastroenterol mapped 1 cM proximal to the DM locus, and pEFD4.2, mapped 13 Hepatol (Paris) 1988;24(l):21-22]. We report the cM distal to DM. Diagnosis of DM was defined by the presence cases of two male infants with Down syndrome who of unequivocal myotonia (by clinical examination or were found to have gallstones. electromyography) and/or risk for DM greater than 95% on Patient 1 was found prenatally to have duodenal haplotype analysis. Exclusion of DM was based on a risk for atresia, and amniocentesis showed a 47,XY,+21 DM less than 5% by haplotype analysis in the absence of karyotype. Duodenoduodenostomy was undertaken on myotonia. Diagnosis of DM was made in 33 members and the first day of life; a preoperative abdominal excluded in 51. The sensitivities of bilateral iridescent radiograph did not show unusual opacities. lens opacities, posterior cortical lens opacities, Abdominal sonograms obtained at 4 and 4-1/2 months orbicularis oculi weakness, low intraocular pressure and of age demonstrated echogenic material within the ocular myotonia were 46.7%, 50.0%, 66.7%, 57.6% and 3.0%, gallbladder and a gallstone lying within the while their specificities were 100.0%, 100.0%, 98.0%, 94.1% cystic duct. Patient 2 was found prenatally to and 100.0%, respectively. In 83.3% of DM patients, bilateral have atrioventricular canal. Stigmata of Down iridescent lens opacities, posterior cortical lens opacities syndrome were evident at birth; karyotype was or both were present. Unilateral iridescent lens opacities 47,XY,+21. Abdominal sonography on the second day occurred in only 4 (13.3%) of our DM patients and 3 (5.9%) of life revealed multiple echogenic foci within of non-DM members showed a few iridescent particles. the gallbladder, consistent with gallstones. No Posterior cortical lens opacities in DM patients always predisposition to gallstone formation (GI tract affected both eyes in this series. We conclude that 1) malformation, hemolysis, cystic fibrosis, bilateral iridescent lens opacities and posterior cortical furosemide therapy, parenteral nutrition) could be lens opacities are highly specific for DM and useful for identified. Neither patient has had signs establishing clinical disgnosis of DM, 2) unilateral referable to cholelithiasis, and management has iridescent lens opacities are infrequent in DM and are seen been conservative. in some non-DM members and 3) ocular myotonia is rare in DM, These cases suggest that cholelithiasis may be unlike myotonia congenita and paramyotonia. more common among infants with Down syndrome than has been recognized previously. Clinical Genetics A47

(0177) 1.92 (0178)

Male to male transmission in a second family supports autosomal Holoprosenoephaly in a cxcaine-exposed infant. P.I. Bader and dominant inheritance in Nager acrofacial dysostosis. A.S. Aylswort W.J. Lewis. Parkview Memorial Hospital and Indiana University and A.E. Lin. Univ. of North Carolina, Chapel Hill and West Penn School of Medicine, Ft. Wayne, Indiana. Hospital, Pittsburgh, Pa. Prenatal cocaine exposure is associated with prematurity, Nager acrofacial dysostosis is characterized by mandibulofacial growth deficiency, microcephaly, developmental delay, intra- cranial hemorrhage, nonduodenal intestinal atresia, limb re- dysostosis and radial dysplasia. The occurs phenotype usually renal aplasia sporadically but a few familial cases for both duction defects, cardiac anomalies, anomalies, provide support cutis and hemangicmata. Fetal vascular disruption autosomal recessive and autosomal dominant hypotheses. We have congenita reported one previous instance of father to son transmission and accaspanying maternal cocaine abuse may lead to cavitary describe here a second family with an affected father and two affected central nervous system lesions and genitourinary anomalies. sons. All five affected individuals have moderate expression of the Although the incidence of holoprosencephaly among live- phenotype. The proband in this second family presented as a newborn borns is one in 5000, this defect has not been associated with with vomiting and abdominal distension. He had a low anterior teratogens in man. We report here a newborn female with hairline with tufts of hair extending down each cheek, mild hypoplasia holoprosenaly who was exposed to cocaine. of the zygomatic arches, micrognathia, small external auditory canals, The patient is a newborn black female born to a 28 year old short palpebral fissures, absent lower lashes and hypoplastic, mother (G4,P3) who had been hospitalized at 3-4 weeks of ges- pedunculated thumbs. Hirschsprung disease was diagnosed and a tation because of cocaine use. Birth measurennts were 2090g colostomy performed. A younger brother, first examined at 2 months, (25%), 42 cm (10%), OFC 27.5 an (-3S.D.). Apgars were 3,8. had growth retardation, hair tufts extending down each cheek, Gestation was estimated to be 34 weeks. On physical exam, hypoplastic zygomatic arches, short, downslanting palpebral fissures there was proptosis, hypotelorism (I.C.D.=0.8an), and a mid- with virtually absent lower lashes, small pinnae, external auditory canal line nasal structure was flat without bridge or tip. There stenosis, flat philtrum, thin upper lip, thick palatine ridges with a appeared to be a single nostril, although a high hypoplastic narrow palatal groove, and severe micrognathia resulting in upper septum or membrane was seen on further exam. There was an airway obstruction. There was radial deviation of the right hand. underdeveloped maxilla, flattened nasolabial area, small nRuth Rudimentary thumbs had been removed. This child is more severely with high palate, malformed ears, redundant neck skin, hir- affected than his older brother and bears a striking resemblance to the suitism and hypotonicity. father who has short stature, mild facial asymmetry, hair tufts extending The CAT scan showed semilobar holopresencaphaly, pachygyria and agyria, absent corpus callosum. The abdominal ultrasound down the cheeks, sparse lower eyelashes, short, downslanting palpebraf fissures, hypoplastic anthelices, small external auditory canals, marked and chramoscones were normal. Cocaine screen was positive. malar and mandibular hypoplasia and ankylosis of the This is the first report of holoprosencephaly in a cocaine- temporomandibular joint for which he had surgery on two occasions exposed infant. Holoprosen aly results fran a primary dumng childhood. His thumbs were congenitally absent bilaterally. defect in the prechordal mesoderm at about 21-25 days of gestation. Since cocaine use is documented at about 3-4 Nager acrofacial dysostosis appears to be an evolving community of weeks gestation, we hypothesize that the vasocmnstrictive ef- syndromes. The association of acrofacial dysostosis with Hirschsprung fect of cocaine resulted in a vascular insult within the disease has not been described previously. There have been reports of prechordal mesoderm leading to the clinical findings of holo- advanced in some cases. paternal age sporadic This observation of prosencephaly. vertical transmission from father to son in two families supports an hypothesis that some cases are caused by dominantly expressed, autosomal mutations.

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The relationship between head circumference and height in Teratogen ingestion in mothers of infants with normal adults and in the Nevoid Pasal Cell Carcinoma aortopulmonary window. J. S. Bamforth and R. L. Syndrome (NBCC) and NF1. SJ Bale . CI Amos1. DM Parry2. AE Collins-Nakai. Division of Medical Genetics and Bale2 1Environmental Epidemiology Branch, and 'Clinical Heritage Pediatric Cardiology Program, Department Epidemiology Branch, National Cancer Institute, NIH, of Pediatrics, University of Alberta, Edmonton, Bethesda, MD. Alberta, Canada. Macrocephaly, defined as a large occiptofrontal Aortopulmonary window (APW), a rare congenital circumference (OFC), is a feature of many human malformation cardiac defect of conotruncal septation, was found syndromes. Several syndromes are characterized by both in 8 cases presenting in the first year of life to increased stature and macrocephaly, but it can be difficult the Heritage Pediatric Cardiology Program between to determine if the large OFC is a primary feature of the 1973 and 1989. There were 3 males and 5 females, syndrome or whether the absolute size of the head is normal 2 native Canadian and 5 Caucasian. There was a relative to height. We have determined the correlation significant history of the teratogen ingestion between OFC and height in normal adults and offer standards during the first trimester of pregnancy in 5/8 for the relationship between these two measurements. Data cases; 2/8 cases had fetal alcohol syndrome, 1/8 from 41 patients with NBCC and 13 patients with NF1 were had a mother who was a known untreated alcoholic, compared with these sex-specific standards. 1/8 mothers admitted to drinking in early pregnancy Analysis of the normal controls showed that OFC was and 1/8 had a mother who was taking sodium significantly correlated with height (r(males)-0.28, valproate for a chronic seizure disorder. p=O.018; r(females)-0.53, p<.0001). OFC to height ratios Associated non-cardiac anomalies were observed were approximately normal-ly distributed in each sex with a in 4/8 cases. 1/8 cases had imperforate anus, mean of 0.326 (sd-0.014) in males and 0.335 (sd=0.012) in rectovaginal fistula and bilateral radial reduction females. Relative macrocephaly (an OFC greater than the 95th defects; 1/8 cases had tracheoesophageal fistula; centile for height) was seen in seven of nine NBCC probands 1/8 cases had microcephally; 1/8 cases had and eight of 32 non-probands with NBCC. Three of four NF1 Beckwith-Wiedeman syndrome. Associated cardiac probands and six of nine non-probands with NF1 had anomalies included patent ductus arteriosus in 5/8, significantly large heads for their height. We conclude that interrupted aortic arch in 2/8, ventricular septal head size appears to be related to proband status in NBCC, defect in 4/8, secundum atrial septal defect in i.e. that there is an ascertainment bias which causes 1/8, pulmonary atresia in 1/8. persons with NBCC who have large heads to be more likely to Although a true association between APW and become probands. However, the evidence suggests that NF1 is teratogen ingestion could only be confirmed by a a true macrocephaly syndrome. Objective detection of case-controlled study involving more patients, and relative abnormalities in head size may aid in syndrome therefore, a multicentre study, the co-existence of delineation and diagnosis. a very rare congenital cardiac anomaly with a significant history of first trimester ingestion of known teratogenic substances (particularly ethanol) suggests a possible role in etiology, as has been demonstrated for ethanol and conotruncal septation defects in mice. A48 Clinical Genetics (0181) 1.94 (0182) 1.95

Familial Inclusion Body Myositis Evidence for Autosomal A feline model for autosomal dominant adult polycystic kidney Dominant Inheritance. L.L. 3aumbach (1), H.E. Neville (1), disease. D.S. Billerl and S.M.V. Pflueger2. 'School of S.P. Ringel (1), C. Garcia (2), and E. Sujansky (1). Univ. Veterinary Medicine, Univ. of Wisconsin, Madison, and 2Tufts Colo. Sch. Med., Denver (1); Louis. State Univ., New Orle.(2). University School of Medicine and Baystate Medical Center, We report the first familial case of inclusion body myos- Springfield, MA. itis (IBM). The disease, a slowly progressive inflammatory Autosomal dominant polycystic kidney disease is a myopathy, is characterized by weakness of proximal extremit- relatively common syndrome in man, but until now there have ies, diminished deep tendon reflexes, dysphagia, and mixed been no good animal models. We present a kindred of cats with myopathic and neurogenic changes on electromyography. All a syndrome which closely mimics the human condition in renal previously reported cases have been sporadic with a 3:1 male: morphology, progression of the disease, and mode of female ratio. The reported family was identified through the inheritance. Kidneys of affected kittens exhibit multiple affected male proband. Family history, clinical, and labora- microscopic cystic lesions lined by columnar epithelium and tory investigations revealed varying degrees of IBM in an focal areas of tubular dilatation on routine histology. The additional five members from two generations of this kindred. condition progresses with age to gross renal cysts with All five affected males had significant clinical findings with replacement of the normal parenchyma and ultimately leads to age of onset at age 20 - 30. The only affected female was renal failure. Ultrasound has proven useful in non-invasive clinically assymptomatic, but showed mild changes consistent identification of affected cats. The proband was a male with IBM on muscle biopsy. The presence of IBM in two gener- Persian who presented with hematuria and irregular renal ations with direct male-to-male transmission and lack of con- enlargement. The cat was subsequently identified by excretory sanguinity is compatible with an autosomal dominant mode of urography and ultrasound as having polycystic kidneys with inheritance. The finding of six affected individuals out of multiple 0.5 to 1 cm. cysts replacing the majority of the ten at-risk family members is also compatible with autosomal renal parenchyma. Ultrasonographic evaluation of the dominant inheritance with full penetrance. While the high proband's offspring revealed additional affected individuals, male to female ratio of affected individuals may explain the from which a line has been established. The renal cystic overall higher incidence of males in this kindered, the mild disease has now been followed through four generations as an symptoms in the only affected female raise the question of the autosomal dominant trait. possible influence of sex upon disease severity, resulting in subclinical expression in females. Evaluation of additional at-risk females may clarify this issue. The identification of this family suggests the importance of genetic evaluation of sporadic cases of IBM, including clinical and laboratory evaluation of "unaffected" relatives, in order to detect mildly affected and subclinical cases. DNA linkage analysis is underway with the ultimate goal of identifying the genetic mechanism(s) responsible for this disease.

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Resemblance and disparity between multiple Y syndrome and FAMILIAL ALZHEIMER'S DISEASE IN GERMANS FROM RUSSIA: poly X-Y syndrome. N. Bingol, C. Hahn, L. Miraz, N. Quintela, A CLINICAL, PATHOLOGICAL AND GENETIC STUDY and A. Boafo. Lincoln Hospital, New York Medical College, New York. T.D. Bird, G. Schellenberg, E. Nemens, E. Wijsman, D. Karyotypes that comprise more than two Y chromosomes are Moore, D. Nochlin and S. M. Sumi, University of very rare. Recently, we studied a 22 year old phenotypical Washington, VA Medical Center, Seattle. male with four Y chromosomes. Clinically, this mentally retarded propositus resembled the poly X-Y syndrome, but his We have ascertained 18 families with proven or probable peripheral blood chromosome indicated a 49,XYYYY karyotype. Alzheimer's disease that are all members of the ethnic There were several similarities with the poly X syndrome: group referred to as Germans from Russia. 12 families are Craniofacially the patient had medial epicanthol folds, hyper- from 4 Volga German villages and 6 families are from 6 telorism, low nasal bridge, mandibular prognatism and large Black Sea villages. There have been 124 known or probably low set ears. There was limited pronation-supination at the demented individuals with male to female ratio of 1.3 to 1 elbows, (ns). 7 families had 2-3 demented persons, 7 had 4-10 and clinodactyly of the fifth digits, genu valgum. The 4 had 11-20. Mean age of onset was 60.5 + 9.3 years and patient had inadequate virilization, hypogonadism, azoospermia mean disease duration was 9.5 + 4.4 years. Complete range and infertility. His facial and axillary hair were sparse in age of onset was 40-84 years. Mean age of onset in the and pubic hair was of the female pattern. Developmental Volga Germans (59.3 + 9.4 yrs) was significantly earlier milestones were delayed, moderately severe mental retardation than the mean age of onset in the Black Sea families (66.7 was present. ± 6.0 yrs; p<.001). Mean age of onset and death in males There were also distinct differences between the poly X-Y (58.9 and 67.8 yrs) were significantly earlier than in syndrome and the propositus: The patient had normal stature females (62.6 and 70.9 yrs; p<.01) with no difference in (174.5 cm), and head circumference (56 cm). He had nodular disease duration between sexes (9.7 yrs vs 9.4 yrs). Of cystic acne, penis of normal length, normally developed and 71 individuals with detailed medical records, 79% had pigmented scrotum and normal testicular volume (4.7x3.5 cm). language abnormalities, 20% seizures, 15% tremor and 12% Patient showed behavior problems, aggressive outburst, low myoclonus. There were 17 autopsies in 8 families showing frustration threshold, destructiveness, and impulsivity which classic signs of AD including 0 A4 amyloid plaques, are more characteristic of the poly Y syndrome. neurofibrillary tangles and amyloid angiopathy. Prion Endocrine workup revealed low serum testosterone(T), and antibody stains were negative. Linkage analysis of low free T, low dihydroepiandrosterone(DHEA), and DHEA-S markers from chromosome 21 including D21S16, D21S13, levels, and high normal FSH and LH levels indicating, partial D21Sl/Sll, D21S82 and APP showed no evidence for linkage gonadal failure. In 47,XYY individuals who manifest aggres- to any chromosome 21 marker and excluded the region from sive and destructive behavior, a high level of serum T was D21S82 to D21S16. These families represent clinically and reported by some investigators. In poly Y syndrome the behavior pathologically typical familial AD whose gene locus is problems were likely to lie on some autosome other than 21. There is present but not associated with the increased clinical evidence (onset for serum T levels. The androgen mediated events of sexual age) possible heterogeneity maturation at the between the Volga and Black Sea families and the time of puberty is known to be mediated by occurrence of "sporadic" AD is a potentially confounding the receptor-dihydrotestosterone complexes. Further endocrine variable. studies are progressing to indicate the exact nature of this patient's problems. This is third case of quadruple Y syndrome, and the only adult man reported in the literature. Clinical Genetics A49 (0185) 1.98 (0186) 3.4

CLOUSTON SYNDROME: A rare autosomal dominant trait with palmo- DNA studies in sisters with classic Turner Syndrome (45,X). plantar hyperkeratosis and alopecia. DAVID BIXLER and RAJ- R.D. Blackston and K.B. Armfield. Emory Univ. School of Med. RAJENDRA A. PATEL. Department of Oral Facial Genetics, Indiana Dept. Pediatrics, Div. Medical Genetics, Atlanta, GA. University Medical Center, Indianapolis, IN 46202. The occurrence of classic Turner Syndrome 45,X in sisters The ancestors of this large kindred came to Indiana from is quite rare (approximately 1:10-8). DNA studies to deter- the British Isles (UK) about 100 years ago. Three members mine parental origin are of importance to explore any possible in generation II and five in generation III are reported to predisposition to sex chromosome loss and to determine if the have exhibited alopecia, dysplastic nails, and hyperkeratosis parental origin of the single X affects the phenotype. of palmar and plantar surfaces. This type of ectodermal dys- We report sisters who have 45,X karyotypes and zero Barr plasia, Clouston syndrome, features normal teeth with severe bodies from right and left buccal smears. Their mother is 46, hair and nail dysplasia. Proband, (III-16), who is 16 years XX, has normal percentages of Barr bodies, has had no abortions old has severely dysplastic fingernails and toenails and gener- and was in her early twenties for both pregnancies. Father was alized alopecia. She has no eyelashes or eyebrows and wears not available for study. a wig. The skin of the hands and feet showed a palmar and The older sister (A.L.) has a typical Turner phenotype: plantar hyperkeratosis best described as rugose and papill- short stature, ptosis, epicanthal folds, blue sclerae, low set matous. She has normal sweating pattern and normal teeth. ears, triangular facies with high palate and small mandible, Proband's father, (II-7) was born bald with a generalized short neck with redundant skin and posterior hairline, congen- alopecia. He has no fingernails or toenails. Remaining family ital heart disease, hypoplastic nipples, pectus excavatum and members, the proband's paternal uncle (II-4) and his children congenital lymphedema. She is hyperactive, has difficulties (III-10, III-11), a paternal aunt (II-1) and her children (III-2 with attention span, and shows developmental scatter consis- and III-3) were all reported to show full spectrum of anomalies tent with a specific learning disability profile. Renal ultra- seen in Clouston syndrome. Autosomal dominant pattern of sound shows horseshoe kidney. inheritance is established in this family. The younger sister (J.L.) also has physical findings con- Dental defects are considered by some authors to be a feature sistent with Turner phenotype. However, she has normal of Clouston syndrome. In this family no one has been reported developmental skills and is described as being "quicker" than to have congenitally missing or abnormally-shaped teeth. How- her older sister. ever, individuals II-1, II-4, III-2 and III-3 are edentulous Analysis of X-linked RFLPs indicates that A.L. has a single, and wear complete dentures; they lost their teeth due to severe paternally derived X chromosome (xpat), while J.L. is Xmat. dental decay. III-10, III- III-16 all have their natural Previous studies of spontaneous abortions have suggested pheno- teeth. , j 5 l v typic differences between 45,xpat and 45,Xmat fetuses, and it is of interest to note that our xpat patient was more severely affected from developmental, academic and attending skills point of view. Future DNA studies will be important in deter- mining if the parental origin of the single X chromosome has an effect on the academic performance of girls with Turner Syndrome. We gratefully acknowledge Terry Hassold's laboratory for III~s O (t 4 b o conducting the DNA studies. 3 ~~~~10 11 15 *16

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Population and pedigree studies rule out a widespread association between PREDICTIVE TESTING FOR WILSON'S DISEASE WITH TIGHTLY LINKED the dopamine D2 receptor gene and alcoholism. A.M. Bolos. M. Dean*, AND FLANKING DNA MARKERS. A.M. Bowcockl, L.A. Farrer2'3, J.M. G.L Brown and D. Goldman. National Institute on Alcohol Abuse and Hebert4, B.Bonne-Tamir5, I. Sternliebu, M. Giagheddu/, P. St. George Hyslpj, M. Frydman , R. Leez, A.E. Baled, L.L. Cavalli Alcoholism, Bethesda, MD, *Program Resources, Inc., National Cancer -Sforza4. UT Southwestern Medical Center, Dallas, 772ston Institute, Frederick, MD. Univ., MA,3 Harvard Medical School, Boston, MA, 4Stanford Univ. In a population study, Blum et al (JAMA 263: 2055, 1990) typed 35 Stanford, CA, 5Tet Aviv Univ., Israel, Albert Einstein College of Medicine, NY, Universita di Cagliari, Italy, 8Yale Univ., deceased alcoholics and an equal number of controls and found that the New Haven, CT. D2/TaqI allele ("Al") was present in 69% of alcoholics and in 20% of Wilson's Disease (WD) is an inherited disturbance of copper controls. This strong association was unexpected considering the observed metabolism caused by a defective gene on chromosome 13 which clinical genetic heterogeneity in alcoholism and the role of the environment results in toxic accumulation of copper in the liver and brain. in the expression in a We studied five new chromosome 13 markers at 13ql4-q21 in 52 ofalcoholism large fraction of alcoholics. WD families from Europe, North America and the Middle East. We evaluated the D2fTaqI polymorphism and a second polymorphism DNA analyses were performed on samples from 114 affected and generated by polymerase chain reaction of the 3' noncoding region of the 381 unaffected individuals. There was significant evidence D2 gene followed by single-stranded conformation polymorphism (PCR- for linkage between the WD locus and all of the marker loci. in 38 Caucasian 108 Multilocus linkage analysis with a genetic linkage map that we SSCP) analysis living alcoholics, racially-matched had established from reference pedigrees (cen-RB1-.021-D13S25- controls and two Caucasian pedigrees. Two blinded raters diagnosed .015-D13S31- . 016-D13S59-. 045-D13S55-.018-D13S61- .047-D13S26) alcoholism and other psychopathologies using the Research Diagnostic suggested that the WD locus is most likely between D13S31 and Criteria (RDC) and data from a structured clinical interview, medical records D13S59 at distances of 0.4cM and 1.2cM respectively. Our and family informant histories. Patients were subtyped as to severity, age of results suggest that the chromosomal location of the WD gene is the same in all families in the populations studied. onset and psychiatric conorbidity. Evidence that the genetic locus for WD is the same in all No significant difference in D2/Taql or PCR-SSCP allele frequencies families and the availability of many close, flanking and poly- was observed between alcoholics, subpopulations of alcoholics or the morphic DNA markers make possible accurate and informative tes- control population. Except for demonstration of a rare 13 kb polymorphic ting of potential carriers and WD homozygotes in families with at least one previously affected child. An advantage of a gen- fragment, D2JTaql allele frequencies were in good agreement with Grandy, etic linkage test over other laboratory methods for prediction et al (Am J Hum Genet 45:778, 1989), but not Blum et al. Allele of genotype in WD is that a reliable diagnosis can be made at frequencies in the three studies were: (1) alcoholics: 0.21, control a much earlier stage in life including prenatally. In addition, population: 0.22 (present study), (2) control population: 0.24 (Grandy, et DNA testing can be used in place of invasive liver biopsy to confirm a diagnosis in patients with borderline serum cerulopl- al) and (3) controls: 0.13, alcoholics: 0.37 (Blum et al). The D2 TaqI RFLP asmin levels. Presymptomatic identification will also allow and alcoholism also appeared to be segregating randomly in two Caucasian therapeutic intervention to prevent symptoms before irreparable families with an alcoholic "Al" parent and several recombinants. Our liver of neurologic damage occurs. Prenatal and preclinical findings would appear to rule out a widespread association between the D2 diagnosis of WD in two families has been performed and will be receptor gene and alcoholism. presented. A50 Clinical Genetics (0189) (0190) 14.2

Hutchinson-Gilford Progeria Syndrome: Clinical, Chromosomal and Weismann-Netter syidrcme in childhood. *CBre. *M.Oliphant. Metabolic Abnormalities. W.T. Brown. J. Abdenur. P. Goonewardena. *L.Thms. +M.Prbinow. °A.Barjier. *SOJNY HSC, Syracuse, R. Alemzadeh. M. Smith S. Friedman. C. Cervantes. S. Bandyopadhyay NY, -Cildren's Med Ctr, Dayton, OH, oRoyal Children's A. Zaslav S. Kuaorn. A. Serotkin. F. Lifshi Depto Hosp, Melbourne, Aust. Pediatrics, North Shore University Hospital-Cornell University Weismann-Netter syrdrine (W-N) is a rare heritable dysplasia Medical College, Manhasset, New York. of asymptciatic anterior bowing of the tibiae and fibulae, The Hutchinson-Gilford Progeria Syndrome (Progeria) clinical diaphyseal bowing of other lonr bones, short stature, sooliosis features that resemble extreme aging in childhood. The condition is and mild mental retardation. Radiographically the bowing is rare with a reported incidence of 1 in 8 million. A lack of acouianied by cortical hyperostosis on the concave side of the consanguinity, a lack of affected sibs and a paternal age effect, curvature. Bone biopsies have been normal; the etiology is argue it is most likely a sporatic dominant mutation. We have unknown. Although W-N syndrae is believed to be a cornenital established an International Progeria Registry and have examined 30 skeletal dysplasia of AD inheritance, sost patients have been cases. We will present a clinical summary and diagnostic criteria to elderly adults seeking medical attention for other problems. assist with clinical identification. We encountered three unrelated children with this disorder. The underlying genetic defect is unknown. Previously we have X-rays of each revealed the characteristic anterior bowing of reported that urinary levels of hyaluronic acid are elevated in the tibiae and fibulae. TNo had lateral bowing of the femora; Progeria. We have employed a new method of testing for hyaluronic gracile radii, ulnae and fibulae; and slightly bowed ulnae. acid (HA) using a radiometric assay (HA TEST 50, Pharmacia) and Case 1: A thin 11 y.o. white male was evaluated for short confirmed the elevation of HA. A study of 10 Progeria subjects showed stature, multiple well healed fractures, delayed dentition, the average molecular weight of hyaluronic acid was significantly cleft palate and mental retardation. Bowed legs were first higher than controls. noted at 8 years. He walked at 4 years. Case 2: A thin 8 y.o. Two identical twins with Progeria at age 8 became ill due to white male (Am J Med Genet 29: 573, 1988) had short stature, cardiac failure and died within month of each other. A postmortem mild mental retardation and late eruption of secordary teeth. skin biopsy was obtained from the older child. Cytogenetic analysis Bowing was first noted in his 2nd year. He walked at 18 mos. revealed an inverted insertion of chromosome 1 described as 46 XY, Alk. phos. was elevated. A left femur fracture healed inv ins (1;1) (q32;q44q23) in 70% of cells. normally. Case 3: A short, very thin 11 y.o. white female had Endocrine studies in a 4 year old boy with Progeria revealed normal intelligence, a VSD, and delayed permanent tooth severe malnutrition and a high Bal Metabolism (BM) of 99 Kcal/kg eruption. Striking anterior bowing of her legs and right (nl 47) with normal thyroid function. Overnight Growth Hormone forearm was noted by age 2. Six long bone fractures produced by levels (OGH) were high (Mean 8 and 10 ng/ml) while Insulin Growth significant trauma healed normally. Alk.phos. was elevated. Factor-l (IGF-1) was low (0.1 - 0.2 uU/l). Growth Hormone (GH) Skin fibrnblast collagen analysis revealed no abnormality in molecule assessed by chromathography was found to be normal. Therapy the structure and biosynthesis of collagen types I and III. with GH was initiated (0.06 mg/kg TIW). IGF-1 levels increased up to Fran these young patients with W-N syndrme we can begin to 1.8 uU/ml. After 4 months of treatment his growth velocity improved characterize the disorder in the pediatric age group. Patients from 2 to 9.8 cm/year and his BM dropped significantly (69). A are short and thin, have marked painless anterior bowing of the second 5 1/2 year old girl with Progeria also showed severe legs usually evident in the first few years of life, slender malnutrition and a high BM. OGH levels were high and IGF-1 was low, long bones, and mild bowing of the femora and ulnae. They may confirming a similar pattern of apparent bioinactive growth hormone. walk late, have scoliosis, mental iipairnent, delayed secordary Treatment with GH is being started in this subject also. These dentition, fractures which heal normally and elevated alk. results suggest that a gene may be located on chromosome 1 and GH phos. Associated findings may include cleft palate and VSD. treatment may offer an effective therapy in Progeria.

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A reassesanent of Beckwith-Wiedemann syrdrae (WS). M.E. Pigmentary retinopathy, sensorineural deafness, mental Carlin, L.F. Esobar, R.E. Ward and T.Y. !!gel S. Univ. of retardation, obesity, hypogonadism, and acanthosis nigricans: T1i-17MaiMaTCter for Child DevelopiienE, Miami, FL; South A new syndrome in the differential of Usher syndrome. Bend Metrial Hospital, South Bend, IN; and Indiana Univ. S.B.Cassidy, K.Byrne-Essif, and H.E.Cross. Univ. of Arizona School of Dentistry, Irdianapolis. College of Medicine, Tucson. Our study of 53 children with BWS reveals a more optimistic Pigmentary retinopathies and progressive hearing loss natural history than that gleaned fran the literature, where occur together in several single gene disorders. We have seen most cases have severe manifestations. Additionally, none of a young adult woman with an apparently distinct disorder the reported Up abnormalities were seen in our 31 patients which includes pigmentary retinopathy with congenital karyotyped. Although we used the same diagnostic features as nystagmus and progressive visual impairment beginning at previous authors, many of our patients were more mildly birth, childhood-onset slowly progressive sensorineural affected, and pheotypic features improved over time. Consis- hearing loss, psychomotor retardation with current function- tent across all ethnic groups, the most cammon clinical find- ing in the borderline range of mental retardation, scoliosis, ings were: macroglossia (92%), ear creases/pits (80%), nevus moderate obesity since the first decade, acanthosis nigricans, flammeas (77%), umbilical defects (75%), vis egaly (70%) and oligomenorrhea. Retinal evaluation shows pigment plaques and macrosamia (68%). HemihYpertrowhy/growth asymaetry was but not clumping, and mottling ofd the pigment epithelium. noted in 43%. Hypoglycemia, occurring in 55%, usually resolved Photophobia is absent, as is night blindness. Family history or sam quickly. Prolonged hypoglycemia perinatal distress is negative, and there is no consanguinity. ocpourded each of the 15 cases (28%) with significant delays/ These findings are not entirely consistent with the previ- retardation. The spectriz of abdmninal wall defects included: ously well described conditions which include pigmentary distasis recti, umbilical defect/hernia, aiphalocele a and retinopathy and progressive hearing loss, including Usher, prune belly-like defect. Others had an excessively protuberant Bardet-Biedel, Laurence-Moon, Refsum, and Alstrom syndromes, abdanen with decreased muscle tone and/or thickness. Polyhy- either because of age of onset of the two problems, their dramnios, stings with ascites, seen not infrequently in progression, or the associated findings. Literature review may affected fetuses, oontribste to these ventral wall find- revealed a previous case report (Edwards et al, 1976) of two ings. Six (11%) have developed tumors: 2 wilm's tumors in sibs brothers and a sister with simlar ophthalmological symptoms and 4 benign growths. The linkage of Bws to the insulin and and retinal findings, childhood-onset slowly progressive related growth factor genes at llpl5.5 likely helps account for deafness, and obesity. The males had hypogonadism and mental growth, characteristic of the excessive and irregular BWS. retardation; one had diabetes and acanthosis nigricans while BWS is reported to be familial in 15% of cases. In 11 (21%) the other had hyperinsulinism. The female had oligomenorrhea. of our more 2 sets families, than 1 child had BWS, including This apparently autosomal recessive disorder is very of twins (1 MZ; 1 DZ). Additionally, 24 families had one, or sRnetMines both parents, and/or other relatives affected with 1 similar to that in our isolated case, which probably repre- sents the second family with this condition. The association or sore signs of BWS. In most cases, this was and/or macrosamia of progressive visual and hearing impairments in a number of growth asyamtry/hwnhypertrophy; this latter sign may be an recessive conditions, at least one of which is metabolic urderappreciated diagnstic clue for BHB. These findings were (Ref sum syndrome) implies similar but distinct etiologic detected in both aid fathers, in both maternal mothers and and mechanisms. paternal relatives, speaking against current theories of sex mediated penetranos or imprinting. Our pedigrees support AD in- heritance with incomplete penetrance and wide variability, but are also ocnpatible with a polygenic/multifactorial pattern. Clinical Genetics A51 (0193) 3.1 (0194) 14.1

NOUSE, HUMAN BOMOLOGOUS LOCI AND CHRCOSOKE AREAS INVOLVED IN The Weissenbacher-Zweymuller syndrome: a unique syndrome of IMPRINTING. B. Cattanacht. J.H. Edwards2. J.G' Hall3. 1MRC delayed osseous maturation. J. Chemke1, R. Carmi2, Radiogiology Unit, Chilton, Didcot, Oxon OX1l ORD, England, J. Bar-Ziv3. Kaplan Hospital, Rehovot1; Soroka Medical 2Genetics Laboratory, Department of Biochemistry, University Center, Beer-Sheva2, and Hadassah University Hospital Ein of Oxford, Oxfor OXi 3QU, England, 3oepartment of Medical Kerem, Jerusalem3, Israel. Genetics, University of British Columbia, Vancouver, B.C., Canada V6H 3N1. The Weissenbacher-Zweymuller syndrome (WZ) was first The concept of imprinting has attracted a great deal of described in 1964 as "Pierre-Robin with skeletal dysplasia" attention in the last few years. Parent of origin differences and a characteristic rhizomelic dwarfism, metaphyseal and phenotypic expression are being noted in many experimental widening of the long bones and vertebral coronal clefts. and naturally recurring systems. The areas of uniparental There has been confusion in the delineation of this condition disomy identified by translocations in mouse suggest that the since until now it is believed to be a neonatal expression of homologous areas in humans may be involved in imprinting the Stickler syndrome, which also presents with myopia and phenomenon as well. retinal detachment, a progressive metaphyseal dysplasia and This poster will present a current compilation of mouse and is inherited as an autosomal dominant trait. human loci which are thought to be involved in imprinting We present 5 patients in 3 families with the WZ syndrome, phenomenon. It will also suggest other loci in those areas none of whom have symptoms compatible with the Stickler which need to be examined for possible imprinting effects. syndrome. A critical analysis of 14 published patients with "WZ" syndrome shows that in only 7 patients is this diagnosis certain, while the other 7 are probably affected with either Stickler syndrome, dyssegmental dysplasia, OSMED or Kniest dysplasia. Among the features present exlusively in WZ syndrome and absent in the Stickler syndrome, are markedly decreased body length at birth with short limbs, catch-up growth after 2-3 years, lack of progressive deformity, and autosomal recessive inheritance (affected siblings with phenotypic normal parents). We propose that the WZ syndrome is a distinctive syndrome of delayed skeletal maturation, inherited as an autosomal recessive trait. The recognition of its unique characteristics has important implications for genetic counseling and the prognostic evaluation of these patients.

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A syndrome of Mental Retardation, Facial Anomalies MARINESCO-SJOGREN SYNDROME ASSOCIATED WITH DECREASED and Distal Arthrogryposis in sibs. Chen, M-F. GROWTH HORMONE LEVELS. C.L.Christian. R.A.Nagel and Chitayat. D.2 Hodgkinson. K.A.2 Blaichman. S.. J.M.GrahamJr. Medical Genetics and Birth Defects Watters. G.V. Department of Pathology, Royal Center, Cedars-Sinai Medical Center, Los Angeles,CA. of Marinesco-Sjogren Syndrome (MSS) is an autosomal Victoria Hospital , Department Paediatrics, recessive disorder characterized by cerebellar Division of Medical Genetics2, Division of atrophy, hypotonia, nystagmus, cataracts and short Neurology3, The Montreal Children's Hospital and the stature. The etiology of this disorder has not been Centre for Human Genetics2, McGill University, determined. We report the case of a 4 year old Montreal, Quebec, Canada. Japanese-American female with nonconsanguineous Distal arthrogryposis (DA) was delineated by parents who was diagnosed as having MSS and growth Hall in 1982 as a subcategory of arthrogryposis hormone deficiency. Hypotonia had been noted from multiplex congenita (AMC), and is characterized by early infancy along with saccadic eye movements and congenital contractures of the hands and feet. Two nystagmus. An MRI of the brain revealed an abnormal A was groups are defined, type I with isolated distal limb cerebellar cortex. muscle biopsy consistent involvement, and type II with additional with a myopathy. An electron microscopy study of a skin biopsy was normal as were high resolution characteristic manifestations. This latter group chromosome analysis and carnitine levels. The child is subdivided into 5, (A-E) depending on the was developmentally delayed, especially in gross different organ systems involved. All reported motor skills, and was unable to independently familial cases of type II followed an autosomal ambulate. No specific diagnosis was given at this dominant mode of inheritance. time. The child was also followed for decreased We present 2 sisters with a malformation growth parameters. A clonidine stimulation test for syndrome consisting of characteristic facial growth hormone (GH) was borderline. A formal anomalies and DA, the association of which insulin/arginine stimulation test was performed that a hormone deficiency. The categorises them as type II. The oldest child is showed partial growth peak insulin response was a GH level of 10.8 ng/ml 4 years old and is severely mentally retarded. The and the peak arginine response was 7.0 ng/ml. A youngest child died shortly after birth of trial of GH repacement was initiated based on these respiratory failure, but with similar features to findings and the patient's poor growth velocity. her older sister. During this latter pregnancy, Cataracts were first noted 3 weeks into the GH fetal ultrasound at 28 weeks gestation had revealed treatment. The cataracts rapidly increased in consistently clenched fists indicative of distal density going from 10% to 100% opacity in 2 weeks. limb contractures. The GH was stopped after 6 weeks of treatment over The etiology of this syndrome is unknown, but concern of the cataracts, but a review did not use of GH and central nervous support an association between the is probably an abnormality of the cataract formation. The addition of the cataracts system. The occurrence of this potentially lethal to the child's other findings led to the clinical condition in 2 sibs with an unaffected mother diagnosis of MSS. The cataracts have been removed suggests autosomal recessive inheritance. and GH replacement has been restarted. A review of the literature found one case report of an individual affected with MSS who had normal GH testing at 17 years of age. A52 Clinical Genetics (0197) 1.103 (0198) 1.104

Hunter disease (MPS, type II) in a karyotypically normal girl associated with KABaUI MAKE-UP aYNDRNm Izs THRE CAUCASIAN CHILDREE. LA selective X-chromosome inactivation. J.T.R. Clarke. R.D. Pearce. W.L. Clarke. JG Hall. Department of Medical Genetics, University Greer. P. Stasberg. P.N. Ray. Div. of Clinical Genetics, Hospital for Sick of British Columbia, Vancouver, B.C. V6H 3N1 Canada. Children and University ofToronto, Toronto, Canada. The Kabuki make-up (Niikawa-Kuroki) syndrome has been A developmentally delayed female child with macrocephaly, dysmorphic extensively delineated in the Japanese population. facies, hypotonia, hepatosplenomegaly, generalized dysostosis multiplex, and Manifestations include characteristic facial features mucopolysacchariduria was shown to have profound iduronate sulfatase (IDS) consisting of hypertelorism with wide palpebral fissures, activity in serum and fibroblasts. Analyses of IDS activities in heterokaryons eversion of the lower half of the eye lid, sparse lateral formed by PEG-induced fusion ofpatient fibroblasts with other mutant cells third of the eye brow, prominent ears and elevated finger showed complementation with multiple sulfatase deficiency (MSD), but no pads. In addition, patients usually have moderate mental complementation with classical MPS II. Serum and fibroblast IDS activities in retardation, post natal growth deficiency, and occasionally the parents were in the normal range. While the distribution of maternal hair congenital heart disease. root IDS activities suggested the mother of the patient might be a carrier of We describe here the clinical features of three unrelated MPS II, no IDSO clones were found among a total of 9 maternal fibroblast Caucasian individuals with Kabuki make-up syndrome. Important clones isolated and analyzed. Methylation-sensitive RFLP analysis ofDNA phenotypic features include: notched eye brows, wide from peripheral blood lymphocytes using MspI/Hpall digestion and probing palpebral fissures, iversion of the lower palpebral fissure, with M27B, showed that the paternal allele was completely resistant to HpaII mild developmental delay, dermatoglyphic anomalies with loss digestion (i.e. hypermethylated) while the maternal allele was completely dig- of "d" triradius and prominence of the finger tip pads. ested (i.e. hypomethylated) indicating nonrandom X chromosome inactiva- Although Kabuki make-up syndrome has been reported outside tion. Somatic cell hybrid clones were produced by fusion ofpatient fibroblasts of Japan we feel that Kabuki make-up syndrome remains under with hprt0 hamster fibroblasts (RJK88, generously provided by R. Fenwick, diagnosed in the non Japanese population and our patients help Houston) and grown in HAT-ouabain selective medium to select for hybrids to illustrate the phenotype in Caucasians. These patients containing at least one active human X chromosome. DNA from hybrid clones highlite the difficulty in syndrome delineation when varying was digested with TaqI and probed with St14 to identify the source of the X racial groups are considered. chromosome in each case. A total of 11 clones contained both maternal and paternal X chromosomes and 16 contained only the paternal X chromosome. No clone was isolated containing only the maternal X chromosome. Methyl- ation-sensitive RFLP analysis ofDNA from patient-hamster hybrids containing a single human X chromosome showed that ofthe 11 clones that retained the DXS255 locus, all contained the paternal allele (paternal X active) and it was hypermethylated (Hpall-resistant) in all cases. Similar studies on DNA from maternal fibroblasts, leukocytes, or lymphoblasts, using BglIl/HpalI digestion and probing with PGK-1, showed random distribution of hypermethylation of the grandpaternal and grandmaternal loci. These studies suggest that the patient has MPS II as a result ofa mutation, possibly a submicroscopic dele- tion, resulting in disruption of the IDS locus on her paternal X chromosome as well as selective inactivation ofthe non-mutant maternal X chromosome.

(0199) 14.10 (0200) 1.105 The Dopamine D2 Receptor Gene is a Modifier of the Myocardial infarction leading to sudden death in th? Williams Expression of the Tourette Syndrome (Gts) and ADHD Gene syndrome: Report of three cases. E.E. Conway, Jr. Coming&, D-E-. MUhleman. D.. Dietz. G.- Shahbahrarni. B., Tast. D., J. Noonan, R.W. Marion, C.N. Steeg. 1- The Departments Kovacs, B.W. Dept of Medical Genetics, City of Hope Medical Center, of Pediatrics, Albert Einstein Col of Med, Bronx, N.Y. and Duarte, CA 91010. Univ of Kentucky School of Med, Louisville, Kentucky. Blumet all reported a significant increase in the frequency ofthe 1 allele The Williams syndrome (WS) is a disorder combining ofthe TaqI polymorphism ofthe dopamine D2receptor gene2 in patients with developmental abnormalities, characteristic craniofacial severe, treatment resistant alcoholism (69% vs 20% in controls). In a study dysmorphic features, and cardiovascular anomalies, including of 2,392 individuals we observed the presence of severe problems with supravalvular aortic stenosis (SVAS), supravalvular and alcoholism and/or drug abuse in 14.4% ofrelatives of TS probands vs 4.4% valvular pulmonic stenosis, and stenosis of the pulmonary, of relatives ofcontrols (P<0.0005) implicating the Gts gene as a major gene renal and coronary arteries. The vast majority of patients in the susceptibility to alcoholism3,4. We have also noted a significant with WS survive to adulthood, but, because of the associated in abnormalities of the cardiovascular system, the potential increase the frequency of attention deficit hyperactivity disorder (ADHD) for premature death does exist. We report three children, in TS probands and their relatives3.4. To determine if the D2 receptor gene ages 8, 16, and 59 months, who had features of WS and who was either the Gis gene, or a modifier of the Gis gene, the frequency ofthe 1 died suddenly with myocardial ischemia following cardiac allele was examined with the following results: catheterization. Autopsies revealed that each patient had Diagnosis N % 1 p SVAS with dysplasia of the aortic valve cusps, left vent- Controls 114 22.8 ricular hypertrophy, stenosis of the left coronary artery Tourette syndrome 83 43.3 9.4 <.005 and its branches, and regions of recent and/or remote Severe TS 29 55.2 11.6 <.0005 myocardial infarction. Two of the patients also had TS 10 Relatives 85 49.4 15.3 <0005 evidence of focal ectopic calcification. Review of the TotalTS+ 10rel 168 46.4 16.2 <.0005 literature revealed two previous cases of myocardial ADHD only 25 72.0 22.9 <.00001 infarction and sudden death following cardiac catheter- While segregation of the 1 allele indicated the D2 receptor gene clearly ization in children with this disorder. From our was not the Gt gene, there was a significantly increased frequency of the 1 experience, it appears that some patients with WS develop allele in TS probands, Gts gene carriers, and individuals with ADHD only. severe coronary artery disease that may result in The more severely affected individuals were often the ones positive for the 1 myocardial infarction and sudden death, and that in such allele. These results are consistent with the Gts gene, acting on serotonin4, as patients, cardiac catheterization carries an increased risk. the major gene in the production of TS, ADHD and alcoholism and the D2 Since none of our patients had any findings on pre- receptor gene acting as a modifier to increase the severity and expression of cathetcization electrocardiogram suggestive of myocardial these disorders. This is thefirst identification ofa gene defect in TS and ischemia or infarction, identification of at-risk patients ADHD and is consistent with the dopamine-serotonin axis in which agents may be difficult. enhancing dopamine action or suppressing serotonin action have a similar andadditive effect on behavior4. I.Blum, K. et al JAMA 263:2055,1990 2.Grandy et, al Am.J. Hum. Genet. 45:778,1989. 3.Connings, D.E. and Comings, B.G. J. Clin. Psychiatry 1990 (in press). 4.Comings, D.E. Tourette Syndrome and Human Behavior 1990 Hope Press, Duarte, CA. Clinical Genetics A53

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Townes-Brock and Prenatal Phenotypic Overlap Syndrome Exposur Monozygotic twins discordant for Aicardi syndrome Cylophosphamid. JoF, Cordero'. Llscike'. Johnson2. L1ng. T. Costa'. W. Gretr. G. Duckworth-Rysieckil. M. Division Dfects and [.D,Alacdon3 (1) BDrth Developmental Disabilities, Musarella2. P. Ray'. Hospital for Sick Children, Disem Control, Atlanta, Dalton Clnic, Toronto, Ontario, Canada.

Division Medical Genetics, Emory University, Atlanta, Georgia.

an autosomal characterized TheTownes-BrocksyndromeIs by Aicardi syndrome is a developmental disorder cranlofacla abnonralities, hemufadial microsomla, preauricular tags, heart characterized by agenesis of the corpus callosum,

limb nus, and deficit. a abn tes orate hearing report retinal lacunae, seizures and developmental delay. Townes-Brock and strong phenotypic overlap syndrome prenatal It is believed to be X-linked with lethality in exposure cyclphosphamide during pregnancy in pattern males. We report a set of female twins, 19-years malformations ear, heart aso characterized by imperforate of age, one of whom is a healthy university anus. Weevaluated whitefemale born who day year was student while the other has the classical Aicardi treated chemotheptlc agents induding cyclophosphamide nodular phenotype with profound retardation. Family scerosing Hodgkins Disease during High ultrasound history is negative. Both had normal karyotypes. fetal weeks was normal. (induding ehcrography) gestation The Monozygosity was established by blood grouping, normal and infant height weight, several chromosomal heteromorphisms, DNA fingerprint features dyrphi werenoted. They indude widening angle analysis using 6 hypervariable probes, 5 autosomal arched and on right side, tag right high palate, facial and 1 X-linked, and 3 X-linked RFLP probes. We which an initial Townes-Brock asymmetry suggested diagnosis syndrome. tested the hypothesis that preferential analysis and cervical were spine radiographs all inactivation of a different X-chromosome had published reports Townes-Brock syndrome published reports of occurred in each girl. Methylation-sensitive RFLP agents cyclophosphamide and chemotherapeutic during pregnancy. analysis of DNA from EBV-transformed B-lymphocytes data from National reviewed Registry pregnancy using MspI/HpaII digestion and probing with exposures chemotherapeutic agents reports individuais M27beta showed an identical pattern of random X- that clinical Townes-Brock syndrome provided detail analysIs. inactivation in both twins. We conclude that the we case Similarly, reports exposure cyclophosphamide during abnormalities in the affected twin are likely the pregnancy. compared findings significant consequence of a post-zygotic mutation in early significant differences overlaps. were In proportion embryonic development. absence and cardiac defects. Imperforateanus, deafnes, Twofindings helpful in diferential diagnosis two conditions anus Townes-Brodcsyndrome weremore likely ectopic polydactyly. the cas with anus, (50%) TownestBrock syndrome had ectopic whereas cam exposed cylpho nde reported finding (p=0.00016). (44%)of cas Townes-Brocksyndrome had polydactyly, none exposed finding cas cycophosphaide reported (p=0.00004). Cydophoephamide should known lict teratogenic agents that produce re malformations.

(0203) 1.106 (0204)

Variant of Hereditary Sensory Neuropathy Type IV: Arhidrosis, GERODERMA OSTEODISPLASTICA. MOLECULAR ANALYSIS Insensitivity, and Normal Intelligence. K.B.Courtney and OF TYPE AND II COLLAGEN GENES. D.L.Freedenberg. Children's Medical Center and roe University TexasSouthwestern Medical Center, Dallas. D.A Coviello. M. Rolleri. N. Sinelli, Institute of Biology and Medical Genetics. Hereditary Sensory Neuropathy Type IV is a rare autosomalreces- disorder characterized y pain insensitivity, anhidrosis, C. Bellini, E. Bonioli, Pediatric Clinic. mental retardation. State University, Genova, Italy. evaluated a four year old girl with pain insensitivity, anhidrosis, and normal development. She was the term product The proposita (mother 24, father 37 year-old, not third pregnancy of non-consanguineous non-Jewish parents. consanguineous) was born at term (birth weight months of age she developed breath holding spells result- ing hypoxic seizures. At eight to nine months of age her 3560 g) after a normal pregnancy; there was no fa- parents noted that she did not sweat after several episodes of mily history of multiple fractures or osteoporo- hyper yrexia. At eighteen months of age a tibial fracture was sis. Bilateral dislocation of the hips was diagno- sustaoned with no evidence of pain. There was poor healing of fracture and exuberant periosteal growth necessitating a sed neonatally. She was referred at 1 year of age: to exclude biopsy the presence of an osteosarcoma. At weight 7394 g (<3rd centile), height 77 cm (90th - approximately eighteen months of age she removed two teeth by on 95th centile); the face appeared droopy, jowly and pulling them with a hanger as a response to anger. Her developmental milestones were normal. Hier physical exam reveal- prematurely aged. There was no psychomotor retar- normal appearing child with full head of blonde hair and dation. SKin was loose and wrinkled; joints were normal appearing nails and teeth. When crying she had profuse tears and hyperextensible and there was severe and generali- consistently developed an erythematous blotching on upper two thirds of her face and blanching of the lower zed osteoporosis. distinct reproducible linear distribution. Her Geroderma osteodisplastica (GO) hereditaria is a neurologic evaluation found her DTR's to be within normal rare familial condition (MIM 23107), whose patho- limits her response to vibration and proprioception. evidence of blood pressure instability. She was genesis is unknown; connective tissue abnormali- have abnormal response to pin prick and was unable ties are suggested by the clinical findings, some distinguish painful stimuli. An intradermal histamine of which are in common with Ehlers-Danlos syndrome challenge revealed an abnormal response with absence of flair. Instillation of 0.0625% pilocarpine resulted in an abnormal (ED) and with Osteogenesis Imperfecta (01). Since miosis of the pupil for a period of approximately five days. defects of collagen genes have been reported for Stanford Binet intelligence testing revealed intellectual in all 01 and ED, we investigated the type and IlIl functioning areas within the normal range with a composite score of 104. A sweat chloride test confirmed her collagen genes in this patients. inability sweat. An attempt to evaluate her respiratory DNA was extracted from a peripheral blood sample. response to hypercarbia was aborted as the child became terrified and We studied RFLPs of COLiai gene using the uncooperative. f ollowing probes: RMS8 (RSAI), NST7O (RSAI), FG2 the previous patients with HSN IV have presented with (MSPI); RFLPs of COLia2: NJ3 (ECORI), NJi4i pain insensitivity, anhidrosis and mental retardation. Of the previous cases that have been reported all affected individuals (MSPI). Hf32 (RSAI); RFLPs of COL3al: DACI have mild to moderate mental retardation. Our patient is (AVAIl). other individuals with HSN IV in that she has anhi- No abnormal or additional band was observed, drosis, isolated pain insensitivity, poor healing of fractures, normal DTR's. She differs from other individuals with HDIV pointing to the lack of involvement of the above that she does not exhibit developmental delay. The patho- genes in GO, although the available probes are genetic mechanisms of Pain insensitivity and anhidrosis are Known not to cover completely the coding but region. uncertain, our patient clearly demonstrates that there is different mechanism involved in the etiology of developmental Northern blot analysis and collagen protein delay. She extends the s1ectrm of HSNIV to include pain studies are in progress. sit ivity, angidrosi and normal intelligence. A54 Clinical Genetics (0205) (0206)

EPIDERMAL NEVWS SYNDROME: A NEUROLOGICAL VARIANT Atypical phenotypic expression of X chromosome WITH HEMIMEGALENCEPHALY, GYRAL MALFORMATION, MENTAL monosomy. D. B. Domek. G. B. Schaefer. A. D. RETARDATION, SEIZURES AND FACIAL HEMIHYPERTROPHY. Garnica. and 0. M. Rennert. Oklahoma Univ. College W.B. Dobyns. L. Pavone. P. Curatolo. R. Rizzo. G. of Medicine, Oklahoma City, Univ. Nebraska Medical Micali. G. Incorpora. B.P. Garg and D.W. Dunn. From Center, Omaha, and Georgetown Univ. Medical School, Indiana University School of Medicine, Indianapolis, Washington, D.C. University of Catania, Catania, Italy; and University of Rome, Rome, Italy. The phenotype most often associated with X The epidermal nevus syndrome (ENS) is a sporadic chromosome monosomy includes characteristic facies, neurocutaneous disorder which consists of epidermal short stature, gonadal dysgenesis, lymphedema, nevi and congenital anomalies involving the brain webbed neck, and cubitus valgus (Turner Syndrome). and other systems. From among over 60 patients with We report three cases which have in common ENS presenting with neurological manifestations, we karyotypes with monosomy X but exhibit three identified 17 who had hemimegalencephaly based on different phenotypes that do not coincide with pathological or radiological studies. Associated Turner Syndrome. A.K. was found to have a 45, XO brain and neurological abnormalities included gyral karyotype on prenatal amniocentesis; physical malformations in 11/11, mental retardation in 13/14, examination is remarkable for mesomelic shortening seizures in 16/17 including 9 with infantile spasms, of the long bones, with bowing of the radius and and contralateral hemiparesis in 6/11. All had ulna, but normal spine, pelvis, ribs and hands; ipsilateral epidermal nevi of the head, and several findings suggestive of dyschondrosteosis. S.P. had ipsilateral facial hemihypertrophy. We presented in the neonatal period with the features concluded that these abnormalities comprise a of acrofacial dysostosis of Nager. Chromosome recognizable NEUROLOGICAL VARIANT of ENS which we analysis revealed a mosaic karyotype; 46,XX (8%) and believe represents the full expression of primary 45,XO (92%). M.B. was evaluated shortly after birth brain involvement. with multiple anomalies including growth retardation Several patients also had evidence of acquired cerebral palsy, and radial hypoplasia. Karyotype brain lesions such as infarcts, atrophy, revealed a 45,XO chromosome constitution. Facial porencephaly and calcifications, which are best features along with severe radial hypoplasia were explained by prior ischemia or hemmorrhage. Given suggestive of Cornelia deLange syndrome. Subsequent repeated observations of blood vessel anomalies in intellectual development has been marked by severe ENS patients, we hypothesize that underlying retardation and autistic behaviors. The conditions vascular dysplasia predisposes to these acquired associated with the above phenotypes are not known lesions. The same cause may be invoked to explain to be associated with loci on the X chromosome. The the wide variety of neurological symptoms reported occurrence of these phenotypes with X chromosome in ENS patients without hemimegalencephaly. While monosomy could be explained by chance. It is also the cause of ENS remains unknown, several possible, however, that the aneuploidy may be observations suggest a somatic mutation. causally related either by producing a predisposition to spontaneous mutations, or by abnormal gene-gene interactions.

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Missense mutations within the rhodopsin gene in patients [UNMA DISEASE LCI WITH NOUSE BOOGIIS: J. H. Edwards. JG with autosomal dominant retinitis pigmentosa. Hall2. A.G. Searle3 1Genetics Laboratory, Department of T. P. Dryja, T. L. McGee, L. B. Hahn, G. S. Cowley, J. E. Biochemistry, University of Oxford, Oxford OX1 3QU, England, Olsson, E. Reichel, M. A. Sandberg and E. L. Berson. 2Department of Medical Genetics, University of British Berman-Gund Laboratory for Retinal Degenerations and Columbia, Vancouver, B.C., Canada V6H 3N1, 3MRC Radiobiology the Howe Laboratory of Ophthalmology, Massachusetts Eye Unit, Chilton, Didcot, Oxon OXll ORD, England. and Ear Infirmary, Boston, Massachusetts. The identification of homologous loci in man and mouse has many implications. One practical application is the We report three missense mutations in the human development of animal models for human diseases. The loci of rhodopsin gene, each of which occurs exclusively in the well over 100 human diseases with homologies in mouse have affected members of some families with autosomal dominant been defined and the development of mouse models for these retinitis pigmentosa. Two of the mutations are C-to-T diseases is helped by a knowledge of the position of these transitions involving separate nucleotides of codon 347; loci. High resolution linkage studies to define order and the third is a C-to-G transversion in codon 58. None of approximate position after the rough mapping possible in human these mutations was found in 106 unrelated, normal families is also feasible in the mouse with the very large individuals who served as controls. Summing the incidence 'families" which can be produced related to mutation at a of these three mutations with the missense mutation single locus. involving codon 23 that we reported previously (Nature This poster will present a current compilation of the human 343:364-366, 1990), we find that 27 of 150 unrelated disease loci in which the homologous mouse segments have been patients with autosomal dominant retinitis pigmentosa mapped in the form of chromosomal displays, the Oxford grid (or 18%) carry one of these four defects in the rhodopsin and listing of disease loci. gene. All patients with these rhodopsin gene defects have abnormal rod function as monitored in their electro- retinograms. Evidence from intragenic polymorphisms shows that most of these patients probably descend from a small set of distant ancestors, each of whom carried one of these mutations. Our data support the idea that some patients with autosomal dominant retinitis pigmentosa have one of a variety of missense mutations of the rhodopsin gene as the etiology of their disease. Clinical Genetics A55

(0209) (0210) 1.107 Combined facio-auriculo-vertebral spectrum and situs Genetics and the relationship to behavior of the low inversus totalis in a viable newborn voltage alpha resting EEG trait in alcoholics and controls. M. H. El-Foulv1, M. A. Portman and D. S. Sciamanna M.A. Enoch, J. Rohrbaugh. V. Moore, M. Eckardt, D. Goldman. Department of Pediatrics and Human Development, College of Hunan National Institute on Alcohol Abuse and Alcoholism, Medicine, Michigan State University, East Lansing, Michigan Icurrent address: Centerfor Medical Genetics, The Johns Hopkins University School Bethesda, MD 20892. ofMedicine, The Johns Hopkins Hospital, Baltimore, Maryland. It has been suggested that certain resting EEG A term newborn girl presented with situs inversus totalis ant phenotypes are genetically determined and that one, the malformations consistent with the facio-auriculo-vertebral (FAV) spectrum low voltage (LV) alpha, may be transmitted in autosomal (Goldenhar syndrome). The facial anomalies included microtia of the right dominant fashion and associated with characteristic behavioral auricle with external canal and traits, notably alcoholism. This study aims to missing auditory associated sensorineural further elucidate the population frequency, deafness, hypoplasia of the right half of the maxilla and mandible, high transmission, and behavioral correlates of the LV trait. arched palate and hypoplastic right side of tongue with midline clefting of the More than 100 individuals including 10 alcoholic and alveolus. The neck had right-sided torticollis and fused C6 and C7 control families were examined clinically using the hemivertebrae with right-sided concavity. The thorax showed a T4 Schedule for Affective Disorders-Lifetime Version, the hemivertebra, hypoplastic first, second and third ribs on the right and Michigan Alcoholism Screening Test and a structured missing twelfth rib on the left The right lung had two hypoplastic lobes, the family history, for personality using the MMPI, EPQ-R and left had three lobes with an air cyst in the upper lobe and atelectasis of the TPQ, and also for cognitive function. For the resting EEG, power spectra were computed and an autoregressive middle one. The cardiac anomalies included dextrocardia with L-loop model of analysis was applied. relation of the great arteries, total anomalous pulmonary venous return Results indicate that the LV variant is transmitted as connecting to the left sided inferior vena cava via the ductus venosus. The an autosomal dominant and occurs in 12.5% of the study right pulmonary artery and veins were severely hypoplastic. The abdomen population. Preliminary analysis suggests that this trait showed situs inversus of all organs and structures with marked hypoplasia may be associated with a higher prevalence of on the right side compared to the left and with missing right kidney. The alcoholism, generalized anxiety and major depressive patient's prophase karyotype was normal. This is, to our knowledge, the disorders. Genetic linkage analysis is being undertaken first reported case that combines both situs inversus totalis and the FAV on several informative families. spectrum The events that resulted in the defect in lateralization leading to failure of normal asymmetry in morphogenesis appear to have preceded the development of FAV anomalies.

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Familial Occurrence of Progeria (Hutchinson- Premature diffuse atherosclerosis in two siblings with photo- Gilford Progeria Syndrome: O.J. Fatunde. L.B.O. myoclonic epilepsy, diabetes mellitus, deafness, and neptro- Benka-Coker and A.B. Scott-Emuakpor, Departments pathy: A genetic syrdrae distinct from Hernnann syrdrame. of Child Health and Radiology, University Benin A. Feigenbauml, R. ftAme3, R. aRhadson:a2, S. latchie3, Teaching Hospital, Benin City, Nigeria. B .Whrrne.t , BR. ..Jq eL. Dept. of Genetics Hopital for Sick Children, Dept. of Int.Med2, Pathology3 and The progeria syndrome of premature aging was Neuology4, Toronto General Hospital, Thronto, Canada. first described by Hutchinson and later by We present a new syndrcme involving severe preanture athero- Gilford. It is a rare disease made up of a sclerosis in two brothers. They both developed deafness, combination dwarfism and precocious senility. rqhropathy, diabetes, yoclonus, and a degenerative Death from diseases of old age, such as coronary neurological disease. The proland presented at 22 years with Artery Disease, may occur before the age of ten. deterioratiog cognitive function, sensorineural deafness and proteinuria. At 27 years, diabetes and renal artery stenosis There are a few reports of the syndrome in were diagnosed. Renal biopsy sowed glomurular sclerosis and siblings. Gabr et al. reported affected sisters mesangiolysis. Progressive neurological deterioration with who are the children of first cousins. Rava cerebellar syuptons and photcyoclonic seizures ensued. Death found six affected among offspring of two occurred at 31 years. Laboratory results were not diagnostic sisters. DeBusk reported that in three cases out of any disorder of lipid metabolism or mitochordrial disease. of 19 patients in home consanguinity was sought, The karyotype was normal. The autopsy revealed severe athero- the parents were related. Though familial sclerosis of renal, coronary and cerebral vessels and aorta. clustering exist, most cases are sporadic. There was diffuse neuronal loss and gliosis of the cerebral deep grey matter, cerebellum and dentate nuclei. In addition, We report here a family of six children in there were scattered infarcts throsghout the brain. A male sib home three have the progeria syndrome. There was died at 26 yrs with similar clinical and pathological fiadings. a seventh child (dead prior to the description) EEG, audiograms and renal function tests on both sisters and that may have been affected. This family parents were normal. Thus, the mode of inheritance in this probably represents the largest number of family is likely to be either X-linked recessive or autsmnal reported affected siblings. Radiographic recessive. evaluation of the patients revealed severe The clinical findings in these two brothers are similar to a generalized osteoporosis. syrircune scribed in 1964 by Hermann et al (Neurol. 14:212). He reported a family with diabetes, deafness, nwopathy, Full clinical and radiographic descriptions as photyoclonic seizures and, variable cerebral dysfunction. well as a suggestion that this syndrome might be Autosonal dominant inheritance was sgted. However, patho- heterogenous will be presented. logy on their prvband revealed PAS-positive deposits in the dentate and olivary nuclei and the renal collecting tubules. Based on the different inheritance patterns and the distinctive pathology, these two families likely represent different genetic diseases. Studies on cells froy our proband may be of interest in the elucidation of metabolic abnormalities leading to presature atherosclerosis and its associated clinical presentations. A56 Clinical Genetics (0213) 1.110 (0214) 1.111

Inverted duplication of 8p: an identifiable syndrome? A clinical and DNA database capable of drawing G.L. Feldman, M.J. Worsham, V.R. Babu, L. Weiss, pedigrees and communicating with the LINKAGE D.Van Dyke. Henry Ford Hospital, Detroit, Michigan. analysis package. We evaluated 6 patients whose karyotype revealed an Iain Fentonr , Lodewijk A. Sandkuil1, Michael J. inverted tandem duplication of chromosome 8p. One proband Aldred. Institute of Medical Genetics, University had a monocentric recombinant of a paracentric of Wales College of Medicine, Cardiff, U.K. inv(8)(p12.2p23.3) carried by her mother. The duplication Over recent years a number of software appeared de novo in the other 5 patients. packages have been developed to aid the geneticist The physical features in these individuals were in both clinical and scientific areas. Programs markedly similar. All six had significant hypotonia at have been written to draw pedigrees, organise DNA birth, resulting in difficulty in feeding, and had data within a database and to ease the process of significant developmental delay. Recurrent otitis media was working with linkage analysis packages such as found in 3 children, requiring the placement of typanostomy LINKAGE and LIPED. Until recently there have been tubes. In two individuals who have been studied absence of few attempts to integrate such software packages the corpus callosum as well as other structural brain into a cohesive system that is capable of all the abnormalities were noted. Common facial features found in tasks necessary for a full analysis of pedigree 3 or more patients were: micrognathia, high arched palate, data or indeed a study of the disease in question. large mouth with a thin upper lip, ear abnormalities, and We have developed a database that is able to facial asymmetry. Skeletal abnormalities were frequent. record both clinical and DNA data, draw pedigrees Three had joint laxity. on-screen of those families, and communicate Tandem duplications of chromosome 8 have been reported effectively with the LINKAGE programs. infrequently and no specific syndrome has previously been The program is presented as a disease- identified. Advanced mean parental age has been noted for specific register with a number of clinical inverted duplications in general; our results are consistent 'pages'. Any number of markers can be added to with this finding. All six known familial tandem the database, and typings of these markers can be duplication cases, including our case, were recombinants of viewed on-screen in either a text or pedigree a paracentric inversion inherited from the mother. An mode. This aids the clinician in visualising the unusual meiotic recombination event can account for such structure of the pedigree and by being able to familial cases. move interactively around the pedigree in a The identification of 6 cases of inverted duplication graphical mode this greatly facilitates of 8p makes this the most common duplication observed in our recognition of problems such as individuals laboratory and ranks in frequency with the commonly seen without samples, identification of possible deletion syndromes, such as 4p- or 5p-. Variation in the recombination events and potential cases of non- clinical phenotype may in part be explained by the different paternity which are difficult to recognise from a chromosomal breakpoints. Recurrence risks of de novo text-only screen. The program's ability to write rearrangements are very low but for the recombinants the files for, and read files from, the LINKAGE recurrence risk is unknown and may be significant; thus it programs in a intuitive way simplifies both risk is important that all parents of such cases have chromosome assessment and mapping tasks such as ordering of studies performed. loci and gene location.

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Fragile X syndrome associated with Niikawa-Kuroki syndrome: A Concurrent expression of three genetic disorders in one child: case report. Implications for gene assignment for craniometaphyseal dysplasia G.S. Fisch. G. Cousins. P. Slobogin. R. PeBenito. R.S. Verma. J. and microcolon-megacystis syndrome. D.B. Flannery, M. Southgate, W.H. Hoffman, C.E. Schwartz, Loh. E.C. A.S. Bernstein, A. Ciccone. G. Hotson. J. Jenkins. Yeager. M.C. Phelan, P.C. White, Medical College of Georgia; Greenwood J. French. Kings County Hospital Center and SUNY/HSC, Genetics Center, Greenwood, SC, Cornell University NY. Brooklyn, NY. Simultaneous occurrence of more than one genetic disorder In Fragile X [fra(X)J syndrome is the most common cause of a patient is an uncommon event, and may represent an "experiment inherited mental retardation (MR) in males. Niikawa-Kuroki or of nature". We report an infant affected by three genetic dis- Kabuki makeup syndrome (KMS) is a disorder with multiple orders. She was born at 36 weeks gestation, weighing 2880 gm congenital malformations: unusual craniofacial features, MR, skeletal to a 25 year old G2P1AO mother after a pregnancy complicated by abnormalities, unusual dermatoglyphics, and postnatal growth deficit. polyhydramnios and hydronephrosis with bladder distension, We present a case of a 13 year old black male who shows stigmata detected by level II ultrasound. Postprandial blood sugar was associated with KMS and is cytogenetically positive for fra(X). 118 during the pregnancy. At birth the baby had a distended a across the nasal Our subject was brought to our inpatient psychiatric facility after abdomen, prominent bony protrusion bridge, and clitoromegaly. Hypaque enema demonstrated a microcolon. he attempted to strangle a 9 year girl. Initial impressions were that Upper GI series demonstrated no peristalsis. IVP demonstrated was but below normal he alert, attentive and cooperative IQ. hydronephrosis, hydroureters, and a large bladder. Skull X-rays Striking dysmorphic features were noted: long (4 cm) upslanted demonstrated focal thickening of the frontal bone, occipital palpebral fissures; high arched eyebrows; prominently protruding bone, and planum sphenoidale, and enlarged nasal bone. X-rays ears; possible scoliosis; fingertip pads; unusual dermatoglyphics. A at 11 months demonstrated bulbous ends of long bones. Rectal high field MRI revealed a large lesion in the left lateral basal biopsy was normal. 17-OH progesterone was 2900ng/ml, androst- ganglia. Cerebellum and posterior fossa were unremarkable. Two of enedione 320, DHEAS 1208, and deoxycortisol 4.6. Based on the 3 cytogenetic evaluations for fra(X) were positive. above f indings, the child was diagnosed as having the microcoln- Speech and language tests indicated deficits in receptive and megacystis-intestinal hypoperistalsis syndrome, 116 hydrozylase expressive language. IQ tests noted mild MR with PIQ higher than deficiency, and craniometaphyseal dysplasia. Consanguinity was tests evoked about violence and ruled out by extensive pedigree. Continguous gene syndrome wSu VIQ. Projective responses sex, searched for. High resolution chromosomes with attention to he to have hallucinations. death. Occasionally, appeared auditory 8q21 were normal. DNA studies did not detect deletions of or Given his perceptual difficulties, we examined his orienting response, in the 11i hydroxylase gene. Minisatellite probes did not which is a good index of attentional processes. A 60 dB tone elicited detect loss of any particular parental alleles. Pulsed field gel sharp skin conductance response without a concommitant change in electrophoresis to date has not demonstrated alteration of the finger pulse volume. Gaze aversion, a frequently reported finding 116 hydroxylase region nor loss of parental alleles. Despite among fra(X) individuals, was never observed. the inability to document a submicroscopic deletion or uniarent- Considering the many clinical features in common to fra(X) and al disomy, we feel probablistically tht the concurrence of 3 KMS, and the low probability that these two syndromes would occur genetic disorders in this child is evidence of possible linkage 119 has been in a single person, we suggest that KMS individuals be examined for of the three genes. Since hydroxylase assigned previously to 8q21, it is possible that craniometaphyseal dys- fra(X). plasia and microcolon-megacystis-intestinal hypoperistalsis syndromes are located on this chromosome as well. Clinical Genetics A57 (0217) (0218) 1.113

Proposal for a code of ethics in medical genetics, composed of A new autosomal recessive syndrome of sparse hair, dysmorphic facies, seven bone hypomineralization an4 mental retardation in Mennonite sisters. working approaches. I J.C. Fletcher and D.C. Wertz. University of Virginia Medical A. M. arritv' aler. H. J. Sternl. K. N. Rosenbauml. B. M. Center and Boston University School of Public Health. Orrisonl. J. C. Marini!-. and H. M. Saatl. 'Department of Medical On the basis of a 19-nation survey of working approaches to Genetics, Children's National Medical Center, Washington, D.C., ethical problems, we propose the following seven standards for 2Human Genetics Branch, NICHD, NIH, Bethesda, MD. a code of ethics: Two Mennonite sisters (21 and 12 years old) were referred for evaluation of sparse hair, short stature, hypotonia, delayed 1. Fairness of Access to Genetic Services. An estimated 25% development and facial dysmorphism. Their healthy parents are half of all mothers in the U.S. (939,000 annually), receive late first cousins whose five other offspring include three who died in or no prenatal care. Women who receive prenatal diagnosis infancy with osteogenesis imperfecta type II and two who are are disproportionately white, well educated, and physically and intellectually normal. The sisters described here have financially well-off. If this trend continues, being each suffered two skeletal fractures after minor trauma and show genetically handicapped coul become a mark of social diffuse bone hypomineralization on radiographic examination. Blue class. sclerae are present in the younger of the pair. The 12 year old child 2. Full Disclosure of All Clinically Relevant Information. has a high frequency hearing deficit and a seizure disorder. Neither Psychologically sensitive information, such as XY genotype has dental abnormalities consistent with dentinogenesis imperfecta, or in a female, should be disclosed only in the context of ectodermal defects of the teeth, nails or skin. full and supportive counseling and patient education. Results of chromosome and metabolic studies were normal. 3. Respect for Parental Choices, Including Decisions to Abort Preliminary analysis of collagens synthesized by cultured fibroblasts or to Carry to Term a Fetus with a Malformation or Genetic from the younger sister revealed overmodification of type I cc,, and a2 Disorder. chains. Collagen analysis in the parents and other affected sister is 4. Protection of Patients' Privacy from Institutional Third in progress. Parties, such as employers and insurers. Alternatives The constellation of abnormalities in these sisters appears to would be a) to permit access to information but to have represent a previously undescribed autosomal recessive phenotype, extensive legal protection for individuals that would perhaps unique to the Mennonite population or to this particular prevent discrimination, or b) government underwriting of family. A relationship between this syndrome and the type II health insurance for those at genetic risk. osteogenesis imperfecta affecting their siblings remains speculative. 5. Use of Prenatal Diagnosis only to give Parents Information about the Health of the Fetus. Any other use, such as for sex selection (except for X-linked disease), should be avoided. 6. Voluntary, Not Mandatory, Screening-Except for Newborns When Early Treatment Is Available. The primary purpose of screening should be to help the newborn; carrier detection is secondary. 7. A need for Further Study and Discussion of the Complex Issue of Patient Confidentiality in Cases Where there is High Risk of Serious Harm to Relatives at Genetic Risk.

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Prenatal detection of recurrent SLOS type 2. Z. Gelman-Kohan, Clinical and Molecular Studies of Craniofacial Anomalies in R. Nisani, J. Cheake, Z. Appelman, S. Rappaport, E. Hegesh(l). the Philippines. 1C. Geoggiou. lit, Murra1. 2K.H. Buetow. Kaplan Hospital, Rehovot, and Sheba Medical Center, 1N. Leysens. 2B. Miller. A. Rare C.S. ChUna. and 5W. Tel Hashomer(1), Israel. McGee. University of Iowa, Iowa City, IA, Fox Chase Cancer 3East Tennessee State 2" Center, Philadelphia, PA, University, "Smith-Lemli-Opitz syndrome type (SLOS type 2) was Johnson City, TE, 4University of Hawaii-Honolulu, HI, and proposed by Curry et al. in 1987 for the association of early 50peration Smile International, Norfolk, VA. lethal anomalies-male multiple congenital pseudohermaphrodi- Under the auspices of Operation Smile, we have initiated tism. This is considered to be the severe form of phenotype a project to study the spectrum of craniofacial anomalies in "classical" SLOS. Since at least 47 of SLOS 1964, patients the Philippines. Over the last two years, over 600 have been cases with and reported, including early lethality individuals with craniofacial anomalies have been seen as The "new" pseudohermaphroditism (Jones, 1988). syndromes part of the evaluation process for surgery for craniofacial reported by Rutledge et al. (1984) and Donnai et al. (1986) anomalies in the Philippines. Blood samples for DNA are now believed to be cases of SLOS 2. type preparation have been obtained on 450 individuals and their We a in which SLOS type 2 was detected present family family members. 105 individuals at one site in one year were prenatally. A phenotypic female infant was born to young examined in detail. Of 85 individuals with cleft lip and/or of Jewish-Yemenite At birth non-consanguineous parents origin. cleft palate, 65 were non-syndromic with no associated a severe hypotonia, microcephaly, microretrognathia, anomalies. Of the remaining 20, there were three cases of congenital heart disease (CHD), ectopic anus and other hemifacial microsomia, five with associated limb reduction anomalies were found. Her karyotype was 46,XX. The infant defects and/or polydactyly/syndactyly, two siblings with lived for 42 A was not days. post-mortem performed. In the bifid nose and a child with Van der Woude syndrome. In fetal a following pregnancy, echocardiography detected severe addition, six cases of nasal encephalocele were seen. and complexCHD. Consequently, the pregnancy was interrupted Association studies of DNA polymorphisms for transforming at 24 weeksgestation. The fetal karyotype was 46,XY. A growth factor alpha (TGFa) were undertaken in the non- phenotypic female fetus was delivered, with unilateral cleft syndromic cases, based on previous reports of associations of lip and palate, unilateral postaxial polydactyly and particular TGFA haplotypes with non-syndromic clefting in malposition of feet. There was a complex CHD, abnormal lung Caucasians. A new HinfI RFLP was identified. No such lobulation, normal mullerian derivatives and completely association could be demonstrated in this population from the undifferentiated gonads. The findings in both cases are Philippines (N-45, p-O.33). Additional studies using probes consistent with the diagnosis of SLOS type 2. To our specific for other genes felt to play a role in craniofacial knowledge, in only 4 other instances has familial recurrence development are underway to look for evidence of association been shown in this syndrome, and none has been detected or detectable deletions or rearrangements causing prenatally. craniofacial anomalies in particular individuals. This study SLOS types 1 and 2 are mostly sporadic but recurrence has demonstrates a similar spectrum of craniofacial anomalies been reported. Concurrence of both types of SLOS has not been seen in a large population from Southeast Asia. In addition, seen in the same family. The possibility of genetic it demonstrates the feasibility of obtaining population-based heterogeneity has to be taken into account: whether there biological samples for studies of genetic and environmental exist more than 2 alleles for both types of SLOS or other etiologies underlying craniofacial anomalies. intragenomic factors affect the phenotypic expression. A58 Clinical Genetics (0221) (0222)

De novo chromosome abnormalities in two patients. P.F. Giampi- Phenotypic Variability in Craniosynostosis Syndromes: Dilema etro, M. Shaham, N.S. Avigdor, M. Lipshitz, B.R. Miller, K.S. in Classification and Counseling. Stern and J.G. Davis. New York Hospital-Cornell Medical Cen- G.R. Goldberg, M.L. Walsh and E. Sujansky. Univ. of ter, New York. Colorado School of Medicine, Denver. We report two patients, with de novo chromosomal abnormal- Two siblings with different degrees of craniosynostosis ities. J.P. is an 8 year old male with short stature, fail- and accompanying malformations are presented. The first ure to thrive, microcephaly, developmental delay and dysmorphic appeared to have isolated craniosynostosis the second, facies including prominent forehead, broad nasal bridge, left Carpenter Syndrome. The proband was identified at 29 weeks epicanthal fold, long philtrum, thin upper lip abnormal denti- gestation by ultrasound (US) which revealed mild polyhydram- tion and mandibular hypoplasia. Chromosome analysis from per- nios, increased abdominal girth, and cloverleaf skull. A ipheral lymphocytes revealed the following karyotype: 46,XY,-5, follow-up US at 32 weeks also revealed a 2-vessel cord and +mar. The marker chromosome consisted of chromosome 5 from hydrocephalus. A male infant was delivered at 32 weeks ges- band p15.1 to q35. Parental peripheral blood lymphocytes re- tation and expired at 9 hours. He had a cloverleaf skull vealed normal chromosome constitution. Therefore, the origin with fusion of the sagittal, coronal, and part of the lamb- of the abnormal region on 5p could not be determined. This doid sutures; dysmorphic facies; brachydactyly and campto- patient is monoaomic for the distal end of the short arm of dactly of hands with tapering of the distal phalangies; chromosome 5 and trisomic for an unidentifiable segment. Recent radial deviation of the distal phalangies of the thumbs and studies have shown that there appears to be a specific region bilateral duplication of the great toe with medial deviation within band p15 that is common to the deletions in patients of all toes. The proband's sister had surgical correction with cri du chat syndrome. Since this patient does not have of craniosynostosis of the metopic and both coronal sutures. cri du chat it is possible that the unidentifiable segment re- She has normal extremeties and no other major malformations. presents an insertion and the segment of DNA which is respon- A mild expressive language delay was present at 4 years of sible for cri du chat is present. Molecular probe analysis for age. She was considered to have non-syndromic, isolated the 5pl5 region would help resolve this issue. The second pa- craniosynostosis and parents were counseled accordingly. tient, B. B. is a female infant with symmetric intrauterine Both parents were clinically normal, however, declined growth retardation, facial asymmetry, micrognathia, low set radiographic examination. left ear, high arched palate, U shaped cleft of the soft pa- The specific diagnosis of a craniosynostosis syndrome is late, heart murmur, proximally placed thumbs, contracture of based on a constelation of associated malformations. Over the right index finger and abnormal dermatoglyphics. Chromo- 50 different craniosynostosis syndromes have been identified. some anaylsis from peripheral blood lymphocytes revealed the Our experience of significant intrafamilial phenotypic following karyotype: 46,XX,16p+. Chromosome analysis of pa- variability in this family and similar cases from the liter- rental blood lymphocytes revealed normal karyotypes. Thus ature, suggest caution when assigning a specific diagnosis this patient is monosomic for the distal region of the short and recurrence risk. Thus, a sporadic case of seemingly arm of chromosome 16 at band 13.3 and trisomic for an unident- isolated craniosynostosis in a family may represent a mild ifiable chromosome band. In addition because of the abnormal expression of a syndrome with significant morbidity and thumb position the cytogenetic analysis included a study of recurrance risk. spontaneous and dieoxybutane induced chromosome breakage rates. These were found to be normal with respect to controls. Using genomic DNA probes we plan to determine the origin of the un- identifiable material which is present.

(0223) (0224) 1.115 Sporadic patterns of anomalies due to maternal hyperthermia: Shprintzen-Goldberg Syndrome: further delineation of Moebius syndrome and Amyoplasia. J. M. Graham. Jr.. Matthew phenotye and investigation2for molecular defsct. Marie T. 3 J. Edwards. A. K lafolia J. B. Moeschler. and Mrshall J. Greally Anthogy Cousineau , Dianna McGookey , Peter Byers1, Edwards. Medical Genetics-Birth Defects Center, Cedars- i.J. Shprintzen , Rosalie B. Goldberg , and James W. Hanson Sinai Medical Center, Los Angeles, CA; Division of Pediatric Department of Pediatrics, Division of Medical Genetics, Genetics and Metabolism, Duke University Medical Center, University of Iowa Hospitals; 3Department of Pediatrics, Durham, NC; Department of Maternal and Child Health, University of Iowa Hospita~s; Department of Pathology, Dartmouth Medical School, Hanover, NH; Department of University of Washington; Center for Craniofacial Disorders, Veterinary Clinical Studies, University of Sydney, Sydney, Montefiore Hospital, New York. NSW, Australia. unrelated Moebius syndrome and Amyoplasia both occur sporadically In 1982 Shprintzen and Goldberg described two and there is evidence to suggest vascular disruption and males with craniosynostosis associated with arachnodactyly hemorrhages that affect central nervous system and limbs as and abdominal hernias. Other associated anomalies included an underlying pathogenetic sequence (Lipson et al., exophthalmos, maxillary/mandibular hypoplasia, hypertrophy Teratoloy40: 33-350, 1989; Hall et al, Amer. J. Med Genet of the palatal shelves, low-set ears and thoracic anomalies. 591-599,1g83). Limb reduction eifects and neurogenic Sugarman and Vogel and Furlong et al. reported two further congenital contractures are commonly associated with both cases. All four patients were characterized by the unusual conditions. These defects can be induced experimentally association of a marfanoid habitus and craniosynostosis. through hyperthermia which results in two types of problems: We report a 12 year old girl with features of Shprintzen- 1.) cell death and delay in cellular proliferation, and 2.) Clinical findings include dolicocephaly, vascular disruption. We now have seen 7 cases of Moebius Goldberg syndrome. syndrome and 2 cases of Amyoplasia which were associated exophthalmos, hypertelorism, low-set ears, micrognathia, with a history of febrile maternal illnesses during the late high palate with alveolar hypertrophy, bifid uvula, 1st trimester and/or 2nd trimester. In addition, we have arachnodactyly, joint hyperextensibility, metatarsus seen 8 other children with cortical atrophy, microcephaly adductus and pectus excavatum. Clinical investigation and/or abnormalities of the corpus callosum whose mothers revealed a Chiari I brain malformation, aortic root gave histories of febrile illnesses during the second dilatation and mitral valve prolapse, and mild ectopia trimester. The exact Pattern of anomalies has varied in lentis. Karyotype was normal female. Family history is each of these 17, cases but the features seen are consistent negative. There is no consanguinity and parents were young seen in heating in with patterns experimental animals after and when was born. an incubator or hot water bath. Correlation of healthy patient experimentally-induced patterns of anomalies with similar Clinically the Shprintzen-Goldberg syndrome shares many patterns in humans ascertained clinically suggests a causal features with Marfan syndrome. The main differences association. The effects of the agents inducing the febrile include: absence of tall stature, absence of family history, illnesses remain as confounding influences in the and absence of paternal age effect in Shprintzen-Goldberg interpretation of these retrospective human case reports, syndrome. Hypotonia of infancy, developmental delay, mental but it is clear that heat alone is capable of inhibiting or retardation and obstructive apnea are features of Shprintzen- killing mitotic neuroepithelial cells, and causing infarcts Goldberg but not of Marfan's syndrome. and/or hemorrhages which may destroy previously formed Investigations include fibroblast culture for fibrillin neural tissues. Further prospective studies of febrile or collagen defect. illnesses during pregnancy will help to provide better risk Follow-up data on the two patients originally described estimates. by Shprintzen and Goldberg will be discussed. Clinical Genetics A59 (0225) 3.3 (0226) 1.116

SMITH-MAGENIS SYNDROME (DELETION 17p112) AS A NEW A unique type of spondylometaphyseal dysplasia in CONTIGUOUS GENE DELETION SYNDROME. nber A three children. G.A. Greenhaw, J.T. Hecht, F. Smith2. S. Richter3. E. Magenis4. V. Guzzetia'. P. I. Patel.J.R. Luski' lBaylor Greenberg, J. Walters, L.O. Langer, S. Berry, R. College of Medicine, Houston, Texas 77030, Children's Hospital, Denver, Colorado Pauli, W.A. Horton. Univ. of Texas Medical School, 80218; 3University of Arizona, Tucson, Arizona 85721; 40regon Health Sciences Houston, Baylor College of Medicine, Houston, University, Portland, Oregon 97202 Tx., Univ. of Houston, Texas, Univ. of Minnesota Smith-Magenis syndrome (SMS) is a recognizable multiple congenital Medical School, Minneapolis, Univ. of Wisconsin, anomaly/mental retardation syndrome due to a deletion of chromosome 17pll.2, Madison. which was first described in 1986. To date, 19 patients with this disorder have been Three children with a unique skeletal described. Primary features include flat midface, brachycephaly, prominent forehead, dysplasia involving the vertebral bodies, broad nasal bridge, prognathism, brachydactyly, short stature, and mental retardation. metaphyses, epiphyses and pelvis have been Many patients have self-destructive behavior. identified. This skeletal dysplasia causes severe Recently, markers linked to Charcot-Marie-Tooth disease type IA have been disproportionate short stature which can be mapped to 17p11.2 using a chromosome 17-retaining hybrid constructed from a SMS observed from birth. Birth lengths of the patient (Patel et aL (1990) Am J Hum Genet 46:801-809). Further analysis has patients were normal but within 6 months indicated that these linked markers are deleted in several SMS patients. To subsequent longitudinal growth was dramatically determinewhether SMS patients had features ofCMT, we re-evaluated six previously reduced. At six years, one child was the size of reported and eight new patients with SMS. Thirteen patients were known to have an average 1 year old. Developmental and language deletion of 17pll.2 and one patient had an apparently balanced, de novo milestones were normal. Two children had visual 2p253;17pll.2 translocation. On examination (by FG and JRL), the phenotype of impairment. One of these had onset of nystagmus the patients was consistent with the previously described SMS phenotype. However, and diminished visual acuity noticed at 1 year of several specific findings were noted. The degree of mental retardation was variable age. Rod-cone dystrophy was diagnosed and at six from mild to severe. Decreased or absent deep tendon reflexes were noted in 79%, years only peripheral vision remained. The other decreased sensitivity to pain in 50%, pes cavus in 28%, pes planus in 28%, gait child had mild hyperopia. This child also had disturbance in 50%, and scoliosis in 25%. Self-destructive behavior including varus deformities of the lower extremities onychotillomania, wrist-biting, head-banging and insertion of foreign bodies in ears, requiring bilateral tibial osteotomies. X-rays was present in 80%. Sleep disorders were reported in 56% and two patients showed showed generalized involvement of the tubular absence of REM sleep on sleep study. bones, spine, ribs and pelvis. The tubular bones Based on these findings, SMS appears to be a contiguous gene deletion were underossified with splayed metaphyses and syndrome which can include signs ofperipheral neuropathy, self-destructive behavior, mildly irregular epiphyses. The bones were and sleep disorders. To date, two patients have had nerve conduction studies markedly shortened. The vertebral bodies were performed which did not demonstrate decreased motor NCV, although the clinical oval and flattened and on lateral projection had features appear to be compatible with a peripheral neuropathy. NCV studies are central anterior ossified protrusions. Contours presently being performed on several additional patients. Molecular mapping of this of the iliac, ischial and pubic bones were very region with correlation to clinical findings will be reported separately. irregular. This type of spondylometaphyseal Supported by a Muscular Dystrophy Association Task Force on Genetics dysplasia is distinctive and more severe than the grant, a Baylor Mental Retardation Research Center grant HD 2406402, an Kozlowski and Sutcliffe types. The epiphyseal Advanced Technology Program grant from the state of Texas and N.I.H. grant RO1 involvement and visual impairment (although NS-27042 to J.R.L. and P.I.P. atypical for SED) raise the possibility of a type II collagen mutation in these patients.

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Prader-Willi and Angelman syndromes in one kindred with Evaluation of families with a single male expressing a expression consistent with genetic imprinting. phenotype of X-linked lymphoproliferative syndrome (XLP) M. A. Greenstein. St. Francis Hospital and Medical Center, using RFLP probes informative for XLP. H.L. Grierson. J.C. Hartford, CT. Skare and D.T. Purtilo. Univ. Nebraska Medical Center, Genetic imprinting, phenotypic variations related to Omaha, NE and Boston Univ. School of Medicine, Boston, MA. parental effects on chromosomal material, has been suggested Diagnosis of XLP requires documentation of fatal as the explanation for the finding of apparently identical infectious mononucleosis (FIM), malignant lymphoma (NHL), or chromosomal deletions in some cases of Prader-Willi (PWS) and hypogammaglobulinemia following EBV infection in two or more Angelman (AS) syndromes. Currently, paternal origin of an maternally-related males. The disorders associated with XLP altered 15q region has been documented in most evaluated also occur sporadically in families without histories of cases of PWS with recent studies suggesting maternal origin other affected individuals. EBV negative males in these of a similar, if not identical, alteration in AS. families must be considered at risk until they seroconvert On evaluation for behavioral and educational problems a 10 normally to EBV. RFLP probes to DXS42, DXS37, DXSl0, DXSl00, year old girl was identified as having PWS with classic DXSl9, DXSl77, DXS99, DXS86, DXSl2, and F9 are informative in physical features and history. Review of the family history families known to have XLP. We have used these probes with revealed that an 11 year old male paternal cousin had been the intent of providing information as to relative risk to diagnosed at another center at age 4 with AS. Evaluation of EBV negative males and potential carrier females in 7 this child confirmed the diagnosis, again with classic families with only one documented affected male. Six of the historical and physical features. familes had one male who died of IM, and the proband in the These cousins are related in a manner consistent with the 7th family developed NHL associated with EBV infection and imprinting hypothesis: the father of the child with PWS and marrow hypoplasia. Seventeen EBV seronegative maternally- the mother of the child with AS are brother and sister. related males, 14 females at risk for being carriers, However, the reasons why these are the first two affected fourteen males who were normal EBV seropositive, and the children in this family and the specific mechanism(s) in mothers and maternal grandparents of the affected males were action have not been determined. Additionally, a review of studied. DNA from lymphoblastoid cell lines, or EDTA- the 4 generation pedigree reveals that almost all of the men anticoagulated blood was studied using the probes listed in this kindred demonstrate a severe reading disability, above. DNA was available from the affected males of 2 despite evidence of normal intelligence. Inheritance of this families. In 1 family, the results were inconclusive because trait is consistent with autosomal dominant, not X-linked, of recombinations on both sides of the XLP locus. We inheritance and raises the possibility that this learning obtained information useful for counselling in the 6 disability may be related to the findings in these two remaining families. In one family, for example, we children. determined that the proband inherited the X chromosome from While it is possible that these children have been his maternal grandfather, who did not have XLP. Thus, if the affected by independent events, their findings and parentage proband had XLP, it was a result of a new mutation in either lends support to the hypothesis that imprinting links these himself or his mother. In another family, the genotype of an two syndromes. EBV negative male was consistent with XLP, and it was recom- mended that he be placed on prophylactic immunoglobulin. Our studies show that this type of analysis can be helpful in providing guidance for physicians treating and counselling these families. A60 Clinical Genetics (0229) 1.118 (0230) 1.119

Congenital Nystagmus in a (46XX,45X) Mosaic Female Clinical Similarity Between Frontometaphyseal from a Family with X-linked Idiopathic Congenital and Mannosidosis II. K.Dysplasia X.J.Hajianpour and Nystagmus. D.H. Gutmann. M.L. Brooks. B.S. Emanuel. ButLer.The Genetics Institute, Pasadena, CA, and D.M. McDonald-McGinn and E.H. Zackai, The Hospital of The Baptist Hospital of Miami, Florida. University of Pennsylvania, The Graduate Hospital, and The The propositus was a 22 year old white female Children's Hospital of Philadelphia, Philadelphia, PA. with short stature, coarse facial features including prominent forehead, thick calvarium X-linked congenital nystagmus is a rare disorder where affected prominent jaw, bony abnormalities such as males exhibit nystagmus and head oscillations. It may occur in scoliosis, spina bifida occulta, and hip fracture, isolation or in association with red-green color blindness, stationary hearing and speech deficiency, mental retardation, night blindness, ocular albinism, adrenoleukodystrophy or and cerebellar dysfunction as ataxic gait. Pelizaeus-Merzbacher disease. Idiopathic X-linked congenital Based on clinical presentation and X-ray nystagmus pedigrees with affected females have been reported and findings the patient was initially diagnosed as ascribed to the effects of random X-inactivation. We describe a having Frontometaphyseal dysplasia (FMP). The pedigree with idiopathic X-linked congenital nystagmus in which Patient had two sisters; 24 and 8 years old. The an otherwise normal (46XX,45X) Turner mosaic female was younger sister had similar facial features, and affected. All affected members in this pedigree exhibited binocular hearing and speech deficiency, and mental uniplanar horizontal nystagmus without a superimposed latent retardation, but no ataxia. The course of the nystagmus component. Null zones, head tilts, and head disease was slowly progressive in both. The older oscillations were noted. There was no evidence for ocular sister was a college graduate with normal stature albinism, nightblindness, red-green color blindness, optic atrophy and a history of speech deficiency, facial or neurological abnormalities. Magnetic resonance imaging of the asymmetry and dental malalignment. The parents are brain of affected members was normal. Karyotype of the affected normal. female in this pedigree demonstrated a (46XX,45X) mosaic pattern Further work-up on the two affected sisters with 12% of the 50 cells counted being 45X. The finding of an revealed alpha-D mannosidase deficiency. The affected (46XX,45X) mosaic female in this pedigree suggests that enzyme testing on the parent and unaffected sister she expresses the affected X chromosome in the majority of cells in is pending. central nervous system tissues responsible for the generation of It is not clear if the older sister is a carrier congenital nystagmus. Future studies will require more families for the disease and if her facial asymmetry and with idiopathic X-linked congenital nystagmus in order to speech deficiency are related to partial deficiency determine the map position of this disorder on the X chromosome. of the enzyme. The phenotypic overlap between FMP and mynnosidosis II (milder juvenile-adult phenotype) seen in these two patients include: coarse faces with wide nasal bridge and prominent supraorbital ridges, high palate, prominent jaw, thick lips,some degree of dysostosis multiplex, hearing deficiency, gait, speech deficiency and psychomotor retardation. Further studies are required to clear the relationship between these two disorders.

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Microgastria and limb reduction defects: a ?ossible Cardio-facio-cutaneous syndrome and de novo t (13; 14).

developmental defect. T. field Ja!es Y. Hanson, Marie S. Havkin(1), Z. Gelman-Kohan(1,2), R. Nisani(2), Greally and Ann Haskins Olney-. Department of Pediatrics, S. Bar-Shalom(2), J. Chemke(2). Barzilai Medical Center, Medical Genetics, University of Iowa 9ivision of Hospitals, Ashkelon (1), and Kaplan Hospital, Rehovot (2), Israel. University ofNebraska MedicalCenter.

Microgastria is a rare congenital anomaly. Fewer than 30 The importance of using clinical information to locate reported in the literature. Asplenia is cases have been the developmental genes was pointed out by Schmickel in 1986. commonly associated abnormality. Upper limb most hypoplasia Known or suspected monogenic disorders associated to a and intestinal malrotation have been described in a number chromosomal anomaly which allows for gene location are known of patients with microgastria while individual case reports as "contiguous gene syndromes". include abnormalities of the hepatic, renal, respiratory, The cardio-facio-cutaneous syndrome was described by gonadal, cardiac, optic and skeletal systems. Reynolds et al. (1986) and defined as "Noonan-like" by We describe a year old male with 2 9/12 microgastria, Baraitser and Patton in 1986. All cases have been sporadic left upper limb hypoplasia and ectopic right kidney. and no consanguinity has been observed. We report on a new the 7 lb 1/2 oz product of a 39 week gestation and family with CFC syndrome and an associated chromosomal result insemination donor followingClomid of by stimulation. translocation. Family history is unremarkable except for a maternal nephew A male infant, born after a full and uneventful pregnancy with unilateral hypoplastic facial development and complex and normal delivery, was detected after birth as having coarse cardiac analysis showed a normal defects. Chromosome male facial features, brachydactyly and pulmonic stenosis. On karyotype in our patient and Diepoxybutane and Mitomycin follow-uphe was small for age, had moderate developmental studies ruled out Fanconi pancytopaenia. Pregnancyhistory delay and showed scanty hair. The clinical features are includes exposure to Cyclohexylamine on days 34 or 35 consistent with the Noonan phenotype or the CFC syndrome. The conceptual age symptoms of nausea and with headache. karyotype of high resolution chromosome analysis showed Microgastria results from a defect in the normal 45,XY,-13,-14,+t(13;14). Parental chromosomes were normal. embryological development of the foregut. The clinical The family is of Jewish-Moroccan ancestry. There is no manifestations depend on the stage at which the development consanguinity and no other affected cases were detected. of stomach is arrested. Since all reported cases the of We postulate here that the association of clinical features microgastria associated anomalies of have had mesodermal consistent with the CFC syndrome and "de novo" t(13;14) by derived organs it possible that this phenomenon is represents centric fusion, point to a possible causal relationship a secondary developmental field defect involving lateral between a submicroscopic deletion and the phenotypic environmental insult such as a plate mesoderm. An vascular expression, as seen in other "contiguous gene syndromes". accident or teratogen could be the specific disruptive influence.

There are seven recorded cases of microgastria with limb defects. A comparison will be made between the varying associated anomalies of these seven patients and our patient. Clinical Genetics A61 (0233) 1.121 (0234)

A three generation family with varying ezpressivity Expansion of the CHARGE Association phenotype: hypoplastic of the gene for eleidocranial dysplasia. left heart syndrome and female genital defects do occur in Hodgkinson. K.A.. Chitayat. D. Department of CHARGE. 0. J. Hood and G. A. Greenhaw. Univ. of Texas Pediatrics, Division of Medical Genetics, Montreal Medical School, Houston. Children's Hospital and the Centre for Human The CHARGE Association is a well described pattern of Genetics, McGill University, Montreal, Quebec, malformations which includes: (C) coloboma of the eye, (H) Canada. heart defects, (A) atresia choanae, (R) retardation of Cleidocranial dysplasia (CCD) first described postnatal growth or development (G) genital defects, and (E) in 1766 by Morand is a rare autosomal dominant ear anomalies or deafness. The heart defects have frequently condition considered to have complete penetrance been conotruncal malformations or the commonly occurring but marked expressivity. The main clinical congenital heart defects. Hypoplastic Left Heart Syndrome features include hypoplasia or aplasia of the (HLHS) has been confirmed in only one previous case of CHARGE clavicles, the late closure of wide fontanelles, and suspected in another. However, the association of HLHS abnormal crowded dentition, hand anomalies, and and CHARGE is likely underestimated because of early hypoplasia of the pelvic area including a wide lethality. Significant anatomic genital defects have been symphysis pubis. A narrow thorax can lead to described only in males although hypogonadotropic respiratory distress in the newborn, and a narrow hypogonadism is seen in both sexes. We report here a female pelvis may necessitate caesarean section in the infant with CHARGE who had both HLHS and an anatomic genital pregnant female. We present a 3 generation family defect. Bilateral choanal atresia, bilateral but asymmetric ascertained after the birth of an affected child. retinal colobomas involving the optic nerve and a profound The proband (newborn) was noted to have sensorineural hearing loss were present. HLHS was proven by features consistent with CCD, including respiratory echocardiogram with atresia of the mitral and aortic valves, distress, and an ability to adduct the shoulders undefinable left ventricle and severe hypoplasia of the anteriorally. X-rays revealed aplasia of the left ascending aorta. Her external genitalia were female with clavicle and a hypoplastic bifid right clavicle. severe hypoplasia of the glans clitoris but preservation of Examination of family members revealed features in the corpus clitoris and uterus. Typical CHARGE facies the mother, aunt and grandmother of the proband including square shaped face, facial asymmetry, abnormal ears consistent with a diagnosis of CCD including 2 and pinched nostrils were present. Chromosome analysis was members with cervical ribs. This family show 46,XX. Although the etiology of CHARGE remains unknown, our marked intrafamilial variation, and illustrate the patient supports the hypothesis that a common pathogenetic need for detailed clinical assessment of family mechanism involving deficient contribution by the cephalic members following the birth of a child with CCD. neural crest to craniofacial and prochordal mesenchyme underlies the craniofacial and heart defects in CHARGE. Defective sacculation of the mitral and aortic valve anlagen or deficient subendocardial tissue in the mitral and aortic annuli may lead to HLHS and would likely be influenced by neural crest cell mass. Thus, infants presenting with HLHS should be investigated for CHARGE. Females with CHARGE should be carefully examined as anatomic genital defects, although subtle, do occur.

(0235) 1.122 (0236) 1.123 Early cirrhosis in survivors with Jeune thoracic dystrophy. The Nosology of Atelosteogenesis I and II and Boomerang L. S. W. J. Dysplasia. A.G.W. Hunter and B.F. Carpenter, Divisions of Hudginsl, Rosenareng, Treeln2, Hyams2 Genetics and Pathology, Children's Hospital of Eastern Ontario, 1 Tucson. Dept. of Pediatrics, Univ. of Arizona College of Medicine, Ottawa, Ontario, Canada. 2Dept. of Pediatrics, Hartford Hospital, Hartford, CT and Univ. of Atelosteogenesis is the term that has come to be accepted Connecticut School of Medicine, Farmington. for the chondrodysplasia first described by Maroteaux et al. Jeune thoracic dystrophy (JTD) is an autosomal recessive (AJMG 13:7-14) and Sillence et al. (AJMG 13:15-25) in 1982. osteochondrodysplasia which is often lethal, either secondary to This lethal condition is characterized by a dysmorphic facies, respiratory insufficiency in infancy or related to renal failure in and marked underossification of the vertebral bodies, distal childhood. Hepatic abnormalities such as periportal fibrosis and humeri and femora, proximal and middle phalanges and pubis. bile duct proliferation have been found consistently on necropsy The skeletal phenotype is variable. Bone histopathology is and liver biopsy, however, patients are reported to be notable for lakes of cell-sparse matrix and nultinucleated wasymptomatic". In 1987, Whitley et al. reported a newborn with giant cells. Kozlowski et al. (Br J Radiol 58:369-371, 1985) and coined the term "boomerang" dysplasia for a condition where JTD who exhibited transient direct hyperbilirubinemia typical facial appearance, underossification of the spine and phalanges pathological findings on liver biopsy. The authors suggested that changes in the pelvis and bone histopathology were similar to clinical liver dysfunction resolves over the first few months of life. atelosteogenesis. Many of the long bones showed no In contrast to their findings, we report two siblings with JTD who ossification and those that were radiologically visible had an have progressive hepatic dysfunction. unusual angtilated shape. In 1987, Sillence et al. (Ped Radiol The first patient, now age 27 months, presented in the newborn 17:112-118) reported four children who had some radiologic period with typical radiographic findings and direct hyper- similarity to atelosteogenesis. However, there were a number bilirubinemia, which resolved by 4 months of age. Serum of differences, including the shape of the distal long bones, aminotransferases remained markedly abnormal, however, and the pattern of ossification of the short bones and pelvis, as liver biopsy at 1 month of age revealed periportal fibrosis with bile well as the bone histopathology. A classification of duct proliferation. Progressive liver dysfunction occurred over the atelosteogenesis I and II was proposed. first year of life and repeat liver biopsy at age 1 year revealed a Based upon published reports and our own case of severe well-established cirrhosis. Her brother, who is also affected wtih atelosteogenesis, we conclude that AT I and boomerang dysplasia are of a continuous of common cause. not direct hyperbilirubinemia, but his serum part spectrum JTD, did demonstrate Atelosteogenesis I and II appear distinct, but details of cases were elevated. fibrosis aminotransferase levels markedly Bridging providing clinical, radiological and pathological data are with cirrhosis and bile duct proliferation were detected on liver required before this conclusion is accepted. biopsy at 2 months of age. At age 11 months, his serum aminotransferase levels remain abnormal. Our findings suggest that not all individuals with Jeune thoracic dystrophy have wasymptomatic" liver involvement, and that progressive hepatic dysfunction associated with cirrhosis may be a significant cause of morbidity in this condition and should be monitored. Prognosis for those who survive respiratory compromise must be guarded in light of significant risk for progressive renal and/or hepatic dysfunction. A62 Clinical Genetics (0237) (0238) 1.124

CHARGE Association with Complete or Partial DiGeorge Vitamin D-resistant rickets and alopecia: three affected Sequence in 4 Patients siblings frn the Dominican Raptblic. M. Jauezz de Gutierrem AK Iafolla, A McConkie-Rosell, S.G. Kahler, Division of and V. B. Penchaszdh. Unidei de Genetica, Hoepital R. Reod Genetics and Metabolism, Department of Pediatrics, Duke Cabral, Santo Daaingo, DIkninican Rerublic and Division of University Medical Center, Durham, NC 27710 Medical Genetics, Beth Israel Medical Center, Maut Sinai DiGeorge Sequence has been reported infrequently with CHARGE School of Medicine, New York. association. We present four unrelated patients with Vitamin D-resistant rickets with alopecia is a rare features of CHARGE association and evidence of complete or sutomomI recessive condition, most cases bea described in partial DiGeorge Sequence. Male:Female (2:2), Meditern people. Cellular defects are het s but White:American Indian (3:1), gestational age <35 weeks all include sane aknorsality in the 1,25(OH)5D receptor (2/4). Karyotype by peripheral blood lymphocytes was properties. We report here three affected siblings (2 obtained in 3/4 patients, profound lymphopenia in 1 patient fe-ales, 1 male) with vitamin D-resistant rickets and necessitated karyotyped by skin fibroblasts. Karyotypes alopecia, ronized in the Dominican Republic. The parents were 46,XX (2/2), and 46,XY (2/2). Features consistent with are stizo from the sume village, although dany CHARGE association included: Colobomas (4/4), Heart Defect coneanguinty. The patients were 9 y.o., 5 y.o., ad 18 (3/4), Atresia Choanae (2/4), Cleft lip and Palate (2/4), manths old, respectively, at the time of their Amet recent Radial Defects (2/4), Renal Anomalies (2/4), Pre and exmination. Their clinical histories were remarkably Postnatal Growth failure with birthweight < 5th %ile (4/4), similar. They apeared noreal at birth and developed Tracheoesophageal fistula (2/4), Malformations of the alopecia by six months. They stood up in the second year of forebrain (4/4). In addition, features of DiGeorge Sequence life but prorssive bone deformities and dmcleweakness included: low set, posteriorly rotated or abnormally shaped prevented them to walk. They received vitamin D treatment in ears (3/4), downslanting palpebral fissures (2/4), profound high doss without io . On examination their height hypocalcemia (4/4), absent thymic shadow on Chest X-Ray was below the 3rd centile. They had generalized alopecia, (4/4), absent parathyroid tissue on autopsy (1/1), abnormal proM inent forehead, sparse sybr and eyelashes, delayed parathyroid function by endocrine evaluation (3/4), and T dentition, enasel hypoplasia and dental decay. There nmre cell dysfunction by immune profile (1/2). All patients had evere bone deformities and wide setaphyses. Skeletal X-rays repeated infections. Two of four had documented candidal showed extensive rachitic changes, radiolucent and deformed sepsis, all had at least one episode of gram negative long bones, coarse trabecular pattern, widening and bacteremia. All died of overwhelming sepsis. DiGeorge destruction of the Metaphyseal plates, epiphyseal chags, sequence may be a more common feature of CHARGE association, platypdyly and scoliosis. The severity of the changes and when present may add to the morbidity and mortality of correlated with the age of the patient. They had sArked the disorder. hypocalcamia, drated hypophomphatemia, very high levels of serum alkaline phosphtase, generalized amino aciduria and deficient urine acidification. Study of the 1,25(CH)XD receptor structure and function at the cellular level will elucidate whether the defect in this family is similar or different to cae of other ethnic origins.

(0239) 3.9 (0240) Linkage analysis demonstrating association of DXYS1 and non-specific Oculodentodigital Dysplasia Syndrome Associated with X-linked mental retardation. KB. Jedele. V.V. Michels. KV. Wagner. Abnormal Cerebral White Matter. J. Kamholz. D.H. D.J. Schaid. S.N. Thibodeau. Mayo Clinic, Rochester, Minnesota. Gutmann, E.H. Zackai. D.M. McDonald-McGinn. and K.H. Mental retardation unassociated with the fragile X syndrome accounts Fischbeck. The Hospital of The University of Pennsylvania and for nearly 1/2 to 2/3 of patients with X-linked mental retardation The Children's Hospital of Philadelphia, Philadelphia, PA. A with various types of non-fragile X non- (XLMR). number of families Oculodentodigital dysplasia (ODDD) syndrome is an uncommon specific XLMR have been investigated using molecular genetic techniques disorder with ocular abnormalities, facial dysmorphism, syndactyly in an attempt to localize the gene(s) for these disorders by linkage and defects in tooth enamel. Affected patients manifest bilateral analysis. As a result of these studies, there have been several reports of microcornca, syndactyly of the 4th and 5th fingers, small nasal alae non-specific KLMR (of varying degrees) with or without associated pheno- with anteverted nostrils, and enamel hypoplasia. In some of the typic anomalies that suggest linkage to DXYS1 using the DNA probe previously reported pedigrees, affected individuals also manifested pDP34. We report another family with non-specific XLMR in which most spastic paraparesis without clear etiology. We describe a patient of the males are mildly affected without other apparent phenotypic who has ODDD syndrome and spastic paraparesis with diffuse abnormalities. We evaluated 31 family members from 4 generations cerebral white matter abnormalities on brain magnetic resonance imaging (MRI). Her family history was negative. This patient including 11 affected and 5 unaffected males. Chromosome analysis on 2 manifested microcornea, syndactyly of the 4th and 5th fingers, were X. A total affected males and 2 carrier females negative for fragile aplasia of the middle phalanges of the toes, a marked depressed of 19 polymorphic markers spanning the entire X chromosome, 15 of nasal bridge with bilateral epicanthus and severe tooth enamel which were informative for linkage, were examined with this family. Of hypoplasia. At twenty years of age, she developed progressive these, pDP34 which defines the locus DXYS1 demonstrated evidence of difficulty walking with frequent falling. Her neurological linkage with a peak LOD score of 2.44 at a max of 0.001. For this examination was remarkable for lower extremity spasticity and analysis, we assumed a penetrance of 100%. Linkage was not demon- hyper-reflexia. MRI of the brain and spinal cord demonstrated strated for those markers (Stl4, 55E, F9) known to be linked to the diffusely abnormal high signal in the subcortical white matter and no fragile X locus. The finding that several families with XLMR, plus or evidence for spinal cord compression. Evoked potential responses minus other abnormal phenotypic features, show evidence of linkage to were abnormally prolonged, also suggesting diffuse cerebral white mattter disease. Her evaluation showed no evidence of HTLV-I DXYS1 suggests that a gene for MR may exist in the region of Xq21.31. infection, multiple sclerosis, or any known metabolic cause of The difference in phenotype between these families could represent leukodystrophy. The association of ODDD syndrome and spastic involvement of other genes in this region as part of a contiguous gene paraparesis (with signs of cerebral white matter disease) in this syndrome. Alternatively, this area of the X chromosome may be prone to patient suggests that the gene or genes which are defective in ODDD mutation resulting in multiple different but closely-linked gene mutations syndrome may be involved in both central nervous system which cause the different types of XLMR which have been linked to myelination and morphogenesis of the face and extremities. DXYS1. Examination of other pedigrees will be important to confirm this relationship. Clinical Genetics A63

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Memory disorder in first degree relatives of optimally healthy very old Clinical overlap of classical Angelman syndrome and Prader- individuals and Alzheimer's disease patients. J. Kaye and H. Paysmi. Oregon Willi syndrome phenotypes in a female with a del 15 (qll-13) Health Sciences Univ., Portland. mat. A.J. Kirkilionisa, A.E. ChudleyE, H.L. Luke3H,M. Toffler2, C.A. Gregory', and J.L. Hamerton. Twenty-five optimally healthy very old individuals and 32 Alzheimee's 'Department of Human Genetics, University of Manitoba, disease (AD) patients were evaluated at the Alzheimer's Disease Center of Winnipeg, Manitoba, Manitoba Developmental Centre, Portage La Oregon by a standardized history, neurological examination and brain CT or Prairie, Manitoba. MRI scan. The healthy very old were recruited for excellent heath, functional The Prader-Willi (PWS) and Angelman syndrome (AS) share memory range to the same apparent cytogenetic and molecular lesions of 15qll-13 independence and absence of complaints of failure (age 85 and yet exhibit distinct clinical phenotypes. The etiology of 101, mean 90.4+4.8; male:female ratio 13:12). They had a standardized 2 hr. PWS or AS appears to depend on the parental origin of the psychometric test examination. Patients were selected sequentially as they aberrant chromosome 15. Clinical overlap has not been reported received a clinical diagnosis of AD by NINCDS-ADRDA criteria. Patient between deletion positive PWS and AS patients. We report a 30- selection was solely based on the diagnosis, regardless of family history or any year-old mentally retarded female with a visible cytogenetic other variable range to mean age at onset range to deletion of 15q11-13 who presents with the typical cranio- (age 47 92, 71.2±9.3; 38 80, facial, behavioral, and neurologic features of AS. She was mean 64.2+10.2; male:female ratio 22:10). Family histories were obtained by placed into institutional care at age 4 years. According to her a standard questionnaire and interviews with the healthy subjects, and two care givers, she had hyperphagia since admission. She was well informants, usually the spouse and a sib, for AD patients. All subjects are known for stealing food from other residents and would eat Caucasian. crumbs from the floor. She would eat anything at anytime. Because of this behavior she was monitored carefully at Only 16% of the old a parent or sib with healthy reported having mealtimes and was placed on a calorie restricted diet. She is progressive memory disorder, whereas 67% of AD probands had a positive moderately obese for her height. Her hands and feet are small. family history. The difference between the two groups is more striking when These latter features are characteristic of PWS and not AS. one considers the prevalence of memory disorders among 1P relatives, rather The molecular studies showed deletions of maternal origin than percentage of probands who have a positive family history (see table). for five distal PWCR loci but normal copy number for the most proximal are a locus D15S13. These findings identical to those Parents of the healthy old individuals have lower prevalence of progressive found in other patients of our AS series who do not exhibit any memory disorder than any other group reported, suggesting that protective PWS features. To the best of our knowledge, this is the first genetic/familial factors can in some cases outweigh the predisposing effect of report of concurrence of classical Prader-Willi and Angelman increasing age. syndrome phenotypes in a female with a del 15 (qll-13) mat. Clinical overlap between PWS and AS has been reported Healthy very Old Alzheimer's Patients previously in a karyotypically normal PWS female patient who No Aff % No Aff % P demonstrated the characteristically stiff and jerky gait of AS Parents 265 yr* 45 1 2.2 15 10-4 in addition to aphasia (Williams et al, 1989, Clin. Genet. 44 34.1 35:303). The clinical findings in these patients suggest that Al parents 50 1 2.0 59** 16 27.1 10-3 overlap in the symptomology of PWS and AS can occur and that Sibs 265 yr*** 66 4 6.1 69 16 23.2 10-4 PWS and AS result from deletions of distinct critical regions All sibs 87 4 4.6 121 18 14.9 0.02 which are strongly influenced by the parental origin of the deleted chromosome 15. *Mean age of parents 265 is 81 for both groups. **Data unavailable on 5 of 62 parents of AD patients.***Mean age of sibs 265 is 75 for AD group and 82 for healthy group. The two groups have similar sibship sizes.

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Sternal Malformation and Vascular Dysplasia Association: The Sex ratio for tics, and attention deficit disorder additional patients, furtheL complications, and dIscussion2 (ADD) in persons with tics, is approximately 1:1 in of inheritanqe. &.L.g~lina D.M.McDonaldtMcGinn . N.Rose I?ichenfed4..L.lnijgA. J.M.Temoleton.Jr.~ and E.H.Zackai-. relatives of Tourette syndrome (TS) probands. EL 2Division of Medical Genetics, Jefferson Medical College. Knell and D. Comings Dept. Medical Genetics, City of Hope Divisions of Clinical Genetics, Dermatology and Surgery, Medical Center, Duarte, CA 91010. The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, PA. Tics are the preeminent symptom of Tourette An association between sternal clefting and vascular dys- syndrome, and are reported as more frequent in males, 3-4:1. plasia was shown by Hersh et al (AJMG,21,177,1985) and felt ADD also has a similarly skewed sex ratio. These disorders to arise from a developmental field defect involving midline may actually be more common in males, due to genetic, mesodermal structures at 6-9 weeks of The vascular gestation. hormonal, environmental, social or other or an dysplasias include cutanous hemangiomas as well as internal factors, lesions. Seventeen cases have been previously reported. The artifact of biased ascertainment. sternal malformations range from partial clefts to complete defects, and the cutanous hemangiomas are found overlying the Sex ratio of people with tics head, face, neck and upper chest. Various complications have TS included subglottic and glottic hemangiomas causing respira- We have examined 338 first-degree relatives of tory distress, multiple hemangiomas in the small bowel and probands by direct interview and questionnaire. Among these pancreas predisposing to sepsis and death, and enlargement relatives, the sex ratio for those with chronic motor and/or of a thoracic aortic aneurysm requiring surgical repair with vocal tics is 1.4:1 (Chi square is 3.03, not significant). Thus, graft placement. The inheritance of this association is unclear. Most in first-degree relatives, tics are not significantly more reports have been single cases in a family. Consanguinity frequent in males. has been noted in Saudi Arabian families with asternia, and there is one such family with two affected siblings. We ADD in first-degree relatives are updating all previously reported cases with respect to ADD as a male prognosis and recurrence. is reported predominantly syndrome. We report two further cases, each born to nonconsanguineous However, when first-degree relatives of TS probands who couples not of Saudi Arabian origin, each with a partial also had tics are studied, the frequency of ADD is high but sternal cleft, a progressive facial hemangioma and an internal equally distributed between males and females (1.27:1, Chi lesion. The first patient developed a complication of the is with our respiratory tract, requiring tracheostomy. The second patient square is 0.70). This finding compatible previous developed a previously unreported complication of this associ- study, that ADD is a pleiotrophic expression of the TS gene. ation: a stroke in the middle cerebral artery with subsequent neurologic deficit, cessation of normal somatic growth and It appears that tics, and that ADD in persons with tics, developmental delay. These cases further confirm the existence are distributed between males and of this association, as well as illustrate the potential for equally non-proband developing life-threatening visceral hemangiomas. females. A64 Clinical Genetics (0245) (0246) 1.127

belated Deects Caused by Human TEratoges_ D. KIer and F. Corder. GMS syndruc: a now dominant dinorder with D ais, Divide. Of Dfet mad for Brth DofdepmeaDlsalities, Keger F37, Comtr mental retardstic and ehrt stature. G. S.Al. hJky Diseae Control, Atlanta, Georgia M. D. admmned F. Gilbertf fBeth IMa1 MAdical Center and Ar ceg-bpattma of _ratbaa beena ba ark for bhmanterato saut Slnai School of Madicine, Mm Yozk. such as hydame ta, feal alcohol syndrome, Ibetreline embryopalby mad other.. We report here on a mother and dom ter ift slr an Litl attenion baa born paid to iceated nefmcta cauoed by teratogene. Westudied unusual eie unly, midygrmwsis, in addition to: the ee et ange majOr among chidren exposed to epicenthal folds, dSaeulanting fisr, S11 asc uat durIog pregnancy. An extendiv review of the literature ears, nicroncrgily, shArt rnos with depre dI bridge, conducted looking for prospective studies of exposure to the borderleo philtrua, m11 hias, short stature and mental a hydanteba,phe-i __a * cb _eeamd Talcvalproica:acid. SledesStudies that1icWnclded retardation. Goni gme-sis, also k n ma the Rige eye a postnatal systematic evaluation for birth defects and presented individual reports malforamtion is a structural defect of the Onterior chd er of prelpancyezpeeur and outcomes wereueed for the review. We found 5 Cohort consisting of iris hypoplasia, a prom t S line ad studie tlat the aelection crierhl. We abstracted data about each pregnancy iris alusions that attach to the Sch'lbe line. Several Including exposures, prancyoucemes, and ap variables.IUS studi ricsxlcular railies he bIe, dscribed in mociation with cuded 379 subjects. 16 usd y, 19 used two acvumnt and 7 this defect, such as hypodntia and failure to involute of udd re.43 (11 )ofhe sbjecb hdati a major mfoaon ofwhom35 the periumbilical skin, Only labeled as the Riger (81%)had an ielated alformatienand 8(19%) had two or Are mmfoMrmtieos. srIr!M. This diagosis is KCluded since our.patients lack The COmmon a among pregnancies ware Congenial hypoditia and auoral periumbilical skin. heart dleease (11 cam), nail bypoplasa (3 cam), urgeseeal defects (4 cases), and short stature aid mental retardation are not usualy oral chb (3 Cases). Thore was no difference the rate of birth dfects by described in Ringersyndrome. syrsiom (tiic tsapy when compared to polytherapy (p=O.S7). There was no signifcant for S short stature, H hyperectensibility of joints or diMl encoIinthe preportien of multiple with the of polytherapy. hernia, 0 ocular depressioi, R Rigger eye awnly, T teething Fding tt most r observed uamn bables exposed to anticonvulsants delayed) is an autosamal recessive conditian different from were odeated defect is abe consistent with the previous findIngs in studies of this report with regards to the inheritane pattern, the diabetes in pregancy. A previous CDC study (Pediatrics 1 ;85:1.9)showed that finding of ocular depression and the levl of intelligence amonguldependnt diabolks, 2@(7M%)of the2malormedbabies hadbolated (lo rnmal in SosyrIT imm). Bevinok et al (as J MAd maIformations. Comuderngas etlogically Mrated, onlythose cas wheremultiple Geet 26:825-831, 1987) reported a aother wl with awf e preset may accoset for only about 241% of the children with nicrocegp-aly, Smll ears, mantal deficieniy, iArt stature malformatons caused byteratogens. faoe fludings haveigmalcant Isplicatots for and eye amamlies duecribed as colob an lo studie of birth defects sic ioolated birth defects nicronor k and nicr alaia. Several of toM fiz may be explaIned by a at lent in a proportion of caes Thy alSo are similar to those in our fully but the eye involvemant ereo the need for cmdh prenatal history for exposure to teratogeon in sem different since hmidygmusis is not peenIt. chilldren with belated malforatims conolude that the mother and dmighter repord here hav a distinct pherntype not previusly deribed. It is libely a ne syndrmew consistent with dminent tre siion. We propose the desigation "GM syrrmA " an a Tmismic for G iAgasis M mental retardation and S shart stature.

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Pathogenetic heterogeneity of open vs. skin- Frontoparietal encephalocele of the entire frontoparietal covered neural tube defects: a population-based cortex: A derangement of neural tube disjunction? G. La- study. E.J. Lammer. G.M. Shaw. and N.G. Jensvold. Trenta, J. Neidich, R. Ward, J. Davis, N. Zellers, and L. California Birth Defects Monitoring Program, Hoffman. New York Hospital-Cornell Univ. Medical Center, NY. Emeryville. Encephaloceles are rare, occurring in North America in one in ten thousand live births. 70% of encephaloceles occur in Recent studies have classified neural tube defects the posterior cranial vault. Sagittal and parietal encephalo- by the most superior vertebral level of the celes, account for only 12-13% of all encephaloceles. In a lesion. This classification scheme presumes that recent series of 13 parietal encephaloceles, all cases present- NTDs above T12 pathogenetically result from ed with degrees of holoprosencephaly ranging from partial abnormal primary neurulation, while NTDs caudal to agenesis of the corpus callosum to lobar holoprosencephaly. or at T12 result from abnormal secondary We report a patient with frontoparietal encephalocele con- neurulation. We hypothesize that the interface of taining the entire frontoparietal cortex. Lobar holopros- the 2 neurulation processes is more caudal, and enencephaly, intracranial lipoma with absence of the corpus that defects of secondary neurulation are dis- callosum, and deviated lateral ventricles were noted. Three tinguished by their closed nature and a different dimensional CT scan at two months of age revealed pancranial pattern of accompanying anomalies. To examine this synostosis with the absence of the sagittal suture. Sugges- hypothesis, we studied approximately 290 tion of a dural constriction band around the base of the fetuses/infants with NTDs who were born during encephalocele implied that spontaneous resolution of the en- 1983-87 in California and were ascertained through cephalocele with growth would be highly improbable. Cerebral a population-based birth defects registry. All angiograms revealed bizarre middle cerebral and pericallosal eligible cases were > 20 weeks gestation and had arteries coursing vertically over the brain mass with a super- spina bifida. Medical records, x-ray reports, ior sagittal sinus capping the abnormal brain mass. Gestation- surgical notes, and autopsy findings were reviewed al and family history was positive for cocaine use during preg- for this study. Cases were classified by the nancy and negative spins bifida or hydrocephalus. Cranial level of the lesion, the presence or absence of a vault expansion with duraplasty was performed and the findings full-thickness skin covering, and by accompanying will be discussed along with photographs, three dimensional anomalies. In contrast to open NTDs, skin-covered CT analysis, angiograms, and two year follow up. NTDs were: 1) much less common (83% vs. 13%); 2) A derangement of neural tube disjunction has long been less frequently associated with Arnold-Chiari theorized as the primary cause of neural tube defects (spinal malformation or hydrocephalus (79% vs. 33%); and dysraphism). Embryologic research has shown that caudal neuro- 3) frequently associated with contiguous abnormal- pore closure occurs at 30 days, two days after cranial neuro- ities of differentiation (e.g. lipomas, pore closure. Failure of neural tube disjunction has been teratomas). Our findings suggest that low skin- theorized to result in adherence of dermis (dermectoderm) to covered NTDs may result from abnormal different- the nervous tissue (neuroectoderm), and if intraneural mesen- iation of the caudal cell mass and that the skin- chymal rests are present, lipomatous tissue may be encountered. covered nature of the NTD, rather than its These features were directly observed as the operative proced- location relative to T12, may be more useful for ure ensued. We conclude that a derangement of cranial neural pathogenetic classification of NTDs. tube disjunction was the primary lesion causing the fronto- parietal encephalocele in this patient. Clinical Genetics A65

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A genetic study of creniosynOstosis. Phenotypic Correlations of Non-Syndromic Ocular Coloboma. M. La Msrrer, V. Ledinot, D. Ranier, D. Mbrchac and M.L, Briard Unite K.A. Lepaig and R.A. Pagon. University of Washington and do Rscherches sur les Handicaps G0netiques de 1'Enfant et Departemant de Children' Hospital and Medical Center, Seattle. PFdiatrie, H6pital des Entants Malades, Paris, France. Ocular coloboma can occur as an isolated malformation We report the results of a genetic and epidemiologic study on 657 cases or in conjunction with multiple congenital anomalies (MCA) of craniostenosis made between January 1975 and Decemer 1988. or mental retardation (MR). Coloboma with MCA or MR From 532 non syndrosic craniostenosis index cases. 299 fasilias studies occurs in numerous well-defined monogenic and chromosomal were performed. The relative frequency of the different types is as follows disorders as well as in malformation syndromes of unknown premature fusion of sagital sutures (44 V) and coronal sutures (30 S), etiology. Isolated coloboma is commonly an autosomal oxycephalias (11,7 5). trigonocephalies (7 5) and complex craniosynostosis dominant disorder. In addition, there are patients with (3.3 %). non-syndromic coloboma (NSC) with or without MCA or MR who For scaphocephaly. the sex ratio was significantly high with a sale excess have normal chromosomes and a negative family history. (4,66). We performed a retrospective review of 53 patients with Associated malformations were reported for 12.5 among the cases of NSC from our institution to determine the incidence of scaphocephaly but no syndrome could be recognized .18 among 32 families MCA and development delay (DD) or MR in this population were multiplex and 6.08 p 100 of the sibs had a craniostenosis. but only (Table). The 53 NSC patients were compared with 11 1.3 p 100 of the parents. patients with familial, isolated coloboma by coloboma The premature fusion of the coronal sutures affected rather the girls (sex phenotype (1- iris and/or chorioretinal coloboma; 2- disc ratio for plagiocephaly 0,54, and for brachycephaly 0,96). Paternal age coloboma, cystic eye, anophthalmia). Phenotype I usually mean was significantly high for plagiocephaly and brachycephaly (32,75 has normal vision, whereas phenotype 2 usually has visual and 32,38 for control sample 29.10). impairment. In 42,5 p 100 of the brachycephaly families and 20 p 100 of plagiocephaly TABLE there were two cases at least. NUMBER OF PATIENTS BY Brachycephaly and plagiocephaly could be observed in a same family this NON-SYNDROMIC COLOBOMA PHENOTYPE finding suggests that these two affections my be variable expression of COLOBOMA PATIENTS NO. x 1 2 a sam pattern i.e. a premature fusion of coronal sutures. Among 85 sibs + MCA + MR/DD 13 25 10 7 of children with brachycephaly or plagiocephaly, 15 sibs were affected - MCA + MR/DD 2 4 2 2 and 14 parents among 176. - MCA + nl IQ 21 40 19 12 Trigonocephaly is more prevalent among boys (sex ratio 4.751. 6.5 p 100 + MCA + nl IQ 11 21 10 7 of sibs were affected and 4.2 p 100 of parents. MCA* 5 9 3 3 For oxycephaly and complex craniostenosis, the data were not too sufficient TOTAL 53 to be included in the survey. All these data put foward that "pure craniostenosis' will be more familial FAMILIAL COLOBOMA than it could be estimated from the literature. We suggest that premature PATIENTS coronal fusion without any other malformation will be a dominant autosomel TOTAL 11 8 8 trait. *Children less than 1 year age at the time of evaluation or death.

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Growth study in Chinese children with Down syndrome Synuromes, associations, a deaopental fields: Delination throuh in Taiwan: 1 month to 18 years of age. S.J.Lin, statistical properties of depeueoce and itiity. M. S. At . S.C.Hu', S.F.Hsu',J.W.Ho , P.C.Chio', M.C.Chao* and Divisim of Genetics, Medical ColWe of Wiscouen,o? T.R.Wang2. Cheng Kung Univ. School of Medicine(l), Models of terat is suggest different relationships betwen multiple Taiwan Univ. School of Medicine(2), Veterans General COMoital SO lis with different types of causes. Tese are reflected Hospital (3) Kao Shung Medical School(4), Taiwan, in tH statistical properties, Depenene and Tsitivity, tiet cm be R.O.C. exmied using clinicsl material. (Note: In exuale: a,b,c are aemlis, Growth charts specific for Down syndrome is de- F(x)m frequemy of fimry x.) sirable for continuing care of these children. An- 1. is die influn of on admaly on the frequeny of thropometric studies of Down syndrome had been pre- anther, or the ratio of F(a) with c to F(a) witshut c. sented in previous publications. However, body size 2. For titive a duelies if a associates with b, and b with c, a has been known to have significant ethnic difference associates proporticietely to c, or: F(afb toether)F(a) x F(aft . Each different ethnic group needs their own growth togetdr)/F(a) - F(bec together). Trasitivity is ewed/expected for data. When compared growth of normal children in F(bfc together). It is unaffected by andd effects Taiwan with NCHS growth charts of normal American relatiombipe to all other aimllies quslly. children, Taiwanese children are shorter. We pre- lue properties differ for synuromes, assoction., and developmtel sented here data for Chinese children with Down fields: syndrome based on 1208 observations on 497 children ociati are dsrivatis of teratogmic eints acting on eJbo . Centile charts were presented for two age inter- ormoised in tie spare. Such te may not overlap. lDWefore, a ai! vals, 1 to 36 months and 2 to 18 years. And when c my both associate with b, but ot with eah other. If a, b, Ad c am compared with growth charts of American children lif : 1. The associatio of b with a or c is dEcressed by piresss with Down syndrome (Cronk et al 1988), we didnot of c or a. 2. The pree- of a 6 c ttep inrs tie frlgmey of find significant difference. This might indicate b. 3. The presses of b ineres the associatin of a ai! c. that the influence on stature is more evident from In opmetal fields appewm of an anly a on die field aneuploidy than from ethnic factors. (This study is being pertubed ax! the naly resultig from diet evnt. The prese supported by a grant from Health Department Exacu- my me field ainomly Increas dithe likeliod diat tde field w affected, tive Yuan, Taiwan,R.O.C.) ad terefore also ncreases the frequley with uAhdu otd caponent asomlies are men ltiple Anmlies in Poetic sind- an gsaly related to an camuse aw ot to e other. Her de, sxent cdnag in fro Iuseeias agest deelopmstel fields or equ within a syndrom Ovrall: syunxrma field asociation titiwa + + - depaxdmt + +

Tee properties sould pmit die objective deliation of multiple malis, distinish mm plilotrW from sequential diaturaness, axd permit ie mapping of aaocatio. A66 Clinical Genetics (0253) (0254) 1.131

Phenotypic Spectrum in Hydrolethalus synrme Autosomal recessive hypothalamic corticotropin deficiency: S. Madan. S. Cao. A. Aiwaebm. M. Ben-Yisavy. A new entity and its metabolic consequences. H. Mandel, M. V. Pulisaal and H.M. Nitckiy. Albert Einstein Oollege Berant. E. Gotfried. and Z. Hochber. Rambam Medical Center of Medicine, Bomx, New York. and Technion Faculty of Medicine, Haifa, and R. Seiff Hydrolethalus syndrme dcaracterized by hydramnios, Hospital, Safed, Israel. (Intro, by M.G. Blitzer) hydroo ly, sultiple malfozmttics and lethality, is a rare Eleven children of a consanguineous kindred were affected AR disorder first descrbed in the Finnish ared later in with autosomal recessive hypothalamic corticotropin defi- other ethnic grups. A 24 year-old G2POO10 Caicasian wman ciency, the hypothalamic origin of which is confirmed here was referred during the third trimester with a presuaptive for the first time. Seven infants died undiagnosed. From diagnosis of hol l y an ultrasound. itesis review of the available clinical information, findings inclu- revealed a rmal 46,XX karyotype, confirmed postnatally by ded lencephalopathy', hypoglycemia, hepatitis, and facial dys- high resolution banding. Massive lhy ly, brain morphism. Four affected patients were studied extensively, dysplasia, shortened lits and oligiydaos were cbsierved two of whom were diagnosed prenatally. The first diagnosed on sAgram and MI. Delivery was by C-section for breech patient presented at age two months with hypoglycemia, hepati- presentation and salocranium; birth weight was 2455 gms., tis, facial dysmorphism, convulsions, and agenesis of the cor- length 52 ac. and occipitofntal cirmfere 45 cm. pus callosum. Plasma ACTH and serum cortisol (F) were unde- Anomalies included ceplex micrciha with cpacified tected during hypoglycemia and following ACTH stimulation. coeas, short palpa fissures, telecanthus, beaked nose, Corticotropin releasing hormone (CRF), 1 ug/kg, failed to

cleft palate, grade III microtia, eic , heart raise ACTH. Repeated CRF administration for five days in- defect, single umbilical artery, simin creases, creased ACTH to 83 pg/ml, and 5 u of vasopressin induced a trihialaneal thumbs with har-like nails and increased rise to 36 pg/ml. Primary hypothyroidism, diagnosed in the joint laxity. Death occurred within 48 hours. Autopsy second patient at age four years, proved to be secondary to aled rocqaly, aqxdctal stenosis, rudimentary organification defect, as perchlorate suppressed iodine uptake corpus calloew, flat posteri foesa, hypoplastic from 9% to 3%. His growth hormone response to GRH was blun- tentorium, T-E fistula, tetralogy of Fallot, hypoplasia of ted; glucagon administration resulted in a flat glucose curve. lungs, alcbar left lung and small spleen. Cortisol replacement therapy resulted in clinical improvement Findings are consistent with Hydrolethalus syndrome with and normalization of thyroid functions and growth hormone re- a transitional containing of Goldenhar sponse to stimulation. In two at-risk pregnancies in the kin- syndroge, VAS/VAC1R asciatin ad retinmic acid dred, prenatal diagnosis of cortisol deficiency was suggested rycpathy. MEu genic as well as sporadic sy anrdesad by low maternal urinary estriol, and confirmed at birth by associaticn(s) are e rgxassed within the spectrum of Axial undetectable levels of cortisol and ACTH. Immediate institu- Mesoderal DLsplasia, (AND). Hydrolethalus may be another tion of cortisol results in normally developing infants. AND and my reflect an aberrant farmation and/or deranged We conclude that this familial hypocortisolism is of hypo- interactio of growth factors and cellular rtors in an thalamic origin. The GH deficiency and thyroid organification early embryo in respose to mutant genes and teratogens. defect are secondary to adrenal insufficiency. This condition appears to be inherited as an autosomal recessive trait. Pre- natal diagnosis is essential for institution of immediate management of an affected newborn.

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Long-term survival in the Jarcho-Levin syndrome is Ablepharon Macrostomia Syndrome: report of a case and related to lack of disIress in the neyborn clinical delineation. D. Markouizos, U. Siddiqi, S. Siddiqi, Iespiratory Methodist of period. R W Marion , B.A. Mttzkin, J.A. Neidich, K. Raziuddin, B. Nangia. Hospital Brooklyn, J.G. Davis and E.C. Traegej Albert Einstein College of Brooklyn, New York. believed to Medicine, Bronx, Hel York; Cornell University Medical Ablepharon macrostomia syndrome (AMS), College, New York; Yale University School of Medicine, represent an ectodermal mesenchymal induction defect, New Haven, Conn. comprises ablepharon (complete absence of all eyelids) in The Jarcho-Levin syndrome (JLS) is an autosomal association with abnormalities of mouth, ears, nose, skin, recessively inherited bone dysplasia in which abnormalities hair, subcutaneous fat, nails, genitalia, and physical and of the thoracic spine and ribs lead to a markedly psychomotor development. Only five cases have been reported constricted chest cage. The majority of infants with this thus far. disorder die in early infancy as a result of respiratory Proband was a full term white female born to a G3 P0 insufficiency, but a small number have been known to survive mother. Parents, in their late twenties, are healthy and not beyond one year of age. We follow three patients with JLS, related. Ultrasound examination had shown oligohydramnios. two males and one female, all of Puerto Rican ancestry, who The infant's measurements were: weight 3.1 kg, length 51 cm, are currently 3, 16, and 23 years of age. In an attempt and head circumference 32.5 cm. Multiple abnormalities were to identify predictors of long term survival in infants noted including severe shortening of all eyelids with corneal with JLS, we compared a number of clinical parameters in exposure, hypertelorism, circumoral wrinkling of mouth, ears with these patients with those of other JIS patients who had macrostomia, high arched palate, low-set died during the neonatal period. No significant differences rudimentary pinnae, broad-tip nose with deficient alae nasi, were noted in birth length or weight, or head or chest absence of lanugo hair, total scalp alopecia, absent circumference. The only striking difference between these eyebrows, absence of subcutaneous fat, redundant folds of two groups was that none of the long-term survivors had skin of the neck, retroauricular areas and back, contractures significant respiratory distress during the neonatal period, of the fingers, hypoplastic convex nails, simean creases, while all infants who ultimately died required mechanical absent nipples, umbilicus without dimple, hypolplastic labia ventilatory assistance. We conclude that lack of majora and minora, and anteriorly displaced anus. At ten respiratory distress during the neonatal period is the weeks infant's weight gain was suboptimal, while head growth only reliable predictor of long-term survival in infants and neurological development were normal. Hearing tested with JLS, and suggest that all steps necessary to medically adequate, but visual acuity could not be assessed. support such individuals be immediately taken. Conversely, The mode of inheritance of the AM; is not known. the presence of ventilatory insufficiency during the Moreover, the term AMS is open to debate. With the exception neonatal period in affected patients suggests that the of macrostomia, absence of lanugo hair, and better visual infant will not survive infancy; this observation should prognosis, the AMS shares many of the features of be carefully considered when managing such patients. cryptophthalmos (hidden eye) syndrome, which is inherited as an autosomal recessive trait. Furthermore, one of the monozygotic twins reported by Azevedo et al (J Med Genet 10:389, 1973) had bilateral cryptophthalmos, while the other had ablepharon on the right and cryptophthalmos on the left. Clinical Genetics A67 (0257) 1.133 (0258) 1.134

A new familial syndome of upper eyelid coloboma, aberrant anterior hairline pattern The neurocognitive phenotype of winen with fragile X. and anal anomalies m M~anioba Indians. M. M. M. Mazzoxco, B. F. Pennington, A. E. Cronister, and SI. Marles CR. Greenberg. TMN Persaud, EP. Shuckeq and A.E. Chudley. R. J. Hagerman. Univ. of Colorado Health Sciences Center, Children's Hospital and the University of Manitoba, Winnipeg, Manitoba, Canada. Univ. of Denver, and The Children's Hospital of Denver. We derbe 6 3F) Mnitoba Indian childen with hypmtelouism and The variability of learning disabilities seen among females varibe omInaioof eye aberrant anterior forehead hairline, nasal who carry the fragile X mutation is great, ranging frcn mild and anomalies Four children have unidaeal upper eyelid colobomata and child retardation to normal cognitive functioning. In this study, has unilateral anoplithalmia. Nasolacrimal ductobstrucon ofthe affected eye is present we investigated the specificity of the neurocognitive profile in 3 children and is bilateral in one of them. No other structural abnormalities were among these wasen and the relation between cytogenetic preset and visual acuity was preserved. AM 6 chilen have a unilaeral anomalous expression of the mutation and the neurocognitive phenotype. wedge of scalp hair extending onto the forehead, ending at or on the affected eyelid. We tested women who fell into one of three groups: fragile X A veslical groove down the nose or isolated to the tip is present in 3 children. One wasen with 2% or more expression, non-expressing fragile X child has an anteriorly displaced anus, 1 child bas anal senosis of a normally placed obligate carriers, and a control group of wamen who do not anus and 2 children had both anomalies. The remainder of the physical examinations, carry the fragile X mutation but who have one or more children includinghearing, growth and development are noral. Qbandedchromosome analyses with a non-fragile X learning disability. ae normal. Crnoial and renal ubrasounds were tasarkable. C-spine films revealed An extensive battery was administered to each wanan. This no stuctur ab its. battery included intellectual and achievement tests; and These 6 children belong to 4 related familial. In these Icndreds, the children measures of left hemisphere, right hemisphere, long term are this cousins (3 familie) or fourth cousins (1 family). parents of one affected memory, and frontal lobe functioning. The intellectual and boy are fist cousi. Two children repreent isolated cases in their immediat families; achievement scores among women in each group was representa- thee we 2 affected Aibs in each of the other 2 families. The parents and 7 other tive of larger samples of these groups fran other research. sibling ar clinically normal on examination. On tests of neurocognitive functioning, the group data indi- Eyelid coiboma may exist in isolation, in association with other craniofacisi cate that no group had deficits on tests of long term merory m with other anomalies such as anencephaly, ventral hernia or with other or left hemisphere functions. In contrast, the expressing mutipecongenital anomaly (MCA) syndromes such as Fraser,Treacher-Colins.Nager. fragile X wrmen performed more poorly on all measures of Goldenhar or Frmsonasal dysplasia. This is the first description of unilateral upper frontal lobe functioning and on one measure of right hemis- eyelid coloboma and aberrant anterior hairline associated with anal anomalies. The phere functioning than did the women in the other two groups. presence of 2 major malformations in siblings and rented individuals (with unaffected Unlike the expressing females, the non-expressing carriers parnts) from an inbred popuiation suggest the likelihood of a new pleiotpic autosomal did not show neurocognitive deficits. An unexpected finding recessive MCA syndrome was that the expressing fragile X wamen performed better on tests of long term memory than did the waren in the other groups. Finally, performance on the neurocognitive and the achievement tests were not in support of Wolfe's hypothesis that fragile X females suffer fran dyslexia. These data suggest there is a specific neurocognitive profile among expressing fragile X females: a marked deficiency on measures of frontal lobe functioning (e.g, the Wisconsin Card Sorting Test) but above average long term memory skills. This profile may be related to previously reported emotional deficits seen among fragile X wLmen.

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Identification of flanking markers for the Charcot-Marie- Marfan syndrome: altered synthesis, secretion or extracellular matrix Tooth neuropathy 1A gene (CMT1A) on chromosome 17p. P.. incorporation of fibrillin. D.J. McGookey. R.E. Pyertz and P.H. McAlpine', T . E . Fe&&by2, A. F. anW.C.Ye C.R. Byers. Univ. of Washington, Seattle and Johns Hopkins Hospital, Greenberg' and D.R. McLeoQ. 'Department of Human Genetics, Baltimore, MD. 3Department of Medicine, University of Manitoba, Winnipeg, Fibrillin is a 32OkD extracellular matrix protein found in Manitoba, 2Department of Clinical Neurological Sciences, microfibrils, which are structural fibers present in the suspensory University of Western Ontario, London, Ontario, 4Department ligament of the eye and associated with elastin in the aorta We of Medical Genetics, Alberta Children's Hospital, Calgary, examined the synthesis, secretion and incorporation of fibrillin into the Alberta, Canada. extracellular matrix by fibroblasts from 21 probands with the Marfan Autosomal dominant Charcot-Marie-Tooth neuropathy syndrome. Dermal fibroblasts obtained from biopsies were labeled with characterised by markedly reduced nerve conduction [35S]cysteine either continuously for 20 hours or pulsed for 30 minutes velocities (CMT1, also known as hereditary motor sensory and then chased for periods up to 20 hours. Control cells synthesized neuropathy HMSNI) is genetically heterogeneous. Mutations fibrillin as a 350kD protein then secreted it from the cell within 4-6 causing this phenotype have been mapped to chromosome 17 hours. The protein was cleaved in the extracellular space to remove a (CMT1A) and to chromosome 1 (CMTlB). We have investigated fragment of approximately 3OkD, and the remaining 320kD protein was genetic markers which map to the short arm of chromosome 17 incorporated into the extracellular matrix. Cells from 7 probands in a series of eight Caucasian Canadian families synthesized about half the amount of fibrillin when compared with segregating CHll mutations and tested the phenotypic data controls but secreted the protein normally. Cells from a second group for linkage. Pairwise lod score analyses gave e of 0.02 of 7 probands synthesized the same amounts of fibrillin as control cells for CMT1A and D17S58, 0.06 for CMTl1A and D17S71, 0.07 for but secreted the fibrillin less efficiently, with a pool of fibrillin CMTlA and HYH2. Inspection of the segregation information remaining within the cells for up to 20 hours. Five out of 7 of these from meioses informative for MMY2, CMT1A and D17S58 or individuals had severe clinical phenotypes. Cells from a third group of D17S71 give a gene order of 17pter-MYH2-CMTlA- 4 affected individuals synthesized and secreted fibrillin normally but did (P17S58 ,1l7S71) -cen. Although we cannot determine the not incorporate the molecules into the extracellular matrix. We were order of D17S58 and D1.7S71 relative to CMT1A unequivocally, unable to detect evidence of altered synthesis, secretion or matrix we have identified flanking markers for the CMTlA locus. incorporation of fibrillin by cells from 3 of the 21 probands. Family Now that boundaries of the region containing the CMTlA gene members of 8 of the probands were also studied. Affected individuals have been identified, this region can be analysed intensely in the same family had the same biochemical defect and unaffected to determine the precise location of the CMT1A gene. family members showed no biochemical defect These results indicate that fibrillin is the major candidate protein Funded by Muscular Dystrophy Association of Canada for defects that explain the phenotypic features of the Marfan syndrome, that the majority of individuals with the syndrome have mutations that affect the synthesis, secretion or matrix incorporation of the molecule, and that only a small proportion of individuals with the syndrome are likely to have mutations in other genes. A68 Clinical Genetics (0261) 1.136 (0262) 1.137

Anthropometric study in the Prader-Willi syndrome with emphasis P p tasia: Clinical, card on hand and foot measurements: Sex, age and chromosome effects. biodwasical irc s in a secord cwe. CJooe FJ. Meaney. J.L Haynes5. and M.G. Butler*. Arizona Dept. of IS fmner. SP Ocean. LAQuivor. KN Ad. and MP RAte. Irdiana Uhiv. Stol of Medicine, dian o, Ft. Wayne Med. Health Services, Phoenix, *Vanderbilt Univ. Ctr., Nashville, State Hospital, Ft. Wayne, IN, C iell Itit, C n, IU, TN. Washirqton Univ. and Shiners Hospital, St. Aiis, MD. Age, sex and chromosome effects on weight, height, sitting (PsIP), a rare decm wi ted height, three head dimensions (circumference, length, breadth), five convirningly in only nae subject (N 281:604,1969), is hand (length, palm length, middle finger length, breadth, wrist by normal serum alkaline pSatase AcUivy (ALP) in the face of clinical riogr ad bioemical breadth) and three foot measurements (length, breadth, ankle evidence of hyp atsia (HYP). In this variant of HYP, breadth) were analyzed in 57 individuals (35 males and 22 females) erdogei accnmlaticn of (FM),i(rE with the Prader-Willi syndrome (PWS). Ages ranged from 0.05 to ganic pyr i te (PPi) and pyridoaml-5'1 J5'ate (PP) 38.6 years with a mean of 12.7 years No significant differences suwort-a funstional ALP deficiency not reflected by clin- ical assays that use artificial substrates at alkaline pH. were observed in anthropometric data between PWS individuals with chromosomes. We report a white feimle infant whio also awears to have the 15q chromosome deletion and those with normal PsHYP. she presented at age 5 so. with a bulging fontanel, Preschool children were found to have dolichocephaly while hand mild lib shortening ard hypotonia. At age 6 mo. she had a and foot measurements, stature and sitting height were within craniectomy for icrmeased ICP; at age 10 mo. he weight and normal range. Foot size was smaller than hand size in PWS females length were <3rd %ile ard she had rahSitic duages. X-rays when compared with males. In adult individuals with PWS, sholed generalized hypcuineralizatin, a l fraying and fracures typical of severe HYP. She had elevated senu measurements, excluding weight, head length and anthropometric Ca and Phos and markedly elevated urine PEA ad Plama PIP ankle breadth, were less than -2 SD. These data indicate that hand levels. Total serum ALP was aa-ornIl in the clinical lab but and foot size are generally normal before 10 years of age, while higher than epmcted given her clinical preeentatic; hEi- abnormal measurements (<-2 SD) are observed in the majority of ever, in the Metabolic Pasearch Unit abratory at Shriners Hospital, total senr ALP was 78 IU/L (n1 75-250 IU/L) PWS individuals after 12 years of age. Short stature (<-2 SD) was -distisotly higher than HYP cases of cmparable severity. also observed in the majority of PWS individuals by 12 years of age, Similarly, ALP in ntes of cultured fibreblasts was while weight was >2 SD by 4 years of age. No apparent difference not subnormal with artificial substrates at alkaline pH. was identified between males and females or between deletion or Her parents are asnptcatic. fhe mother's senup ALP is nondeletion individuals in the onset of these abnormal borderline low bit her plasma PIP is 4X the uwr limit of measurements. Abnormal growth patterns apparently exist in PWS nonral in controls. 7he father has norml serum ALP ad plama PIP. Both parents showed markedly elevated PIP levels as with age, evidenced by significant negative correlations after an oral load of pyridoxine (0.3 i dkd for 6 days). particularly in males, for height, sitting height, head circumference, *other the mechanisem of PsHP in this patient an the and hand and foot measurements. previassly reported case is the sne is unknown; however, these two patients had a very similar early clinical coure. Identification of a secoad patient with POP further illustrates the p'wo*ype of this ural variant of HYP that may present diagnostic and ounselimg difficulties.

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DeBarsy Syndrome. Differential Diagnosis. Brain imaging in variants of the holoprosencephaly malformation sequence: The face is the best predictor of C.A. Horris and E.G.I. Clark. University of Nevada the brain. B.A. Motzkin, R. Goldberg, M. Wechsler, and School of Hedicine, Las Vegas, NV. R.W. Marion. Centers for Congenital Disorders and Cranio- We recently evaluated a child with a neonatal facial Disorders, Montefiore Medical Center, Albert Einstein progeroid presentation most consistent with DeBarsy College of Medicine, Bronx, New York. syndrome (AR), which has been reported in 15 previous The holoprosencephaly malformation sequence (HMS) is cases. The sale infant had intrauterine growth defined as a heterogeneous spectrum of anomalies of the face retardation, bilateral corneal clouding, progeroid and brain which results from impaired midline cleavage of facies, hypoplastic ala nasi, small nasal tip, thin upper the prechordal mesoderm. DeMyer et al. described the lip, long smooth philtrum, transluscent, lax skin, significance of median facial anomalies as a clue to the extreme deficiency of subcutaneous tissue, narrow and existence of underlying midline brain malformations, as seen hyperconvex nails and extremely hyperextensible on autopsy. Clinicians have come to rely on methods of peripheral joints. At 3 1/2 months, postnatal growth brain imaging such as CT scan and MRI to confirm the deficiency, hypotonia and athetoid posturing of the presence of developmental cerebral malformations in the extremities was present. Chromosome studies were normal. living infant to determine appropriate medical management. Light microscopy of the skin biopsy showed few elastic We describe five children with midline craniofacial defects fibers which were thin and frayed. characteristic of HMS in whom CT imaging of the brain We compare selected clinical features of DeBarsy failed to reveal an associated cerebral malformation. The syndrome (DB) with other similar syndromes: Cockayne craniofacial abnormalities noted on physical examination in syndrome (CS); Cutix Laxa (CL), Hallermann-Streiff these patients included midline cleft of the lip and palate syndrome (HS); Hutchison Gilford syndrome (HG); and (4 patients), ocular hypotelorism (3 patients), and Wiedemann Rautenstrauch Neonatal Progeria syndrome (WR): hypoplasia of the nose (3 patients). Medical problems in these individuals were multiple and varied in nature and CLINICAL FEATURES DO Cs CL HG. +R included respiratory distress, seizures, hypoglycemia, and Post mortem evaluation Progeroid Facies + + hyponatremia, hypothyroidism. Sparse Hair + of the brains of all patients in whom autopsies were Hental Retardation + performed revealed midline defects varying from isolated Cataracts + absence of the hypothalamus and pituitary to lobar holo- with arrhinencephaly. Our experience showv Retinal Degeneration + prosencephaly _ be considered in Hearing Loss _- that the diagnosis of HMS must strongly _ _ of midline Joint Limitation +_ individuals with facial findings suggestive of normal CT Joint Hyperflexibility + R brain abnormalities even in the face imaging. Neonatal Teeth MRI performed in one patient did reveal central nervous F system evidence of HBKS. Although MRI may be the diagnostic Abnormal Elastic Fibers + + test of choice in such patients, further studies are Neonatal Onset + R required. R-rare Clinical Genetics A69 (0265) 14.4 (0266) 1.139

The phenotype of X-linked torsion dystonia (XLTD) L. M~iller,. Monozygotic twins discordant for tuberous sclerosi L.V. Lee. G. Viterbo. J. Arancillo. F. Caballar-Gonzaga, H. NorthruDp. J. W. Wheless'. R. A. Lewis2 and T. K. Bertin. M.Hebron-Ortiz. K.G. Kupke The Children's Hospital and Univ. of Texas Medical School-Houston': Baylor College of Harvard Medical School, Boston, MA (U.M. and K.G.K.); The Medicine. Houston. Texas': Univ. of Texas Graduate School of Philippine Children's Medical Center, Quezon City, Republic Biomedical Sciences. Houston3. of the Philippines (L.V.L., F.C.-G. and M.H.-O.); Capiz Tuberous sclerosis (TSC) is an autosomal dominantly Emaanuel Hospital, Roxas City, Philippines (G.V. and J.A.). inherited disorder of hamartomas with an incidence of 1/10,000 The natural history of X-linked torsion dystonia (XLTD) individuals. Approximately two-thirds of cases are the result was documented in 42 affected individuals from 21 families. of new mutations and one-third are familial. One member of a All patients had a positive family history of XLTD consistent set of twin boys was diagnosed as having TSC at age seven years with an X-linked recessive mode of inheritance. In 8 six months. Findings consistent with TSC in the patient families, there were 10 sibahips with two or more affected included a 5 x 5 cm shagreen patch in the right lumbosacral individuals available for evaluation. The mean age of onset region, a small achromic patch in the right eye, a hyper- of XLTD was 34.8 ± 8.1 (S.D.) years. First manifestations pigmented patch in the left eye and several cortical tubers. were noted in head and neck in 39%, in the lower extremities The parents and twin brother were also investigated. in 33%, the upper extremities in 24%, and in the trunk in 9% Evaluation included careful physical examination with Wood's of the cases. Some individuals had initial symptoms in more light examination of the skin, slit-lamp ophthalmological than one body segment. Dystonic manifestations became examination, CT scan of the brain without contrast, renal generalized rapidly (mean duration after onset 5.1 + 2.8 ultrasound of the kidneys, and echocardiogram. The twin had years) in 90% of the cases and the severity of dystonia was an MRI scan in addition to the other testing. No signs of TSC independent of age and site of onset. 36% of the cases could be detected in either parent or the twin. The finding displayed at least one "parkinsonian symptom" (bradykinesia, that the twin had no stigmata was unexpected because the boys rigidity, loss of postural reflexes and "fine" resting appeared identical. Zygosity was tested by performing genetic tremor). Within families, some affected males had marker analyses of twenty independent systems. Based upon the parkinsonian symptoms but others did not. X-linked recessive opportunities for differential segregation of marker alleles, inheritance of parkinsonism without dystonia was never the probability of monozygosity ranges from a minimum of 99.97 observed. Thus XLTD is a nosologic entity which in addition to a maximum of 99.99. Several explanations could account for to dystonic symptoms may also display parkinsonian features. two individuals with identical genetic material being discordant for TSC. Even with extensive testing, estimates are that 2% of cases of TSC have no stigmata of the disease. Also, stigmata of TSC are often not present at birth but appear as the individual ages. Both of these explanations would be dependent on environmental factors causing gene expression in one twin but not the other. A third possible explanation would be that the mutation occurred after the twinning process in one twin only. After the TSC gene or genes are cloned, it will be important to determine at a molecular level the difference between the twins. This illustrates the difficulties encountered in making the diagnosis of tuberous sclerosis for counseling purposes and for gene linkage studies.

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DNA analysis in four generations of a family with X-linked Growth hormone treatment of short stature in Prader-Willi syndrome. Mental Retardation and Agenesis of the Corpus Callosum. E.B. V. A. Pallante. R. D. Lovinger. and J. Bodurtha Medical College of Oto. N.J. Carpgntej.. and V.K. Proud. Div. of Medical Virginia and Children's Hospital, Richmond, VA. Genetics, Dept. of Child Health, Univ. of Missouri-Columbia, Short stature and decreased growth velocity are characteristic features of and Chapman Institute of Medical Genetics, Children's Medical Prader-Willi syndrome, the most common syndromal cause of marked Center, Tulsa, OK. obesity. We report the response to human growth hormone (GH) therapy of We report four generations in a family with three living 4 prepubescent male Prader-Willi patients. In patients 2 and 4 high males, three males who died in the first year of life, and two resolution cytogenetic analysis was performed and revealed the deletion females with neurological impairment and partial agenesis of characteristic of Prader-Willi patients. The karyotype of patient 2 is the corpus callosum (ACC). Clinical features in the surviving 46,XY,del(15)(ql lql3) and that of patient 4is 46,XY,del(15)(qllql2). In males include microcephaly, distal limb contractures, patients 1, 2, and 4 GH levels were evaluated using sequential GH scoliosis, tapered fingers with hyperconvex nails, arid a stimulatory agents: L-arginine and L-dihydroxyphenylalanine. Patients 1 characteristic facies with large eves, prominent: supraorbital and 2 had blunted responses to the provocative stimulation with maimum ridges, synophrys, optic alveolar ridges, and micrognathia. GH levels of less than 10 ng/ml. Patient 4 had a supranormal level of GH Urologic anomalies include renal dysplasia, cryptorchidism, (55-60 ng/ml) upon provocative stimulation suggesting the production of and hypospadias. Prometaphase and Fragile X chromosome biologically inactive Goa The bone ages of these three patients were delayed analyses on one of the affected males are normal. Pedigree from 9 to 21 months. Patient 3 was evaluated at Stanford University and analysis suggests an X-linked form of ACC. The features of was reported to have a very poor response to GH provocation. All 4 patients severe microcephaly, seizures. mental retardat ion. sensorv were treated with synthetic GH at a dosage of 0.3 m/kgtweek and all deficits, large eves. distal contractures and severe scoliosis demonstrated an excellent response which has been sustained as long as the are not consistent with PG Syndrome or Aicardi Syndrome. patient continued GH therapy. Patient 1 began GH therapy at the age of 12 Neurologically normal obligate carrier females have appar(cnt years and has continued treatment for the past 3-4/12 years during which he hypertelorism. a widow's peak. and large eves;. It is has grown 23.5 cm. Patient 2 began GH therapy at the age of 14-3/4 years difficult to propose a mechanism to explain the extreme and has continued treatment for the past 2-1/12 years during which he has variability of expression in the neurologically impaired grown 19.5 cm. This patient has also been receiving testosterone enanthate females compared to the obligate carrier females whose in oil (50 mg/month) for the past year and has progressed from Tanner stage children are severely neurologically impaired ma-les. DNA I to Tanner stage 3. Patient 3 began GH therapy at the age of 8 years and analysis is currently being performed. during the next 2-7/12 years he grew 31.5 cm. Patient 4 began GH therapy at the age of 3 years and during the next 1-7/12 years he grew nearly 22 cm. The excellent responses of these four patients to GH therapy is encouraging and supports previous suggestions of a neurosecretory growth hormone * Affected deficiency in Prader-Willi syndrome. It also suggests that all Prader-Willi patients should be evaluated for abnormal GH production and given a 6- pNeuro-e month trial period of GH therapy. Impailred [ >|>- l 4 j A70 Clinical Genetics (0269) 1.142 (0270) 1.143

A Second Case of Sakati-Nyhan Syndrome (Acrocephalopolysyn- Neurofibromatosis 2 (NF2): Groups at high risk. D. M. dactyly with Leg Hypoplasia). HK Park. SG Kahler. NS Prose. Parry and R. Eldridge. National Institutes of Health, A McConkie-Rosell. Duke University Medical Center, Durham, Bethesda, Maryland. NC. NF2 is an autosomal dominant disorder characterized by We report here an infant girl who has trigonocephaly, bilateral acoustic neuromas (ANs) which usually become high frontal hair line, narrow upslanting palpebral symptomatic between the ages of 15 and 40 years. The natural fissures, ptosis, bulbous nose, simple ears, high arched history of NF2 is variable so that some older individuals palate, micrognathia, multiple skin dimples on the face and with AN can retain useful hearing for years. As many as 10% trunk overlying bony prominences or depressions, linear of al l AN may resul t from NF2. Because the diagnosis of clefts on the chin, areas of alopecia and cutaneous atrophy unilateral AN can lead rapidly to surgery with risks of on the face and scalp, nevus sebaceus on the scalp, severe deafness and facial nerve palsy, it is imperative to hypoplasia of the tibias, bifid great toes, and club feet. distinguish a patient with AN from NF2 from a patient with She is the second child of unrelated healthy parents. sporadic unilateral AN. Five groups of individuals are at Pregnancy and delivery were uncomplicated. Blood lymphocyte high risk of NF2 (N. Engl. J Med. 318:684-688, 1988): karyotype is 46,XX. Her growth and development have been 1. An identical twin or 1st degree relative of an affected. normal for the first 3 months. Her features are very 2. A person under age 40 years with unilateral AN. similar to those of an 8 year old boy reported by Sakati et 3. A youth or young adult with mild NF, but not NFI. al. (J PEDIATR 1971; 79:104-9). In addition, this boy 4. A child or youth with meningioma and/or schwannoma. had a heart murmur. This disorder has been called 5. A person with multiple meningioma or low grade astro- Sakati-Nyhan syndrome, Acrocephalopolysyndactyly (ACPS) type cytoma. III, or ACPS with leg hypoplasia. To our knowledge, there Seventy-five individuals evaluated by us for NF2 were have been no other reports since the original report. Both originally ascertained as follows: children are isolated cases. The identification of a second case confirms this disorder as a recognizable pattern of Risk No. at No. with Ave. Age Median Age Age Range malformation. Advanced paternal age (37 years in our case, Group Risk NF2 NF2 dx. NF2 dx. NF2 dx. 40 years in original case) supports new single gene mutation 1 61 28 30.1 25 18-70 as the cause. However, autosomal recessive inheritance 2 5 3 41.3 39 38-47 cannot be ruled out. 3 6 6 18.8 17.5 12-37 4 2 2 22.0 22.0 11-33 5 1 1 45 45 45 Screening of such individuals should include: Review of personal and family history for hearing loss, brain tumors and balance problems; skin exam for cafe-au-lait spots and cutaneous neurofibromas; audiology and brainstem auditory evoked responses of both ears; brain and spinal cord MRI with gadolinium with emphasis on the posterior fossa; and slit- lamp exam of both lenses with pupils dilated seeking posterior capsular opacities.

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Molecular genetic studies of alcohol subtypes: #- and - PFEIFFER SYNDROME and/or acrocephalosyndactyly variant. RAJ- Alcohol dehydrogenases (ADH). A. Parsian. C. R. Cloninger. RAJENDRA A. PATEL and DAVID BIXLER, Department of Oral Facial R. D. Todd. and E. J. Devor. Departments of Psychiatry and Genetics, Indiana University Medical Center, Indianapolis, Genetics, Washington University School of Medicine, St. IN 46202. Louis, MO, USA. II-2: born in 1962; brachycephaly with a wide, high flat That alcoholism is a familial disorder has been forehead; bilateral coronal and partial sagittal synostosis; established by family, adoption, and twin studies short, broad, rotated thumbs and mildly broad great toes; hear- (reviewed in Devor and Cloninger, 1989). Numerous ing loss. III-1: 1959; not examined; photographs & previous candidate genes have been proposed to account for the skull Xrays show nasal septum deviation, abnormal orbital con- increased familial risk of alcoholism. Among these are tour, and plagiocephaly suggesting unilateral coronal synos- the alcohol dehydrogenase genes (ADH) which are involved tosis. III-2: 1958; bilateral coronal, bilateral lambdoidal, in the oxidative metabolism of ethanol. Gross assessment & sagittal craniosynostosis; history of craniectomies; acro- of ADH activities determined by enzyme isoforms have cephalic head; bilateral broad thumbs; slightly broad big toes failed to link these enzymes to increased risk. To bilaterally; slow in reading; hearing loss. III-3: 1959; further test whether ADHs are involved in the development brachycephaly; moderately flattened forehead; short broad of alcoholism, we have completed a linkage study using thumbs; brachydactyly; moderate hearing loss. III-5: 1963; RFLPs detected by ADH-DNA probes. A sample of 21 two and brachycephaly with wide, high forehead; downslanting palpebral three generation families (n 205) ascertained through fissures; short, broad thumbs. IV-1: 1974; not examined; alcoholic probands was screened with the fi-ADH probe history of craniosynostosis surgeries; hearing disability; pADH12 and the L-ADH probe pADH74. The sex averaged lod slow in reading. IV-4: 1987; hypertelorism; proptosis; maxil- scores are shown below: lary hypoplasia; brachycephaly; normal thumbs and toes. IV-7: 1978; brachycephaly with wide forehead; downslanting palpebral Recombination Fraction (8) fissures; thumbs are short & broad; history of bilateral coronal Loci 0.00 0.05 0.10 0.20 0.30 0.40 craniectomies; slow in reading. IV-ll: 1982; breech delivery; femur & skull fractures; respiratory distress; hydrenancephaly; ALC vs. A-12 0.399 0.336 0.276 0.167 0.079 0.020 coronal craniosynostosis; broad, high, flat forehead with ALC vs. A-74 -0.078 -0.030 -0.002 0.019 0.014 0.004 brachycephaly; extreme midface hypoplasia; depressed nasal A-12 vs. A-74 -a 7.421 7.459 5.520 3.028 0.874 bridge; barrel-shaped chest etc.; death after few hours. The remaining individuals were not examined. All individuals in While neither pADH12 nor pADH74 provided unequivocal III are children of II-2; III-1 is parent of IV-1,2; III-2 evidence of the presence or the absence of linkage to of IV-3,4,5,6; III-3 of IV-7,8; III-4 of IV-9,10; III-5 of alcoholism, we were able to demonstrate strong linkage IV-ll,12, 13, 14 and III-6 of IV-15. between the two ADH probes (LOD - 7.459 at 1 - 0.1). The Many of the cases of craniosynostoses represent well-known results suggest that all of the ADH genes might lie in a syndromes of heterogeneous causes, most commonly genetic, such small region. Currently, we are expanding the number of as (1) Apert syndrome, (2) Crouzon syndrome, and (3) Pfeiffer probes typed in order to more fully map the ADH region. syndrome. The remarkable feature of this family is variable (NIAAA Grants 8028 and 7466). phenotypic expression of the Pfeiffer gene. An unusual range of expression leads one to consider the possibility of multiple alleles for the same trait. Clinical Genetics A71 (0273) (0274) 1.145

Inversion-duplication of 9q21 in a patient with mental reta- Congenital chronic 1yaphedisa: further evidence for an rdation and behavioral problems. R.PeBenito, S. Luke, R.S. autoaceal recessive form. V. B. Penh _,zedeh. M. Jaguez de Verma, M.J. Macera and J.G. Rodriguez.The Long Island College Gutierrez, B. Schofield and D. Pnales-Mreion. Division of Hospital-SUNY Health Science Center at Brooklyn, New York. Medical Genetics, Beth Isarel Medical Center, Mknt Sinai Structural abnormalities involving heterochromatic regions School of Medicine, New York, and Hospital R. Reed Cabral, of the human genome are most difficult to characterize, be- Santo Domingo, DMinican Republic. cause these segments are G-band negative by GTG-technique, a routinely used procedure. Chromosomal abnormalities of such Several forms of genetic lyqphedema have been described, most cases have gone undetected or were incorrectly characterized of which are automoal dint. Sugestion of a recessive because these regions are so called heteromorphisms or vari- congenital fore is based on only three families (2 from ants. We report the first documented case with a so called Germany, 1 from Japan) with rmal parents and two affected highly unusual h region of chromosome 9 which is not a vari- siblings each. We report here a family from the Dominican ation but a structural rearrangement involving duplication Republic with three affected siblings (two males and one and a paracentric inversion. A 17 year old hispanic female female) with lyaphedema present since birth. Parents were was referred for a neurologic evaluation because of mental unaffected and denied gity although they were born retardation and behavioral problem. Cytogenetic findings with in the sawi smal village. One of the affected males died GTG-banding revealed a 46,XX,9qh+ karyotype. By GTG banding shortly after birth and was not exanined by us. The probani, alone, it was suspected that the 9ql3-9q21 region has been a male, presented marked welling in upper and lwaer duplicated. When the DA/DAPI technique was applied, it was extremities at birth; lymphedesa affecting face, extremities clear that the G-negative band included band 9ql3 and also and genitalia persisted chronically, on examination at 21 included a portion of the heterochromatic region (9ql2); this years of age, he appeared well developed and in good geraral entire segment had not only duplicated but was also in a para- health. He had fullness of tha cheeks and mild swelling of centric inversion. This heterochromatic segment was further eyelids end upper extremities, including fingers, hands, characterized by C-banding and restriction endonuclease AluI/ forearma and arm. Chronic chatseis of the conjunctivas with Giemsa and found to be positive with both techniques. It is vascularization s present. ETe lceier extremities highly unusual to note that breakage occured within the hete- were markedly swollen, the right more than the left, with rochromatin of the secondary constriction region. The ab- pitting edema and areas of fibrotic eIatous changes and normal phenotype as noted in this case might have been the trophic skin manifestations; scrotum and penis were markedly result of duplication and inversion of band q21 of chromosome sllen. A lymphatic scintiscan of the loeer extremities 9. This band may contain a few sensitive gene loci and on demonstrated absence of lymph nodes consistent with duplication gained additional active genes. Thus, the trisomy hypoplasia of lymphatic vessels. A 26 y.o. sister is of these active genes (by duplication and the reversal of similarly affected and wes found on examination to have an their order (by paracentric inversion) may be responsible for identical pbotype. Both patients have a peculiar facies these unusual genetic effects. This case may serve as an due to edema of eye lids, cheeks and lips but no dy si example of phenotypic mapping by structural rearrangement dsu strictu. Their physical grovth and nutrition were involving the h region of chromsome 9. nixl and there was no evidence of visceral lymphaniectasia. The recognition of this sibehip lards further support for an autososal recessive form of congenital lynxids.

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Familial lethal cystic hygroma: An inherited nuchal bleb Congenital cardiac malformations: Excess in offspring of women with syndrome. S.M.V. Pfluegerl, K.S. Kagan-Hallet2, and G.S. hyperphenylalaninemia. M.E. Pieront. C. Firkins-Smith. S.A. Berry. Khodr3. 1Tufts Univ. Schol of Medicine and Baystate Medical R.O. Fisch and H.K. Berry. Variety Club Children's Hospital, Univ. of Center, Springfield, MA, The Univ. of Texas Health Science Minnesota, Minneapolis and Cincinnati Children's Hospital, Univ. of Center, San Antonio, TX, and 3Southwest Genetics, San Antonio, Cincinnati, Cincinnati, Ohio. TX. Previous reports have suggested that infants born to mothers with Cystic hygroma is a not uncommon fetal malformation, hyperphenylalaninemia (HPA), usually phenylketonuria, can be at higher especially in association with Turner syndrome, and is not risk for congenital cardiac malformations (CCM). In this study, we report generally thought to have a high recurrence risk. We present four infants born to mothers with HPA, 2 with CCM. In addition, all an unusual lethal syndrome characterized by massive cystic available literature reports from 1962 to 1989 were reviewed as to the hygroma and single umbilical artery. The consultand is a pregnancy outcome and presence of CCM. gravida 6 who lost three gestations to this condition, Information was available on 392 pregnancies of 153 women with including a markedly hydropic male stillbirth with massive HPA. There were 352 liveborn children and 44 miscarriages or stillbirths. cystic hygroma at 39 weeks, a fetal demise at 14 weeks of a Of the liveborn infants 29 of 352 (8.2%) had documented CCM and a male showing what was thought to be an encephalocoele on further 4% were suspected of having CCM because of cyanosis or ultrasound but was seen to be cystic hygroma following murmurs. Complex cardiac disease or multiple cardiac malformations, delivery, and a macerated fetus at approximately 16 weeks including 8 infants with hypoplastic left heart syndrome, 1 with with massive cystic hygroma. This last gestation revealed a transposition of the great vessels, 5 with tetralogy of Fallot and 8 infants 47,XXX karyotype from placental culture. Full autopsies were with more than one CCM, occurred in 22 of 29 affected children, while performed on the first and third affected gestations, revealing single cardiac malformations such as ventricular septal defect or patent no visceral anomalies. Examination of the placentas revealed ductus arteriosus occurred in 7 of the 29 affected children. When the only two umbilical cord vessels. Although the patient was maternal blood phenylalanine (MBP) was <10 mg/di, none of the 66 initially counseled by her physician that the anomalies seen offspring had CCM. For MBP from 10-15 mg/dl, there were 2 of 35 in the first two losses were not thought to represent an children affected by CCM, while MBP >15 mg/dl was associated with inherited syndrome, the subsequent history suggests that the CCM in 27 of 266 children. Maternal dietary treatment initiated in the first condition is indeed inherited, possibly as an autosomal trimester did not prevent the occurrence of CCM in 6 of 21 treated recessive or X-l inked trait. The significant features in pregnancies. This study indicates that maternal HPA is associated with a this family include cystic hygroma similar to that seen in much higher risk of CCM than exists in the general population. Particularly, monosomy X and single umbilical artery. The case illustrates the occurrence of hypoplastic left heart syndrome in 2.3% of the liveborn the need for full evaluation of pregnancy losses with genetic infants of this study group is considerably higher than the 0.016% expected follow-up through subsequent gestations. for the general population. Consideration should be given to measurement of MBP in mothers of infants with hypoplastic left heart syndrome. A72 Clinical Genetics (0277) (0278)

A Case of Cerebral Gigantism (Sotos Synedrae) A Sequence Endocrine implications in Hallermann-Streiff syndrome: first report of a patient Variant? with hypopituitarism. F K. Pivnick, S. Burstein, J. C. Ward, and R. S. Wilroy. M.C. Pinto,* L. Pereira da Silva,** J.A. Antunes," M.E. Univ. of Tennessee, Memphis. - 7F Z nteOiMW N. Qrdei Fer.ira."" *Div' of Oculomandibulodyscephaly with hypotrichosis (Hallermann-Strelff of Chenistry, University of Syndrome, HSS) is a rare syndrome characterized by these cardinal Genetics, Institute Physiological abnormalities: dyscephaly and bird-Ike fades, dental anomalies, proportionate Lisbo, Portugal, **Dona Estefania Pediatric Huspital, Faculty dwarfism, hypotrichosis, atrophy of the skin, bilateral microphthalmia, and of Medical Sciences of Lisbcn, Portugal. congenital cataract. Less common anomalies seen are microcephaly, Sotos Syxnrmne is characterized by large size at birth, colobomata, developmental delay, and hypogenitalism. Although one of the excessive growth during the first four years, macrocehaly and originally described patients did not have growth retardation, the majority of distinct ofacial anoailies. reported patients with HSS have had short stature (2 SD below the mean for Recent data suggest that, at least in sane families, the age). Eighty cases of HSS have been reported in the literature, and In none gene seregates as an autoscnal dominant with variable ac- has hypopituitarism been found as a contributing cause of growth failure or pressin. We report a 4 year old female patient with neonatal rnicrogenitalia. Russel et al (1970) presented an HSS patient with short stature and post-natal gigantin, x retardation, character- who was not deficient in growth hormone. Few additional patients have had istic facies with acimgaid features, including frontal endocrine studies. bossing, high forehead, jaw, We evaluated a 2-7/12 year old black male who had the following typical prninent pseuzb-hypertelorism, features of HSS: proportionate short stature, brachycephaly with separated arched palate ocular findings as and high and hyperetropy cranial bones, micrognathia, delayed dentition, unilateral cystic eye, left eye exctrcpy. Deratoglypic pattern displayed bilateral contralateral microphthalmia with coloboma and cataract, hypotrichosis, external diplac t of the 2rd interdigital triradius. Cr microphallus, cryptorchidism, psychomotor retardation, and generalized scan showed diffuse dilation of lateral and 4th ventricles. osteopenia. Ketotic hypoglycemia was found on multiple occasions. Because Attention is drawn to muscle mass hype with hypotonia of the hypogonadism, short stature, osteopenia, and hypoglycemia, an and increased levels of Aldolase and Creatine Phosphokinase. endocrine assessment was done. Also to be noted was an asiatio with unilateral pyelocal- Endocrine evaluation of our patient revealed a low T4 (FT41, 3.3 ug/dQ) with icial duplication. Genes for Insulin, Insulin-like Growth a low TSH, suggesting hypothalamic hypothyroidism. This was confirmed by Factor (IGFlU), Differentiation (yo Dl), Parathyid TRH stimulation. TSH rose slowly from 1.2gU/mi to 12.9 at 2 hours and did not Myogenic return to baseline by 3 hours. Metyrapone test confirmed ACTH deficiency as orm}one (PTIG), Becwith-Wiedmann Syffrane (BWS) have been a contributing factor to the ketotic hypoglycemia: both cortisol and 11- mapped to the region e i fran lp13 to llpter. Glucose deoxycortisol were low the morning following a midnight dose. A superagonist tolerance test, basal hunan growth lhrmone, thyroid arnd GnRH test showed a low LH response at 1 hour and no testosterone rise aiter adrenal functios were normal. 24 hours, suggesting hypothalamic GnRH deficiency as being responsible for Cytogenetic analysis showed no ch sme duplication on his microphallus and cryptorchidism. Growth hormone deficiency is suspected lUp. Fragile X studies have not yet been done. Analysis of on the basis of marked short stature, osteopenia, and ketotic hypoglycemia; this patient at the molecular level may help to clarify the additional studies are continuing to confrim growth hormone status. seen in precise correlation between Sotos Sequence and the nuscular We propose that 1) at least some of the complex features HSS, dystry such as hypogenitalism and short stature, may be related to pituitary axis findings. dysfunction; and 2) HSS patients be evaluated for pituitary abnormalities. Since the majority of cases are not known to be severely retarded, aggressive evaluation and treatment is warranted.

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Migraine: A clue to the pathogenesis of Hereditary Haemorrhagic Secular trends in genetic expression of complex Telangiectasia. human diseases: An example of human obesity. R. Arlen Price, Kathryn Lunetta and T.I.A. M.E. Porteous, U. Nath, J. Steele, S. Proctor, J. Burn. S~rensen. Dept. of Psychiatry, Univ. of Univ. of Newcastle, Newcastle Upon Tyne, England. Pennsylvania, Philadelphia, PA & Dept. of Hereditary Haemorrhagic Telangiectasia is an autosomal Medicine, Hvidore Univ. Hosp., Copenhagen dominant disorder of the vascular wall. We have seen and During the past half century there have been interviewed 102 affected individuals in 21 families. 61 large secular increases in prevalences for affected individuals fulfill Olesen's diagnostic criteria for several complex human diseases that are known to migraine . Using these criteria 2-20% of the general pop- be influenced by genes, e.g., obesity, diabetes, ulation may be diagnosed as suffering from migraine. This and some psychiatric disorders. Extreme obesity difference is significant at P<0.05. in humans is strongly influenced by genetic Saxena has recently postulated that arterio-venous anasta- factors, including both polygenic and major gene moses have a central role in the pathophysiology of migraine. inheritance. Yet the recent secular increase in Hereditary Haemorrhagic Telangiectasia is associated with obesity in several populations must have an abnormalities of arterio-venous anastamoses and is the common- environmental origin. For example, in two est cause of multiple pulmonary arterio-venous malformations. overlapping samples of Danish young men (Na - Investigation of this association may provide further 16,557 and 21,747), the overweight component information about the pathogenesis of Hereditary Haemorrhagic increased in size over a ten year period, Telangiectasia. beginning in cohorts born in 1942 (Chi square (30 1. Olesen, J. et al. (1988). Classification and diagnostic df) = 128, p < 0.001, and Chi square (15) - 28, p criteria for Headache disorders, cranial neuralgias and facial < 0.05, respectively, for the two samples). This pain. Cephalagia 8; supp.7: 19-28. pattern is consistent with an interaction of 2. Saxena, P.R. (1990). in Migraine: a spectrum of ideas, genotype with environmental change, such that eds Sandler, M. and Collins G.M., Oxford University press. penetrance of susceptible genotypes increases pp 191-199. over time. Quantitative indices of disease (e.g., body mass index or glucose tolerance) make it possible to distinguish change in disease gene penetrance (genetic interaction) from a more general increase in penetrance for all genotypes. It is important that the nature of such secular increases in disease prevalence are understood, otherwise statistical "corrections" for sex, age and birth cohort effects cannot be appropriately applied. Our concern is broadly applicable to a number of complex human diseases and to different human populations. We are currently examining secular trends for obesity in other populations. (Supported by NIMH Grant MH43409). Clinical Genetics A73 (0281) (0282) 1.148

Abnormalities of fibrillis In the Mafran syndrome: Phenotypic Variation in the Marden-Walker Syndrome: Report of Immunoassays are inadequate for routine diagnosis. Three New Patients. J.C. Ramer, R.L. Ladda, C.A. Frankel, R.E. Pveritz. G. Stetten. G.J. Anhalt & L.Y. Sakai. Johns Hopkins M.J. Mascari, P.N. Mowrey. PA State Univ., Col. of Med., Univ School Med, Baltimore, MD & Shriners Hosp, Portland, OR. Hershey, PA. Three unrelated male infants were initially evaluated in Fibrillin is the major constitutent of extracellular microfibrils, and the newborn period because of multiple contractures involving is a prime candidate protein for the fundamental defect in the Marfan both large and small joints, blepharophimosis, high-arched syndrome (MS). Previous studies have shown that immunofluoresence of palate, prominent forehead, scoliosis, camptodactyly, and skin sections and fibroblast cultures, using monoclonal antibodies severe hypotonia. Two of the children also had a cardiac against fibrillin, detected the majority of patients with MS and misclas- anomaly and cleft palate. One had severe stridor with a cyst sified few patients with other connective tissue disorders. We have present in the supraglottic region. Craniofacial peculiarities expanded these studies to more patients with MS, families with MS, and varied somewhat among the children, but all fit within the patients with a variety of skin disorders. In blinded assays of full- described phenotype of Marden-Walker Syndrome. Two of the thickness sections of skin, 35% of patients with MS had apparently normal children died of respiratory insufficiency at ages 2 mos. and distribution of fibrillin in skin; there was intrafamilial variation, 27 mos. Detailed autopsy of one child showed structurally and with younger patients occasionally appearing normal while older relatives microscopically normal brain and spinal cord. Severe develop- were abnormal. Samples from 12 acquired skin disorders often showed mental delay was present in the two children who survived abnormal distribution of fibrillin, occasionally in patterns indistin- early infancy. guishable from MS. Fibroblast cultures of 18 MS patients were examined Comparing the children we describe with the nineteen for incorporation of fibrillin into the matrix, most multiple times: 8 previously reported individuals, the following features were (including all of the sporadic cases) showed no incorporation, while 10 present in >50% of the group: blepharophimosis (100%); micro- showed incorporation indistinguishable from controls. There was no gnathia (82%); upturned nose (68%); small mouth (53%); high- variation between 2 blinded observers and no intra-sample variation. arched palate (937); multiple contractures (95%); scoliosis Results were consistent among affected relatives. Samples from patients (86%); camptodactyly (627.); arachnodactyly (67%); talipes with homocystinuria, MASS phenotype and annuloaortic ectasia were equinovarus (63%); poor growth (height, weight and head normal. Cultured amniocytes and CVS cells produce abundant fibrillin; circumference) (60%); psychomotor retardation (84%); attempts at prenatal diagnosis of MS were thwarted by the affected hypotonia (88%). mothers being among those patients whose fibroblasts incorporate some Review of the diagnostic investigations performed on all fibrillin into the matrix. Thus, these immunoassays, while pointing to the described individuals revealed variable findings implying defects in fibrillin in some MS patients, are neither sensitive nor that the Marden-Walker phenotype may occur by several specific enough to be useful in routine diagnosis. Quantification of different mechanisms. Radiographs have demonstrated variable the fibroblast assay and analysis of the nascent fibrillin itself, for subtle abnormalities including radio-ulnar synostosis, erosions example, by electrophoresis, will be explored. of the ribs, long phalanges and osteoporosis. Studies of brain structure have shown mild ventricular enlargement in several patients. One of our patients had normal neuropatho- logical exam. A particularly significant difference among the children is the presence of either myopathic or neuropathic changes documented by electromyography and histologic examination of muscle.

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Acrodysontosie in an Afghani girl with apparently normal The impact of prenatal diagnosis on the epidemiology of intelligence. L.l. Randolph. P.A. Lee and R.C. Elliott. The osteochondrodysplasias. SA Rasmussen, FR Bieber, B Genetics Institute, Pasadena, CA; Huntington Memorial Benacerraf, LB Holmes. Depts. of Newborn Medicine, Obstetrics Hoepital, Pasadena, Ca; and Cigna Healthplans, North Hollywood, CA. and Gynecology, Pathology, and Radiology, Brigham and Women's A 3-year-old girl of Afghani descent was referred for Hospital, Boston, Massachusetts. evaluation of a flat nasal bridge and short limbs. She was The osteochondrodysplasias are a heterogeneous group of born after a normal pregnancy to a 27-year-old Afghani al PO disorders characterized by abnormalities in cartilage and woman and her nononeanguineous 29-year-old Afghani husband. bone growth and development. These conditions are now Developmental milestones were normal, and she was considered above average in her preschool. detectable during the second trimester by sonographic tech- Clinical evaluation revealed a weight at the 75th Cile and niques. To study their epidemiology, we ascertained cases of height and head circumference at the 50th tile. She had osteochondrodysplasias in elective terminations, stillbirths brachycephaly, a flat facial profile with a flat nasal bridge greater than 20 gestational weeks, and livebirths diagnosed and small, upturned noe with midfacial hypoplasia. She had ocular hypertelorism with e*ophorLa. Her chin was prominent. by the fifth day of life. Cases were identified as part of an Her arms were short. Her hands wer very small, measuring at on-going malformation surveillance program by staff members the 50th tile for a one-year-old. Her lower extremities were who make frequent visits to the regular and intensive care slightly short without bowing. Ber spine was straight. Her nurseries, pregnancy termination service and the autopsy speech was fluent and hypernasal. Laboratory evaluation revealed 46,XX chromoscee. service. 47 cases of osteochondrodysplasia were identified Radiological evaluation of the skull revealed early closure among approximately 112,000 births at Brigham and Women's of the sagittal suture. There was slight interpedicular Hospital during a 14-year period (Feb. 16, 1972-Feb. 15, 1975, narrowing of the lower lumbar spine, mild concavity of L and Jan. 1, 1979-Dec. 31, 1989). The overall birth rate was L5 posteriorly and early synostosis of the sternal 4.2/10,000 births and increased over the time period studied, ossification centers. The lower long bones pelvis, hips and because findings noted on routine prenatal ultrasound resulted were normal. Rhisomalic shortening and thickening and bowing care center. When of the humeri ware seen. slight shortening and bowing of in transfer of patients to this tertiary radii were also present. Most striking wer findings in the index cases born to women who were transferred to this hands and feet; all tubular bones wer short and wide with hospital because of an abnormal prenatal sonogram were closure of the epiphyses of the middle and distal phalanges excluded, the birth rate was 2.3/10,000 births. The likeli- and cone-shaped epiphyses of the distal metacarpals. Bone age hood that a case was suspected prenatally has increased from was advanced; wrist age was approximately 8 years and hand age about 14 years. The great toe was wide. CT scan of the head 0% during 1972-1975 to 89% during 1988-1989. During the 1988- was negative for hydroelu 1989 time period 44% of pregnancies ended in elective termina- We believe this patint fulfills the criteria for diagnosis tions, compared to none during the 1972-1975 period. Despite of acrodyaostosis and note the relatively unique findings of evaluations histologic, pathologic and bowing of humeri and radii and premature fusion of the sternal complete including a definitive was not ossification centers. Intelligence seams unimpaired contrary radiographic studies, diagnosis possible to the 75-90% incidence of mntal retardation reported in this in 15 patients (32%). Prenatal diagnosis of the osteochondro- condition; formal develometal testing is underway. dysplasias has altered the epidemiology of these conditions and complicates diagnosis because of an increasing frequency of pregnancy terminations, often by destructive methods. Biochemical and molecular genetic methods of diagnosis will continue to become more important if these trends continue. Supported by funds from the New England Regional Genetics Group. A74 Clinical Genetics (0285) (0286) 14.3

Epispadias in a girl with Down syndrome. J.Rau. Autosomal recessive variant of nephrogenic diabetes insipidus (NDI). P.N. L.X. Randolph. J.Paul. R.E. Krauthamer and S.S. Ray. T. Selander. J.W. Balfe. J.M. Langley. J.T.R. Clarke. Div. of Clinical Swillev. Torrance Memorial Hospital Medical Center, Genetics and Nephrology, Hospital for Sick Children and University of Torrance, CA, and The Genetics Institute, Pasadena, Toronto, Toronto, Canada. CA. Two sisters born to consanguineous Pakistani parents presented at 6-10 We describe what appears to be the first weeks of age with moderate dehydration associated with polyuria, vomiting, published case of epispadias in an individual with hypernatremia, and hyposthenuria. The urine specific gravity was consistently Down syndrome. <1.003. Urinalysis was otherwise normal. Ultrasound examination of the Baby D. was born to a 32-year-old white G3 P1 kidneys and renal radionuclide scans were nonnal in both. A kidney biopsy on Abl woman and her 33-year-old white husband at 38 the older sib at 6 mons of age showed immature-oking hypereellular glomer- weeks' gestation via cesarean section for breech uli, but no evidence ofmedullary cysts. Short term trials ofvasopression presentation. Features of Down syndrome and stimulation (65, 200, or 400 mU/kg subcut.) produced no change in urinary malformations were noted. The family history was specific gravity. A 2-hour infusion ofvasopressin produced no change in negative for genitourinary abnormalities. urinary specific gravity or urinary cAMP excretion. Prolonged vasopressin Chromosome analysis revealed a 47,XX,+21 stimulation (30 mU/kg/day by IM injection for 3 days) also produced no karyotype. Cardiology evaluation revealed a small change in urine specific gravity. Cytogenetic studies, including high resolution inlet ventricular septal defect and transitory banding, of peripheral blood lymphocytes showed no abnormality. The parents pulmonary hypertension. and a male sibling were all clinically normal and showed normal urinary con- Urological evaluation revealed exstrophy of the centrating ability following 16 hours offluid deprivation. Southern analysis of urethra; divided, hypoplastic and anteriorly placed DNA extracted from peripheral blood leukocytes was carried out by digestion labia minora; divided, hypoplastic and normally with Taq I, agarose gel electrophoresis, transfer ofDNA fragments to nylon placed labia majora; and an anteriorly placed membranes, and probing with St14, a highly polymorphic probe localized to vagina. The anus was normal. Abdominal ultrasound and revealed normal kidneys and probably normal uterus. Xq28 tightly linked to the X-linked NDI locus (od 10.35, 0=0, Knoers et The On voiding cystourethrogram, the bladder was small al., 1988). mother of the patients was found to be heterozygous for one of the systems and voided rapidly, and a bladder neck was present. polymorphic revealed by Stl4. The RFLP patterns of the girls in- dicated that each a A vaginogram revealed no abnormality. There was no inherited different maternal Xq28 chromosome region. The communication between the bladder and the vagina. unaffected brother as well as one ofhis affected sisters inherited the same Although hypospadias, dorsal urethral duplica- Xq28 region of a maternal X chromosome. The findings rule out a diagnosis tion and other genitourinary abnormalities have ofclassical X-linked NDI and, along with the history of parental consan- been described in males with Down syndrome, this guinity, were concluded to indicate the existence of a previously unrecognized represents the first reported occurrence of epi- autosomal recessive form ofNDI. spadias in either sex in Down syndrome. Of note is that epispadias itself is rare (1:35,000 births) and is more common in males. Given that dorsal urethral duplication is nonrandomly associated with Down syndrome in males and that epispadias involves dorsal location of the urethra, the abnormality seen in this patient may reflect an extreme variant of this pattern of malformations.

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Mast cell stabilization to minimize the symptoms of The Dyggve-Melchior-Clausen Syndrome: A Report of Two Cases enlarging neurofibromas. Vincent M. Riccardi, and Expansion of the Phenotype. J.Rinsky and A.Shanske. Alfigen - The Genetics Institute, Pasadena, CA. Albert Einstein College of Medicine, Queens Hospital Up to the present, there has been no effective Afiliation, Long Island Jewish Medical Center, Dept. of medical therapy to decrease the tumor burden of NF. Pediatrics, Jamaica, New York. Here I present data from two separate studies to The Dyggve-Melchior-Clausen syndrome is a rare, usually document that the mast cell stabilizer, ketotifen, autosomal recessive skeletal dysplasia characterized by can decrease both the symptoms associated with short-trunk dwarfism, mental retardation and characteristic neurofibroma growth and the rate of tumor growth. radiographic changes. These manifestations do not include One study has been open-label, in progress since cardiac defects. August 1983 and involving 25 patients over 1,125 Case 1: D.O. is a 10 year old hispanic female born to patient-months of treatment. With follow-up peri- unrelated hispanic parents. The family history is negative ods of up to 6.5 years, using a rating scale of 1 for birth defects or skeletal anomalies. She is a good- to 10, I have compared the rates of tumor growth looking short-trunked child whose height 110.5 cm was and/or the presence and severity of neurofibroma- more than 4 S.D's below the mean. The only other skeletal associated itching and pain. With mean values of anomaly was bilateral genu valgus. Her development has been 8.0 and 7.0, respectively, the results demonstrate delayed and she attends a special education class. Radio- clearly that ketotifen is strongly correlated with graphic survey revealed mild to moderate decrease in decreased neurofibroma growth and a marked decrease vertical height of vertebrae, hypoplasia of the odontoid or total ablation of associated symptoms. A second "lace-like" fraying of iliac bones and generalized meta- study, performed in a double-blind manner from physeal irregularities. The metacarpals were short and November 1988 through May 1990, involved 27 sub- the bone age was delayed. jects in two groups: Group-l, those with early or Case 2: S.K. is a 13 year old Pakistani male who is the mild symptoms, and Group-2, those with severe younger of two sons born to first cousin parents. His body symptoms. For Group-l patients where the primary proportions altered during his infancy in Pakistan and he concern was neurofibroma-associated itching or mild was noted to have a murmur. The family history is other- pain and tenderness, both I and the patients accu- wise unremarkable. The physical examination showed a hand- rately predicted whether they were taking ketotifen some short-trunked boy with a waddling gait and severe or the placebo. For Group-2 patients, the ability kypho-scoliosis. His height was 102.5 cm. In addition, he to distinguish placebo from ketotifen in terms of was noted to have a grade IV-V/VI harsh systolic ejection decreased pain and tenderness was apparent so early murmur. An echocardiogram revealed severe valvar aortic as to obviate the blinding. The predictions of stenosis associated with moderate concentric left ventri- drug versus placebo were confirmed at the time the cular hypertrophy. Radiographs showed severe kypho- blinding code was broken. The results of these two scolosis, flattened vertebrae, short long bones, irregular studies provide both a sound basis for current and metaphyseal and epiphyses, a "moth-eaten" appearance of future treatment of patients with NF and a strategy the iliac crests and atlanto-axial instability. for exploring the cellular interactions involving The addition of these 2 cases will help to further de- mast cells in the pathogenesis of neurofibromas. lineate this rare disorder with only about 45 known cases. The cardiac defect in case 2 may help to distinguish the autosomal recessive disorder from the X-linked form. Clinical Genetics A75 (0289) (0290) 1.151

Aniotic Bard Setaenae and Visceral Anomalies. G. Riscile. M. Benign Familial Neonatal Convulsions in a Newfoundland Kindred. Torres, G. Jervis. Division of Medical Genetics, University of T.O. Rosales and Mary Connolly. Department of' Pediatrics, Div. South Florida, Tampa. of Genetics, Janeway Child Health Centre and Memorial Univer- Amniotic bande are a cocponent of the asniotic bard sequence sity School of Medicine, St. John's, Newfoundland. but not its cause. They result frc- a sultifactorial process in Benign Familial Neonatal Convulsions (BFNC) is an autosomal which vascilar cprmse appears to be the principal dominant disorder that is important to recognize clinically pathcenetic factor with the formation of fibrous bands being a and may also give a clue to the genetics of seizures. Within late, secondary event (ILockood et al., Am J Cbstet Gynol, 1989;160:1030). a 6 year period, 68 individuals in 6 generations were found to Patient #1: A 3440 g girl born to 21 year old G2P0010 by have this disorder in a Newfoundland kindred. Thirty-three vertex vaginal delivery. Apgar scores were 5/6. The pregnanoy had available satisfactory medical documentation. The other 35 was complicated by "heavy bleeding" at 7 weeks gestation. were diagnosed retrospectively based on pedigree analysis and Birth length was 50.0 an, head circumference - 32.0 cm. clinical histories from the affected individuals and/or first Anomalies included midface hypcplasia, sloping forehead, low and second degree relatives. anterior hairline, brachycephaly, midfacial cleft, absent nose, Clinical information confirmed previously published features bilateral arthalia, distal syspalangism of the right and in addition other variable characteristics and patterns of second, third, fifth with a distal constriction and fingers seizures, attributable to this disorder, were defined in this band on the fourth; fusion of the left third, and fourth kindred. To date, among the 33 documented and 29 historical fingers and constriction band on the index. Chrorosases: 46,XX. Head Cr: alcbar hol. Echocardiogram: cases, 12% (L4) and 23% (8) respectively, had and/or still ex- three chamber heart and complete AV canal. Chest x-ray: partial perience later seizures for an overall of 18% (12/68) inci- absence of first and second ribs on the left and partial fusion dence of subsequent seizures. of the seventh, eight, and ninth bilaterally. Several consanguineous matings were noted but none involved Patient #2: A 4395 g boy born by Caesarian section at 42 partners where both were clinically affected. Those individ- weeks gestation to a 29 year old GlPO white female with a uals examined ( 46) were physically normal with intellectual history of infertility and clasid therapy. Pregnancy was ability grossly appropriate for family and enviromental back- crmplicated by first trimester bleeding. MSAFP was normal. grounds, with the exception of a 67 year old female who had Birth length was 51.0 cm. Anomalies included occipital mild hand tremors. encepthalooele with 21.0 cm circumference, sloping forehead, Due to heightened awareness among medical practitioners, bilateral cleft lip and palate, short neck, low set ears, constrictions at the distal IP joints of the right second at our centre and in the province generally, BFNC is invariably through fifth fingers, hemangicma and sacral dimple. Karyotype a consideration in the evaluation of neonatal seizures. When was 46,XY. Renal ultrasound: left caliectasis. Head Cr: brain the diagnosis is most likely BFNC "reasonable evaluative tissue within the encephalooele and disorganized cerebral studies" are suggested. The majority of these families have cortex. EEG: subclinical seizures. The visceral adopted a benign attitude toward these "frights/fits/spells". abnormalities of these two patients corraborate the In contrast, medical practitioners unfamiliar with BFNC view pathogenetic model prcposed by Locbwood et al. Further studies it with understandable concern and intensive investigations. are warranted to elucidate the cause of the presumed vascular A collaborative study has been prompted by recent reports of insult and to determine if a relationship exists between markers linked to the BFNC gene on the long arm of chromosome maternal first vascular This trimester bleeding and accidents. 20, in a previously reported family. Blood samples have been would help to identify wcsen at mncreased risk to deliver obtained from relevant family members to investigate the pre- infants with amniotic bard sequence. sence of the same and/or other DNA markers in this kindred.

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Tethered cord in Rubinstein-Taybi syndrome. K.N. Computerized pedigree drawing in the SCHESIS Rosenbaum. D.L. Johnson. C.R. Fitz. D.C. McCullougch. risk calculation and linkage package. A. P. Round. Children's National Medical Center, Washington, D.C. 52, Dartmouth Court, London SE10 8AT, UK. (Intro. by Pi. Bobrow). Rubinstein-Taybi syndrome (RTS) is a well-known Computer-based genetic risk and linkage dysmorphic syndrome of unknown etiology. Affected calculations using a general model, perhaps with patients have microcephaly, characteristic facies, linked markers, mutation, consanguinity, and short stature, broad thumbs and toes, and mental variable penetrance or other parameters, are retardation. Most have significant delay in walking complex and may be difficult or impracticable to and an unusual gait. Bowel and bladder dysfunction check even for small pedigrees. It is therefore are common. Although a small number of RTS patients vital to prevent, as far as possible, the have CNS malformations (agenesis of corpus callosum, occurrence of errors at the input stage, and to Dandy-Walker cyst), anomalies of the spinal cord ensure that the pedigrees for which calculations have not been described. We report 4 cases of RTS are performed are those intended. with clinical and radiographic evidence of spinal In developing the SCHESIS package a high cord tethering. 2/4 patients have had successful priority has always been Civen to the surgical release. Patient #1, a 10 10/12 year old comprehensive checking of pedigree input data, female, developed acute neurological symptoms at age and so the decision was made to develop an 6 with severe leg pain and bowel/bladder algorithm which could plot this on the screen. incontinence. MRI of the spine showed a low-lying Ideally its form would be very close to that of cord with a thick filum terminale. Surgical release the plot which a user would have drawn (at least produced reduction in symptoms and return of mentally) prior to allocating identification continence. Patient #2, a 9 2/12 year old male, codes or numbers to each pedigree member. developed weakness and fatigue with gait Coping with consanguinity, multiple and deterioration and encopresis. WRI of the lumbar intergenerational marriages makes the production spine showed the conus at a mid-L2 level. Release of such a plot a difficult task. The SCHEDIAST was carried out with improvement in symptoms. algorithm has two phases: in the first a Patient #3, a 10 1/12 male, presented with gait conceptually best plot is produced, in which deterioration, fatigue, and bowel dysfunction. MRI allocations to generations and left to right showed a low-lying cord with the conus at L2-L3. orders within each are made; and then actual Surgical release is planned. Patient #4, a 3 3/12 screen coordinates are assigned using an year old female, presented with hyperreflexia and iterative technique in which so far plotted parts severe constipation. MRI showed the conus low at may be 'moved' prior to adding others. L3. The radiographic finding of a low-lying cord in 4 patients with RTS suggests that tethering of the spinal cord may be the cause of gait disturbance and incontinence in this syndrome. MRI of the thoracolumbar spine is an essential part of the routine assessment of RTS. A76 Clinical Genetics (0293) 1.154 (0294) 1.156

Lisch nodules in neurofibromatosis-l are not Genetic implications of the Robin sequence. dependent on sympathetic innervation. H.M. Saal. K.N. Rosenbaum. H.J. Stern, and M. A-E- Rubenstein. J-C- Halgerin. Mindel. S. yal*iaEn. Children's National Medical Center, Wallace and A Aron- Mt. Sinai School of George Washington University School of Medicine, Medicine, New York. Washington, D.C. Lisch nodules are melanocytic hamartomas which The initiating event in the Robin sequence is are one of the most consistent clinical findings mandibular hypoplasia prior to the ninth week of in adults with the neurofibromatosis type 1 (NFl) gestation, resulting in posterior placement of the gene. Most of the cells which proliferate in NFl, tongue and impaired fusion of the lateral palatine including those which constitute Lisch nodules, processes. Since 1978, 81 patients with the Robin are neural crest derived. This has led to sequence have been managed in the Department of speculation as to whether sympathetic neurons, Clinical Genetics and the Children's Craniofacial which are themselves neural crest derived and Center at Children's National Medical Center. Of which exert considerable control over the growth these 81 patients, a diagnosis of a specific and development of neural crest derived cells, dysmorphic syndrome has been made in 50 patients, have an influence on the proliferative capacity of including 34 patients with Stickler syndrome. neural crest derived cells in NF1. Clinical Eight patients have been diagnosed as having rare examples to document this theoretical possibility disorders, including Smith-Lemli-Opitz syndrome, have been few. We report a case of iris cerebro-costo-mandibular syndrome, and fetal heterochromia due to unilateral congenital alcohol syndrome. Nine patients have been found Horner's syndrome in a 22-year old woman with NF1 to have the Robin sequence as part of what are in which normal iris pigmentation from stromal presumably unique multiple congenital anomaly melanocytes was absent in the denervated eye. disorders. Of the 34 patients with Stickler Lisch nodules were present in equal numbers in syndrome, 18 have positive family histories for each eye. Iris stromal melanocytes which produce this disorder, and 16 patients are felt to normal iris pigmentation are sympathetically represent fresh mutations for this autosomal modulated and fail to develop in congenital or dominant condition. Thirty of the patients with early onset sympathetic denervation. Melanocytes Robin sequence have isolated cleft palates which constituting iris nodules in NF1 appear not to are felt not to be related to any specific be sympathetically controlled. Escape from the dysmorphic syndrome. normal developmental influences of the sympathetic These data suggest that in a large proportion nervous system may be a component in the of patients with the Robin sequence there is an pathogenesis of the abnormal proliferative underlying recognizable etiology for the disorder. phenomena observed in neural crest derived cells It is recommended that all patients with Robin in NFI. sequence have thorough genetic evaluations at the time of initial diagnosis for the purpose of appropriate genetic counseling and improved medical and psychosocial management.

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Immortalized Werner syndrone and progeria fibroblasts. Morphometric Trait Markers in the Detection of Gene Carriers H. Saito and R. E. Moses. Baylor College of Medicine, of X-linked Hypohidrotic Ectodermal Dysplasia (XHED). S.S. Houston, TX 77030. Saksena. I.U. Medical Center, Indianapolis, Indiana. Skin fibroblast cells fron two premature aging The most striking manifestations of XHED disorder are i) diseases, Werner syndrome (AG780) and progeria (C2033) hypohidrosis; ii) hypodontia; iii) hypotrichosis; and iv) a were transformed by SV40 early DNA, and after the characteristic facial physiognomy. Affected males typically "crisis' period, permanently proliferating cells were show the full symptoms, whereas affected females representing successfully isolated and established as immortalized lyonization effects exhibit a wide spectrum of clinical signs, cell lines (WS780 and P2033). WS780 and P2033 have been ranging from subtle to the most severe expression of the dis- passed over 30 PD (population doublings) and 80 PD order. The gene carriers manifest only 1 or 2 or none of the respectively. The lineage of each immortalized cell line 4 clinical symptoms. The probability of carrier detection was confirmed by VNTR analysis. By using these on the basis of clinical findings has been estimated to be immortalized cell lines, two interesting cellular around 60%. Contrary to general opinion, affected females phenotypes of Werner syndrome and progeria were verified: outnumber affected males (1.4:1). The use of currently avail- 1. Slow growth able methods does not allow the routine identification and/or AG780 and C2033 grew much slower than IMR90 (normal recognition of all gene carriers. fibroblast). WS780 and P2033 grew slower than other Facial physiognomy of fully affected patients in both sexes immortal cell lines. Thus, it was confirmed that slow (from a wide variety of ethnic groups) is similar and highly growth, a characteristic cellular phenotype of both diagnostic. The goals of this study are to employ quantitative diseases, was maintained even after the immortalization. methods (roentgencephalometry and multivariate morphometrics) 2. DNA repair ability for the facial pattern recognition and characterization of Sensitivity of WS780 and P2033 to UV (ultraviolet), the fully symptomatic patients and mildly affected gene BLM (bleomycin), MMS (methylmethanesulfonate), CDDP (cis- carriers. dichlorodiammine platinum) and MMC (mitomycin-C) Several two group (carriers vs. noncarriers) discriminant treatment was studied. Both of the cell lines showed and classification functions based 3 or more facial measurements normal sensitivity to BLM, MMS and CDDP, and slight (trait markers) were derived. These functions correctly diag- sensitivity to UV. However, the immortalized Werner cell nosed 100% of the gene carriers. This morphometric method line (1S780) showed significantly more cell killing appears to be a valuable diagnostic tool; the use of a few (D37-0.010*0.002 )s g/ml) by lMC than normal simple objective measurements significantly improves clinical (D37-0.052*0.020 Ug/ml). judgement and brings to one's attention facial anomalies in We conclude that the Werner syndrome cell line WS780 mildly affected gene carriers who would otherwise be overlooked shows the cellular phenotypes of slow growth and MMC- and/or judged as more or less normal, also helps in the objective sensitivity. Supported by USPHS grant AG07123. characterization of facial anomalies in gene carriers as a group. Furthermore, it can be readily replicated and validated by other researchers and/or clinicians. Since carrier females are the source of most affected indi- viduals their correct identification can be extremely useful in not only providing accurate genetic counseling to HED families but also in reducing or preventing neonatal mortality and morbidity in individuals with XHED. Clinical Genetics A77 (0297) (0298) 1.157

Features of Larsen Syndrome in a child with a chromosomal NOR-ALLLC OF FMILALy SPINL abnormality. E.K. Schorry and J.A. Westry. Children's Hospital Medical Center, Cincinnati, Ohio. Larsen Syndrome (LS) is characterized by dysmorphic facies T.A. SeIs, V. Riccordi3, P. Fain4, D. BarkM 4, R. with flat nasal bridge, multiple congenital joint dislocations orenberf, S.-L. Pulse 2 and other limb anomalies. The etiology appears to be genetic- Div. of Neurology and Medical Genetics, Cedars Sinai ally heterogeneous. Most patients with LS who have had chrom- jedical Center, Univ. of California, Los4 Angeles, CA; osome analysis have had normal karyotypes. We are reporting Baylor School of Medicine, Houston, TX; Univ. of Utah, a patient with features of LS who also has developmental Salt Lake City, UT. delay, microcephaly, and an abnormality involving chromosome #15. Ve have identified two families vith Familial Spinal The patient is a black female who was born to a 17 y.o. Neurofibromatosis (FSNF) and have used genetic linkage GlPl mother after an unremarkable pregnancy. She was noted analysis to evaluate the location of the mutations with to have congenital anterior dislocation of both knees and respect to the NF1 locus on chromosome 17 and the NF2 locus bilateral hip dislocations. Other features included flattened on chromosome 22. The first family has three affected facies, very flat nasal bridge, unusual ears and hypoplastic embers with spinal neurofibromas as the only manifestation thumbs. A diagnosis of LS was made at birth. of NF. There are no cafe-au-lait (CAL) spots, cutaneous or The family history revealed that neither parent had any CNS tumors. Genetic linkage analysis with DNA markers features of LS, and there was no known consanguinity. The tightly flanking the NF1 locus revealed a recombination patient was seen for developmental assessment at age 3i yrs. event between the disease trait and informative flanking Her height and head circumference at that time were less than markers thus excluding the NFl locus as the site of the 5% for age. She was found to have moderate developmental mutation with odds > 100,000 : 1. The family was not delays, with overall functioning between 18-24 mos. informative for DNA probes flanking the NF2 locus. Chromosome analysis was obtained and revealed a karyotype The second family has spinal neurofibromas accompanied by of 46,XX,15q+. The extra genetic material appeared euchrom- CAL spots, but no other signs of NF1 or NF2. Using atic and was of unknown etiology. Maternal chromosomes were multipoint linkage analysis with several DNA markers tightly normal, and attempts are presently being made to obtain blood linked to the NF1 we did not observe any recombinants and from the father. obtained a multipoint LOD score of 1.8 at the NF1 locus. Most previously reported individuals with LS have had Further, a DNA probe for the locus D22S1 which is tightly normal intelligence and normal chromosomes. Curtis and linked to the NF2 locus detected a recombination event in Fisher in 1970 reported a boy with LS who had a sex chromo- this family. These results strongly suggest that in this some abnormality. To our knowledge, ours is the first family FSNF is caused by a mutation in the Ni1 locus. Ve reported case of LS in association with an autosomal abnormal- are currently analyzing this mutation with cosmids spanning ity. We hope that further information delineated from these the Ni1 region. chromosomes may be of value in determining the location of one Our results suggest that FSNi may be genetically of the genes responsible for LS. Based on our findings, we heterogeneous and that pathologically identical spinal would recommend that chromosome analysis be considered for neurofibromas may be caused by inherited mutations at selected patients with features of LS, especially those who different loci. Investigation of such families may help show developmental delays. elucidate the basis of tumor formation in Ni.

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Early nephrocalcinosis and renal stones in Bartter syndrome? Congenital rhabdomyomatous mesenchymal hamartomas:? Oculo- J. R. Secor McVoy. J. N. Bodurtha and J. W. Foreman. Medical cerebro-cutaneous syndrome. S.D. Shapiro, S.M. Shields, L.V. Vanner and G.S. Pai. Medical University of South Carolina, College ofVirginia - Virginia Commonwealth University, Richmond. Charleston, S.C. U.S.A. Bartter syndrome is a disease of potassium wasting, characterized We would like to report a black male infant who was born by hypokalemia, juxtaglomerular hyperplasia, polyuria, with multiple facial hamartomas, multiple eye defects, and developmental delay. This child was born to a 32 year old hyperreninemia, and hyperaldosteronism with normal or low blood G4, P2, Abl at 42 weeks of gestation. Prenatal history was pressure. unremarkable and the patient was delivered via spontaneous We report two siblings with some of the features of Bartter vaginal delivery. Apgars were 8 and 9. At birth, this child was noticed to have multiple finger-like projections from his syndrome, including hypokalemic alkalosis, and polyuria. In addition, face and preauricular area. These skin appendages ranged in urinary calcium excretion was markedly elevated, and renal size from a stalk-like projection measuring a few millimeters ultrasound examination revealed mild hydronephrosis, in length to large globular lesions with dimpled ends. These lesions had the very unusual feature of random movement and a nephrocalcinosis and renal stones before one month of age. Both biopsy showed a connective tissue core with striated muscle children were the products of pregnancies complicated by bundles which were parallel to the long axis of the lesion. polyhydramnios, and delivered by emergency caesarean section at 31 The eyes had bilateral sclerocornea but no retinal detachment. The rest of the exam was within normal and 32 weeks, respectively. There was a suggestion of arachnodactyly limits. The child underwent surgery for removal of these lesions in these patients, with hand measurements up to two standard which initially improved facial appearance. However, some of deviations above the mean. However, this may be unrelated to the these areas have had regrowth and there is some concern for above observations, as the mother's hands and feet were also the possibility of malignant transformation. The left eye has had corneal transplant but the right eye was discovered by relatively long. The upper to lower segment ratio was not greater ultrasound to have an intraocular tumor, etiology unknown. than normal in either the mother or the children, and no cardiac or Developmentally the child is delayed and has myoclonic other abnormalities associated with Marfan syndrome were observed. jerking. No sustained seizure activity has been recorded but an EEG was frankly abnormal. There was no evidence for increased oxalate or cystine excretion or Differential diagnosis for this child includes the for rickets. Parathyroid hormone levels were in the normal range. following syndromes: oculo-cerebro-cutaneous, Goldenhar or No overt dysmorphic features were noted in these patients, and both Goltz. had normal karyotypes. Early presentation of nephrocalcinosis and kidney stones with Bartter syndrome is unusual, particularly with features of arachnodactyly. It is possible that this constellation of symptoms which we have observed in two siblings may represent a new syndrome. A78 Clinical Genetics (0301) 3.5 (0302)

COCHLKR DEANES TRANSHITTD IN A AT NOD 0F Erythropoietin in Gaucher Patients. E. Sidranskv1. G. INHERITAM V1TH NUCI GME INVOL IT Shohat*, L Schiller2. J. Younger3. and E. I. Ginns1. 1NIMH, ADAMHA, Jaber*, X. B , H. Y. Yan , S. J.U # J. I. Rotter. Bethesda, MD, 2Univ. of California, Los Angeles and 3Mass. *Beilinson Medical Center, Tel Aviv University, Israel; General Hospital, Boston. Intro by J. Sidbury. #Cedars-Sinai Medical Center and UCLA,Los Angeles, California Gaucher disease, the inherited deficiency of Hereditary deafness is a heterogeneous group of disorders. glucocerebrosidase, has diverse clinical manifestations and Autosomal dominant, recessive and X-linked modes of may include a severe anemia, the pathogenesis of which is inheritance have been implicated. We present a large Arab- often not well understood. The anemia of Gaucher Disease has Israeli kindred vith hereditary deafness which, for the first been treated by splenectomy, chronic transfusion, and time, demonstrates a two-locus mode of inheritance with one pharmacologic infusions with varying degrees of success. In locus mitochondrial(mt) and the other autosomal. an attempt to better define possible abnormalities in In this family, 54 deaf individuals(28M, 26F) have been erythropoiesis and to gain insight into potential therapy for identified and traced back 5 generations to one common female the severe anemia in Gaucher patients, erythropoietin levels ancestor. The deafness is progressive in nature, usually were studied. presenting in infancy and childhood. Two individuals with The glycoprotein hormone erythropoietin was quantitated in remnant hearing skills underwent extensive hearing tests which both anemic and non-anemic Gaucher patients by radio- suggested a cochlear lesion. Transmission only through the immunoassay. Five of the 6 patients with normal hemoglobin maternal line(and ending paternally) initially suggested mt levels (Hgb greater than 13 gm/dl) had normal EPO levels inheritance. This vas, however, an inadequate explanation as (less than 4OmU/ml). The other patient had significant to the many cases of transmission through unaffected females. pulmonary disease and an elevated EPO level of 110 mU/ml. Transmission of the disease occurred from 9 of 15 unaffected Three of 6 anemic Gaucher patients demonstrated elevated EPO female parents [segregation ratio(S)-26/1081 and 6 of 7 levels of 300 to 1600 mU/ml. Although the 3 other anemic affected female parents (S-28/53). In contrast, none of the 11 patients had initial erythropoietin levels within the normal unaffected male parents (S-0/68), and 6 affected male parents range, when their baseline Hgb levels (8-11 gm/dl) dropped (S-0/27) from the maternal pedigree had affected offspring or further by 1-2 gm/dl, erythropoietin levels rose to above disease in subsequent generations. 100 mU/ml. Thus, while some Gaucher patients appear to have The pedigree data formally fit a two-locus mode of erythropoietin levels and EPO response appropriate for their inheritance due to a mt gene and an autosomal recessive gene. degree of anemia, in other Gaucher patients the response may Given the size of the pedigree, all other single locus and be inadequate. two-locus modes of inheritance could be rejected. The size of Two additional adult Gaucher patients were treated with individual nuclear families was such that the distribution of recombinant human erythropoietin (rHuEPO) for their anemia. affected was further consistent with the segregation of an Each demonstrated an appropriate rise in hemoglobin and autosomal recessive in addition to the mt locus. One class of reticulocytes, both parameters falling when the rHuEPO dose mating types(affected mother) allowed prediction of the was tapered. The successful response in these patients recessive gene frequency, which was confirmed by the offspring suggests that certain Gaucher patients may benefit from this frequency in the second mating class(unaffected mother). The therapy. Treatment with rHuEPO may alleviate the need for underlying disease mechanism is likely the dysfunction of the repeated transfusions and/or splenectomy, help support mt multienzyme respiratory chain, components of which are patients perioperatively, and by improving tissue perfusion coded for by both mt and nuclear DNA, resulting in an energy and oxygenation potentially may decrease bony complications. supply problem to the auditory system.

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Craniofrontonasal dysplasia: A disorder with female prepJ Another X-linked mental retardation syndrome. rance J. Sienel-Bartelt. M.D. Hospital for Sick Children. A. Sommer, Childrens' Hospital and the Ohio State Univ., Toronto, Canada 700 Children's Dr., Columbus, 43205. We report two families with coronal synostosis and fronto- We describe two brothers with mental retardation and nasal dysplasia (craniofrortonasal dysplasia). D tic fea- tures incliied 1. Orbital hypertelorism, 2. &oad or bifid multiple congenital anomalies different from previously nose, usually with a midline groove, and 3. Coronal synos- reported cases of X-linked mental retardation. tosis. Associated features incltxed cleft lip ard palate, MEF was born to a 29 year old G4,P3,Abl mother after a broad or duplicated thsta, minor skeletal abnormalities and pregnancy complicated by spotting during the 2nd trimester. bicornate uterus The five affected female meuibers in these Delivery was near term by repeat C-section. While in NICU, families are cxnsistent with the aberrant sex ratio previously he developed seizures. Because of hypertelorism, abnormal reported for this sub-group of frontonasal dysplasia. auricles and hypospadias, the diagnosis of Smith-Lemli-Opitz The first family was identified following the birth of a female child with unilateral cleft lip and palate, marked Syndrome was suspected. By age 4 years, the patient was hypertelorism, and bilateral coronal synostosis. Thunbs were retarded and thought to be 4 months developmentally. He had bifid and unilateral talipes equinovarus was present. Her a normal calvarium with abundant dark hair, bushy eyebrows, mother had surgery in childhood for orbital hypertelorism and a hypertelorism, broad nasal tip and small auricles with No one else in the was midline nasal groove persisted. family superiorly folded helices. He had very small male genitalia, affected but an older male has widely spaced eyes. sibling hypospadias and non-palpable testes. He had camptodactyly of Ihe second family was seen following a bilateral cleft lip and palate repair in a female infant with unilateral coronal all fingers, smooth palms and soles, overlapping toes and synostosis, wide nasal tip with midline nasal groove and marked metatarsus adductus. Tracheostomy and gastrostomy tubes were hypertelorism. Metatarsus aducts and broad thumbs were in place. present. The child's mother had a unilateral coronal synos- MF is the younger brother of MEF and was born at term by tosis repaired in infancy, marked hypertelorism and a median repeat C-section with a weight of 8#5oz and a length of 21". nasal groove, as well as a bicornate uterus. Her maternal Examination revealed: small palpebral fissures, mild ptosis, grardmother had bilateral coronal synostosis, brachyoehaly, epicanthic folds, broad nasal bridge, upturned nasal tip, hypertelorism ard broad thumbs. Affected members had a v- superiorly folded helices on posteriorly rotated auricles. shaped frontal hairline. hese affected females (with mother-daughter transmission) redundant skin at the back of the neck, small penis, add to the female prepcrderance of reported s of cranio- hypospadias, undescended testes, metatarsus adductus and front al dysplasia. He discrepant sex ratio, excess of flexion contractures of all IP joints. Although apparently reported other-daghter diads, and more severe female expres- more alert than his older brother, he also developed seizures sion in reported cases cannot be simply explained. X-linkage and had significant developmental delay. with fetal male loss and sex-limited expression have been The patients have some features of the Smith-Lemli-Opitz previously proposed. Biryonic imprinting may also erhane maternal transmission of this disorder and could account for Syndrome but too many additional problems to be consistent with that diagnosis. They also have similarities with cases the excess of affected mothers over affected fathers. Bicornate uterus in the second family may represent a of X-linked mental retardation described by Holmes and Gang, further midline defect in this disorder. We would eorage but enough features are different to suggest yet another X-linked mental retardation sYndrome. abdosiral ultrasounds in other affected female children. Clinical Genetics A79 (0305) (0306)

Premature Ageing Syndromes as Connective Tissue Disorders Eventration of the diaphragm with colobomatous microphthalmia and corneal clouding-a new Mak W. Steele and John M. Greatly, Div. Genetics, Dept. Pediatrics, Chil- syndrome? R. D. Steiner, K. E. Bove, and P. Hosp. and Univ. of Pittsburgh, PA. St. J. Dignan. Children's Hospital Medical dren's Center, Cincinnati, Ohio. The study of premature ageing syndromes is in the hope that the genes found A 2380 gram female infant was born at 37 to be involved in these disorders might also have effects in physiological weeks gestation to healthy, unrelated parents. ageing. However, there is increasing evidence to implicate connective tissue The child had severe respiratory distress at birth. Chest radiograph revealed bilateral disordersinthe aetiologyofseveralofthe premature ageing syndromes. Achild elevated On examination the is presented whose phenotype falls into a diagnostic category between acro- hemidiaphragms. infant had small eyes with corneal clouding geria and metageria, and who had normal fibroblast senescence, type III and hypoplastic nipples and nails. Respiratory procollagen and breakage analysis, but had elevation of his urinary hyaluronic status deteriorated and the infant expired at acid. As these two conditions appear to comprise part of a range of related 10 days of age. conditions, his phenotype was designated as belonging to the 'acrometageria At autopsy the external examination revealed spectrum.' The phenotypic resemblance between acrometageria and Ehlees- bilateral colobomas, corneal edema, and cataracts. Danlos syndrome type IV is apparent, but in the former there is no apparent The nipples were hypoplastic. Internally, there deficiency of type III procollagen production, as characterises the latter. was pronounced thinning of the central/posterior Increased urinary hyaluronic acid levels in some of these patients provides an musculature of the diaphragm. The liver and indication of abnormal type III collagen metabolism, however, and a previous spleen were deformed conforming to the inference that thedistribution of abnormal collagen in acrometageria is hetero- configuration of the eventration. No other malformations were noted. Phrenic nerves and genous may explain in part our failure to demonstrate abnormalities. Urinary were normal. Cultures and hyaluronicacid is also elevated inthe Wemerand Hutchinson-Gilford progeria skeletal muscles serology for viruses and toxoplasma were negative. syndromes, indicating that even these phenotypically dissimilar premature Eventration has been found in association ageing syndromes may be part of the same biological continuum. How with many different anomalies including syndromes are defined as representative of'premature ageing' is due largely to hypoplastic lungs, volvulus, hypoplastic ribs, thework of Martin in 1974. His criteria areassessed fortheircompatibility with cleft palate, hemivertebrae, and congenital connective tissue disorders, simple analysis supporting the association. The heart disease. Fryns (J Med Gen 24:271-274) question inevitably arising out of all of this speculation is whetherdemonstra- describes a syndrome with diaphragmatic defects, tion of specific genetic aetiologies invalidates the concept of premature ageing coarse facies, corneal clouding, and distal syndromes. A priori this appears inevitable, as such mutations occurring limb hypoplasia. This child lacked the coarse predictably with age does not appear physiologically realistic. On the other facies and severe limb defects seen in Fryns hand, the evolutionary approach to ageing has provided the conclusion that syndrome. Diaphragmatic eventration with has been ageing is the result of the inevitable interplay between genes and environment, colobomatous microphthalmia reported in one other child (Radhakrishnan, J a model in which the mutant genes in may find Ind. Ophthal. premature ageing syndromes Additional case relevance. 28:221-222, 1981). reports are needed to identify the associated defects as a new syndrome or a variant of Fryns syndrome.

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A unique Neurofibromatosis 1 (NF-1) family: two mutations, Aarskog syndrome revisited:I.T. Thomas, MJ. Pettenati. N. Rao. and nonpenetrance or heterogeneity? M.N. Berry. Bowman Gray School of Medicine of Wake Forest E. Suiansky.' and K. Ward2. University of Colorado School University, Winston-Salem, NC 27103. of Medicine, Denverl; University of Utah School of Medicine We have recently seen two families with a four generation Salt Lake City2, pedigree for Aarskog syndrome. One family presented with a 4 yr NF-1 is an autosomal dominant disorder with variable old boy who had a learning disability, short stature, hypertelorism, an clinical expressivity. The recent mapping of NF-1 gene to unilateral frontal upsweep hair pattern, small hands, soft tissue chromosome 17, absence of documented heterogeneity and of and a shawl scrotum. His mother was of short of linked DNA make syndactyly fingers, availability closely markers, accurate had a widow's and soft tissue of the hands prenatal and presymptomatic diagnosis possible. We have stature, peak, syndactyly of extended At least two of her sons identified a kindred with clinical symptoms of NF-1 in 3 with unusual position fingers. years old proband and his paternal aunt. The proband's were affected, as was her mother, one of her brothers, the maternal parents, paternal grandmother and 3 aunts/uncles were grandmother and the great grandfather. The other family presented clinically asymptomatic. Paternal grandfather was deceased, with a 14 yr old boy who had mild mental retardation, hypertelorism, but unaffected by history. small hands, fifth finger clinodactyly with only one interphalangeal DNA linkage analysis was performed on 7 individuals crease, soft tissue syndactyly of the fingers and a shawl scrotum. His from 3 generations, including the 2 affected. Genotypes mother was short, she had a widow's peak, small hands, soft tissue for 8 RFLPs which flank the NF-1 locus where determined for syndactyly of fingers, and fifth finger clinodactyly. Her mother was each family member (marker: disease recombination is <3% affected and had two affected sisters, one had three affected for each of these were constructed similarly markers.) Haplotypes the other had one affected She also had one affected assuming no recombination. The proband inherited a grand- boys, boy. two sons. The maternal was maternal haplotype from his father which was not found in brother who had unaffected grandfather his affected aunt who has inherited the opposite grand- short and had the features seen in the proband. maternal haplotype. Alternatively, this aunt may have The literature contains pedigrees which purport to show sex- inherited a recombinant haplotype. Either explanation influenced autosomal dominant inheritance, autosomal dominant, and would require that a number of other family members have in two families an association with a balanced X-autosome nonpenetrance or recombinant haplotypes. Since penetrance translocation with breakpoints at Xql3 and Xql2. We feel our cases is felt to approach 100% in NF-1, and since recombination support the contention of sex-influenced autosomal dominant has not been previously observed with 2 of the markers inheritance and present molecular analyses of the two families. which would have to show 2 or more recombinants in this family, we feel that this family has 2 individuals each with a de novo mutation. Alternatively, this family may have a less penetrant form of NF-1, perhaps due to a gene on another autosomal location. Sequence analysis of the NF-1 region is in progress. A80 Clinical Genetics (0309) (0310)

Syndromes with renal, radial, and ocular defects: A phenotypic A unique combination of hamartoses in an adolescent. community. E. Torrijos, C. Laqui-Pili, B. McKenna, E. Lieber, J. French. H.V. Toriello, J.B. Moeschler, J.M. Graham, Jr, and J.V. Interfaith Medical Center, Brooklyn, New York, 11238. Higgins. Butterworth Hospital, Grand Rapids, MI, Michigan A 13 year old black female was assessed for multiple anoma- State University, E. Lansing, Dartmouth-Hitchcock Medical lies; bilateral macrodactyly, noted at birth, splenomegaly, Center, Hanover, NH, and Cedars-Sinai Hospital, Los Angeles, and cafe au lait spots (CLS). She was the product of a non- CA. consanguineous union; the mother was drug dependent. There We have recently seen two individuals with renal, radial were no similarly affected biologic relatives. She was and ocular defects, as well as choanal atresia and hearing operated on for a lymphangectatic cyst at 11 years of age. loss. Patient 1 had in addition Duane ocular retraction and The height and weight were 25th %tile; head circumference imperforate anus; patient 2 had lacrimal stenosis and exter- 75th. She was Tanner stage III. A large cluster of irregular nal ear anomalies. Patient 1 likely has the DR syndrome, border CLS (7 x 8 cm) were present on the ventral left thigh whereas patient 2 and his family likely have LADD syndrome and a small CLS (0.5 x 0.5 cm) located at the left side of the with previously undescribed additional features. These mouth additionally involved the buccal mucosa. Healed surgi- patients prompted a literature review which demonstrated that cal scars were present on the left clavicular and right dorso- there are at least nine phenotypically similar conditions lateral chest areas. A smooth firm spleen was palpated 7-8 cm which thus constitute a syndrome community. below the left costal margin. Macrodactyly was present in The concept of a syndrome community was first proposed by both thumbs, as well as the right second and third fingers. Pinsky in 1974, who suggested that syndromes with similar Right ulnar deviation was present. Left upper extremity phenotypic patterns were caused by similar pathogenetic hemihypertrophy was manifested by a left elbow circumference mechanisms. However, recently more attention has been paid of 21.0 cm; the right was 20. No focal or space-consuming to describing differences, rather than emphasizing similari- neurologic signs were present. She was cognitively normal ties between syndromes. We suggest that whereas distin- with a decreased attention span and hyperactivity. guishing between syndromes may be useful for gene mapping X-rays of the hands show bilateral enlargement of the soft purposes, recognizing similarities may aid in improving tissues and bones of the thumbs and the right 2nd and 3rd patient management and determining possible pathogenetic digits, as well as right ulnar deviation. A liver/spleen scan mechanisms. Discussion will focus on this concept. demonstrates spleen enlargement. The EEG and cranial CT scan are normal. Hematologic studies are normal. The phenotype appears to represent one the over 50 phakomatoses (hamartoses) presumed to be paracrinopathies (Kousseff & Medan, Dysmor- phology, Clin. Genet. 2:76, 1988; Clin. Genet. 37:97, 1990). The uniqueness of this case emphasizes the existence of transitional forms of these disorders that continue to cause diagnostic and nosologic difficulties.

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A Nm DOIN CRPTO UTXS SYNON WITH Mental retardation in Turner syndrome: etiology and x cmo B ND DNT ANOIMAL. frequency. D. L. Van Dyke, A. Wiktor, M. J. Worsham, M. Traboulsi. M.D. X Howard M. Sial. M.D. Amy Witt, D. A. Miller, J. R. Roberson, V. R. Babu, L. Weiss. Cstexbaum. N.D. Carol. A. ~anannO-Sru Henry Ford Hospital, Detroit; University of Michigan, Ann fd.D.' and Paul ?avaris. M.D.%. Departments of Arbor; Wayne State University, Detroit. Ophthalmology, "Clinical Genetics, and Although specific learning disabilities are more common in 3Pediatrics, Children's National Medical Center, Turner syndrome (TS) patients than in the population at 111 Michigan Avenue, N.W., Washington, D.C. 20010. large, most have normal intelligence. However, since there The association of cryptophthalmos, syndactyly, has been uncertainty about the frequency of mental coloboma of the alae nasi, unusual hairline, and retardation (MR) in TS, and since some patients with TS have urogenital anomalies is a well recognized unexplained mental retardation, we reviewed our experience to autosomal recessive disorder. We have evaluated a look for a high risk subgroup. Among 174 TS and gonadal mother and daughter with isolated cryptophthalmos, dysgenesis patients who had an X abnormality, twelve had MR. sicrophthalmia, and Peters anomaly. All of the six (100%) who had a small ring X were educable or At birth, the proband daughter was found to trainable mentally impaired (EMI/TMI) - more severe delay have bilateral fusion of the upper and lower lids than expected with TS. Among the 168 with other X with a midline dimple where the cornea adhered to abnormalities, only six had similar delays (two from the upper lid. Small globes were palpable, and postnatal catastrophies). Thus the frequency of mental the infant reacted to bright light. The retardation among those without a small ring X was 3.6%, and extremities were normal as was the genitalia. the of mental retardation was and of the frequency unexplained 2.4%. Ultrasonography computed tomography Polymerase chain reaction studies showed no Y-derived orbits revealed microphthalmia and a lens in each material in two eye; the brain appeared normal. Chromosome r(X) patients, and in situ hybridization studies revealed a normal female karyotype. The confirmed X origin in all five who have been evaluated (using lids were surgically separated on the right side X centromere-specific probes pBamX (tritiated, in 1 case) and and a corneal transplant was done. The lens was DXZ1 (biotinylated, in all 5 cases)). C-banding was fused to the posterior surface of a scleralized consistent with origin from the X in the sixth subject. The cornea. The retina was attached and the optic subjects with a very small ring X (our 6 subjects plus 14 nerve was hypoplastic. The proband's mother was other cases) comprised a clinically distinct subgroup who had found to have the same condition. Bilateral EMI/TMI and shorter stature than expected in TS. Half had a enucleations had been performed for end stage head circumference <10%ile, 37% had seizures or an abnormal glaucoma. The proband's father is blind from EEG, and 25% had simian creases. Strabismus, sacral dimple, retinopathy of prematurity. There is a healthy 17 and heart disease were also common. Hypomelanosis of Ito in month old brother with normal vision. There is no 25% may be a general feature of mosaicism. Neck webbing was consanguinity. less frequent than in 45,X. We hypothesize this syndrome This family represents a unique dominant syndrome of isolated cryptophthalmos with globe results from lack of lyonization of the r(X) chromosome due anomalies and normal neurodevelopment. to loss of the X inactivation center. Thus, excluding the MR associated with presence of a small ring X, MR is not significantly increased in. Turner syndrome. Clinical Genetics A81 (0313) 3.8 (0314)

Residual Ca-mediated chloride secretion In CF rectal biopsies and age at The significance of genetic and nonshared environmental diagnosis In relation to the most common CF mutation. inf luences on human obesity: The Danish Adoption Study. H.J.Veeze1a D.J.J.Halley2. M.Sinaasaapel. H.R.de Jonge3 G. P. Voiler. M. R. Srinivasan. A. J. Stunkard! T. I. A. of Ped. Children's Hospital, Serensen, and D. C. Rao. Washngton Univ. School of M.F.NlermelueW. 'Dept Gastroenterol., Sophia Medicine, St. Louis, Missouri ,-Univ. of Rotterdam. Dept Clinical Genetics, Academic Hospital Rotterdam. Pennsylvania, Dijkzigt, Philadelphia, Pennsylvania, Hvidovre Univ. Hospital, 3Dept of Biochemistry, Erasmus University, Rotterdam, The Netherlands. Copenhagen, Denmark. Electrophysiological studies have demonstrated that the pathological Human obesity, which is considerably familial but which basis of cystic fibrosis Is a defective cAMP regulation of chloride secretion can be ameliorated with appropriate environmental across epitheilal cells. In addition to the defective cAMP regulation the Ca- intervention, represents a significant health problem. This mediated chloride secretion Is also defective In the intestine of CF patients. has been used to justify prevention strategies that focus on The observed frequency of the most common CF mutation deltaF508 in the family environment. However, this focus might be misplaced since Dutch CF chromosomes was 77%. This Implies that about 60% of the there is little evidence that the familial nature of the disorder is due to familial to Dutch CF patients are homozygotes for the deltaF508 mutation. environment any substantial degree. This investigation uses a model of We performed short circuit current measurements In a modified (Isc) biological inheritance and cultural transmission in a full Ussing-chamber on rectal suction biopsies In 29 CF patients who were adoption design (nonrandomly-ascertained adopted child, tested for the deltaF508 mutation. We identified patients with reversed biological and adopted parents, unadopted full- and half-sibs responses to carbachol as well as those with small positive Iac responses. of the adoptee, and intact nuclear families). The model Fifteen out of nineteen homozygotes for the deltaF508 mutation had a permits quantification of genetic heritability; various forms reversed response only. A small positive response, however, was seen In of cultural inheritance including parent-child transmission, 7/10 CF-patlents with at least one other mutant allele. The difference maternal effects, and uterine environment effects; sibling shared between these categories of patients was statistically significant (p=0.03, environmental effects; spouse resemblance; and a test Fishers Exact Test). We conclude that the carbachol Induced chloride for selective placement. The model was applied to the Danish adoption study of obesity, including data on 660 adoptees and secretion Is not completely defective In all CF-patients. Apparently, most of 2302 of their biological and adoptive relatives. Results the homozygotes had mutations that do not affect the non-deltaFS08 indicate that there is no evidence of selective placement or function of CFTR In the Intestine to the same degree as deltaFS08. We spouse resemblance in the sample. The most striking result sought to Investigate whether these differences between the genetic is that detailed scrutiny of cultural transmission and subclasses of patients were reflected In different clinical presentations. We sibling environmental effects indicates that there is clear did observe a significant difference (Wilcoxon's Rank Sum Test, T=3.03, P absence of an effect of the familial environment on < 0.001) In the distribution of the ages at diagnosis between homozygotes resemblance among relatives--all familial resemblance appears for deltaF508 (n-23) and the other patients (n=17). DeltaF508 to result from genetic effects (h = .31). Nevertheless, there is a substantial component of an individual's homozygotes tend to be Identified as patients at neonatal or Infantile age. phenotypic variance that is due to nonshared environmental The age at diagnosis of patients with at least one unknown allele on the effects (e = .69). Thus, nonfamilial environment should be other hand ranged between neonatal and young adult age. Presently, the focus of intervention and prevention strategies. studies are carried out to correlate these findings with specific clinical parameters.

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Y-156 and H-Y antigen In a 46,XY true hermaphrodite: PCR and flow Insulin Dependent Diabetes Mellitus in A Child With Femoral cytometry. S WachteL V.Yaswanr, L.r, U. MflOlBrt, G. Wachtel. Hypoplosia-Unusual Facies Syndrome.M.Wajnrajch, P.Kreitzer, S laas. University of South Alabama', Boston Children's Hospitalt, A.Romano, and A.Shanske. Albert Einstein College of and Unersity of Tennessee. MemphisIN 38163. Medicine, Schneider Children's Hospital, Long Island 46,XY true hermaphroditism, repreing only 12% of the 195 Jewish Medical Center, Craniofacial Center and Division cases oftue hermaphroditlsm catalogued by Van Niekerk and Retlef of Endocrinology, New Hyde Park, N.Y. (Hun Genet 58, 117-122, 1981), was recently diagnosedin a six-month- The increased risk of malformations in infants born to old phenotypic female with mild clitoromegaly and elevated plasma diabetic mothers is well established. The relative risk for testosterone (103ng/dl). Exploratory laparotomy revealed a left testis malformations of the musculoskeletal system is especially a small remnant of ovarian tissue, and a right ovary. The with high. The femoral hypoplasia-unusual facies syndrome karyotype was 48,XYIn testis, ovary, akdn and peripheral blood. There (FH-UFS) is a rare constellation of skeletal was no evidence of mosalcism and the Y appeared normal. Because craniofacial, intermediate levels of aerologlcally-detected H-Y antigen (H-Ys) have and genitourinary defects often seen in infants of diabetic generally been reported in 46.NX true hermaphrodites relative to the mothers. We have been following a 3 year old white male levels of H-Ys in normal males and females, and because H-Ys has not with FH-UFS who has developed insulin dependent diabetes been studied in 468XY true hermaphrodites, we comparedaepession of mellitus (IDDM). H-Ys in the cells of the patient with expression of H-Ys in cells from S.A. was the 2520 gm product of a term pregnancy com- normal males and females. Blood lymphocytes were reacted with plicated by class R maternal diabetes. His mother took 50 monoclonal H-Y antibody gw16 and with FITC-conjugated goat units of Lente throughout the pregnancy.The infant was antimouseig, and 10,000 cells were scored directly for fluorescent label delivered by c/section because of a breech presentation. in the EPICS V flow These tests revealed no difference cytometer. He required respirator support because of RDS and was weaned between expression of H-Ys in the patient and that in normal male to room air after 3 days.He was observed to have a controls. In other tests. DNA was extracted from whole blood of the long upper lip, small broad notched nasal patient with guanidine hydrochloride. The DNA was evaluated for mouth and mandible, tip, ptosis of left third of hard and sequences in the short arm of the Y-chromosome by amplification lid, a cleft of posterior accordig to the PCR method of Perkin-Emer-Cetus. We used the Y- entire soft palate and bilateral inguinal hernias.The 156a and b oligonucleotide primers derived from the Y-156 DNA skeletal anomalies incuded bilateral femoral hypoplasia, sequence. Located among the Y-specific repeats of the DYZ5 regionin dislocation or subluxation of both hips and hypodactyly of the paracentromeric Y short arm, Y-156 is proximal to TDF and the left foot. A gastrostomy was placed at 2 months because probably also to HYS (the controlling gene for H-Ys antigen). No of poor feeding. The hips, palate and hernias have been differncne was noted in the amplification of Y-156 in DNA from the surgically repaired.He has had frequent urinary tract in- patient and of Y-156 in DNA from a normal male. We conclude that the fections because of a neurogenic bladder. He is develop- of the patient is with respect to the regions Y-chromosome normal mentally delayed and developed overt IDDM at 3 years of age by Y-156 and HYS. Further of the Y is planned represented evaluation and currently requires 15 units of insulin per day. in this case, by Southern blotting with a battery of Y-probes, as this Our patient case of a rare could provideinsights in the etiology of 46,XY true hermaphroditism represents another FH/UFS, disorder infants of diabetic whose even though the condition might be caused by autoaomal or X-llnked associated with mothers, factors. complete phenotype has not been fully delineated. The occurrance of IDDM in this patient most likely represents another feature of this disorder, since the recurrence of IDDM, in the offspring of adiabetic parent is in the order of 1%. A82 Clinical Genetics (0317) 1.163 (0318) 1.164

DOES ALOPECIA AREATA (A) PREVENT THE CLINICAL EXPRESSION OF Successful pregnancy in a woman with argininosuccinic aciduria (ASU): biochemical THE GENETIC SUSCEPTIBILITY TO INSULIN DEPENDENT DIABETES investigations and outcome. JL.Wacd.#, C.M.MeyMrs#, V.E. Shi*, M.Drooks#, LE. Si D)? S.J. Wan* T. Shohat#, C. Vadheim*, J. Tyler*, ice#, D.C.Shave#, S.Eljas#. Univ. of Tennessee, Memphis#, and Mass. General Hosp., J. Edwrds-, lu, J.I. Bter*. *Cedars-Sinai and Boston'. UCLA, LA, CA; DIOF, Israel; -HAIR, Inc.; AVA and UCLA. Late onset ASU is a rare autosomal recessive urea cycle disorder caused by absence of Prior studies of family and autoimmune aspects of alopecia argininosuccinic acid Iyase (AL). Accumulation of argirinosuccinic acid and its metaboltes (ASA) areata have demonstrated an increased frequency of thyroid occurs; additionally arginine (arg) becomes an essential amino acid. As with maternal PKU, disease and autoimmunity in patients and a concomitant improved treatment has resulted in affected females surviving to reproductive age. In pregnant increase in thyroid disease in relatives. Of further interest ASU patients it was unknown whether 1) ASA crosses the placenta, and 2) if so, whether ASA was the increased frequency in relatives of diabetes mellitus, exerts a teratogenic effect. We report the first monitored case of maternal ASU, the patient type unspecified, yet paradoxically, no increased frequency of being part of a family previously reported (J Ped 84:85,1974). The patient is a 21-year-old, diabetes in the AA patients. The evidence for autoimmune basis gravida 2, para 1, diagnosed with ASU since age 3 1/2 years. She has been in poor dietary for AA and for IDDM led us to propose that any relationship complience, but has continued sporadic arg supplementation. She has had no documented with diabetes would be specific for the IDDM type. hyperammonemia. Her first pregnancy was electively terminated at 8 wks gestation. ASA was To test this hypothesis, we conducted a questionnaire study detected in amniotic fluid (normally absent) (AF) and fetal cells were in the heterozygous range on 517 Caucasiar patients with AA ascertained through the HAIR for AL deficiency. She presented a year later at 23 wks gestation. A plan was Initiated for (Help Alopecia International Research) organization. Respon- assessment of 1) presence of ASA in AF; 2) the affected state of the fetus; 3) maternal plasma dents consisted of 140 males and 374 fema es, with a mean age and AF amino acids; 4) biweekly maternal ammonia and nutritional parameters; 5) fetal growth of 40.5 + 17.5 years. Information included the type of and development; and 6) neonatal outcome. AF from the second pregnancy revealed ASA present alopecia, age of onset, age at study, frequency of remission, at 25 wks (401M/L), 34 wks, and at term (203 gM/L). Chromosome analysis was normal. Fetal and the frequency of autoimmune and other diseases in first growth was foliowed monthly by ultrasound. Doppler flow studies were normal. Maternal plasma and second deqree relatives. Among the 11 diseases surveyed, arg levels were in the low normal range, but required increased arg supplementation to maintain an increased frequency of thyroid autoimmune disease (14.7%), them. AF arg levels were low normal or below normal throughout Maternal plasma glutamine levels vitiligo (8.9%) and Addison (1%) was found among the AA were normal, but rising near term. Dietary protein intake exclusive of arg ranged from 25-68 patients, compared to the general population. Relatives of AA gm/day. Ammonia levels were normal. A 3310 gm female delivered at 37 weeks gestation. Physical had a higher frequency of the autoimmune diseases as well. The exam revealed head circumference at the 25%ile, weight at the 50Yile and length at the 75%ile. prevalence of colon and other cancers, studied as control An asymptomatic ventricular septal defect (VSD) was the only anomaly detected. AL analysis of diseases, were not increased in either cases or relatives. cord blood erythrocytes was 2.8 gM urea/gm/Hbthr (25% normal), considered heterozygous for The frequency of IDDM was significantly elevated among ASU. ASA was present in cord blood (51pM/L); infant plasma levels decreased to 8g.ML at 2 relatives of patients with AA (e.g., 1.2% in siblings compared days, and were absent at 1 month. Breast milk obtained 3 weeks postpartum revealed 858IML to 0.2% in the U.S. population, p-value 0 004) but not of ASA. Infant plasma and urine taken pre- and post-breast feeding revealed no ASA present. increased among the AA patients themselves (O.2%5. In At 7 months of age, the infant is growing and developing normally. EEG is normal. We conclude 1) contrast, the frequency of NIDDM was not elevated in AA since ASA is recovered in AF of this ASU mother whose infant was unaffected, maternal ASA families (e.g., 1.2% in AA cases, 1.6% in siblings). The indeed crosses the placenta; 2) maternal arg requirements need to be monitored during familial predisposition to autoimmune diseases is consistent pregnancy; 3) pregnancy did not induce detectable or clinical hyperammonemia; 4) reproductive with the interpretation that AA itself has an autoimmune capacity has not been adversely affected in this patient; 5) ASA appears in breast milk of basis. The increased frequency of IDDM in relatives suggests maternal ASU; 6) the unaffected heterozygous infant appears able to metabolize breast milk that this genetic predisposition includes that toward IDDM, ASA; 7) the significance of a VSD in this infant is unknown; and 8) at 7 months, infant growth and yet the lack of increased frequency of IDDM in patients, in development appear normal. direct contrast to their own increased frequency of other organ-specific autoimmune diseases, suggests that the occur- rence of AA uniquely protects against the development of IDDM.

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Quantitative analysis of the face in the Beckwith Wiedemann NEUROBLASTOK AND HIPATOBLASTOMA IN A PATIENT WITH WIDIMANNI- syndrom! and detection of minimnlly affected g~ne iarriers. BECIWITH SYNDROME. D. Warcowskil. D. Chitavat2. V. Ebelt3 RE Ward , LF Escobar, ME Carlin, and JL Haines Indiana J.E. Dimmick. Departments of Medical Genetics1, Pediatrics University Purdue University, Inlianapolis, IN. Indiana Pathology , University of British Columbia, Vancouver, B.C. University School of Medicine. Un4versity of Miami Mailman and Montreal Children's Hospital, McGill University, Montreal, Center for Child Development, FL. Neurogenetics Laboratory, P.Q.2, Canada. Massachusetts General Hospital, Boston. We recently reported a patient with Wiedemann-Beckwith We conducted an anthropometric analysis of the face in syndrome (WBS) who developed neuroblastoma at 7 months. She 20 individuals with the Beckwith Wiedemann syndrome (WBS) is now 4 years old and has developed a hepatoblastoma. She and 75 of their first degree relatives. The findings demonstrate was born at 31 weeks gestation to nonconsanguineous parents. that (1) contrary to expectations affected individuals gener- The family history was unremarkable. The pregnancy was ally have a smaller, narrower mandible than is seen in normal complicated by maternal hypertension necessitating Cesarean controls. They also tend to have shorter nasal length, greater section. Weight, length, and OFC were at the 75th centiles upper facial depth, total facial height, mouth width, ear at birth. Physical findings included macroglossia and width, inner and outer canthal distances. (2) This unique omphalocele as well as a glabellar nevus flammseus, large pattern can also be found in first degree relatives who have anterior fontanelle, creased ear lobules, cleft soft palate, other non-facial traits associated with the condition (e.g. hepatosplenomegaly, and enlarged kidneys with sonographically ear pits or umbilical hernia). (3) The facial pattern appears normal parenchyma. The neonatal course was complicated by to moderate with age as facial parameters grow toward population severe pulmonary disease and hypoglycemia which responded to means. We conclude that this unique facial pattern can be IV glucose infusion. Her neuroblastoma was identified at 7 used in some multiplex families to ascertain minimally affected months of age and partially resected. The histopathological gene carriers. This should enhance the feasibility of molecular findings were indicative of a good prognosis. Prometaphase linkage studies for localizing the genetic defect (s) respons- analysis of chromosome 11 was normal. ible for this condition. At the age of 4 years she developed abdominal pain, dark urine, pale stools and jaundice. The serum AFP and the conjugated bilirubin levels were markedly elevated. Abdominal CT scan showed massive hepatomegaly with abnormal vascular enhancement. Open liver biopsy revealed multiple nodules of tumor. Histopathologic examination showed this to be a fetal type of hepatoblastoma. To the best of our knowledge this is the first report of a WBS patient with both neuroblastoma and hepatoblastoma. Despite the differences in the histopathology and organ of origin of the tumors associated with WBS, the possibility of a common pathogenetic mechanism is suggested by the occurrence of more than one primary tumor in some patients as well as the demonstration of loss of heterozygosity for genes on the distal part of llp in many of these tumors. The propensity for the occurrence of this phenomenon among WBS patients has yet to be fully explained. Clinical Genetics A83 (0321) 14.12 (0322)

Congenital erythropoietic porphyria: characterization of the ge- AUTOSOMAL DOMINANT CRYSTALLINE DYSTROPHY: nomic structure and identification of mutations in the uropor- Lester Weiss, Bradley W. Richards, David E. phyrinogen III synthase gene. C.A. Warner. H.W. Yoo. S.-F. Tsai. Brodstein, Joseph R. Ferencz, Koichi Maeda A.G. Roberts and R.J. Desnick. Mount Sinai School of Medicine, New Julian Nussbaum. MedicalVGenetics and Birth York, NY Defects Center, Department of Pathology, Congenital erythropoietic porphyria (CEP) is an inborn error of Department of Ophthalmology Henry Ford heme biosynthesis resulting in the deficient activity of uropor- Hospital, Detroit Michigan. phyrinogen III synthase (URO-synthase), the fourth enzyme of the heme biosynthetic pathway. The enzymatic defect results in the A black woman was identified with a accumulation of uroporphyrin I in affected homozygotes with this tapetoretinal degeneration with sparkling autosomal recessive disease. Clinical manifestations include he- intraretinal crystals, retinal pigment molytic anemia and cutaneous photosensitivity leading to blister- epithelial and choroidal atrophy, night ing, scarring and deformity. Previously, we isolated, sequenced blindness, color vision abnormalities, and and expressed in E. coli the full length cDNA encoding URO-syn- paracentral scotomas. This constellation thase (Tsai et al., PNAS 85:7049, 1988). We now report the genomic of findings is most consistent with the organization of the URO-synthase gene and identification of the diagnosis of Bietti's crystalline first mutations causing CEP. Using the cDNA to screen cosmid and dystrophy. Eight other family members were XEMBL genomic libraries, three overlapping clones containing the identified with intraretinal crystals URO-synthase sequence were isolated. Restriction mapping and se- similar to those seen in the proband but in quence analysis indicated that the URO-synthase gene is about 45 varying degrees of progression. kb. All intron/exon boundaries and the 5' regulatory elements Transmission electron microscopy of have been determined. To identify the molecular lesions responsi- circulating lymphocytes in several patients ble for CEP, Southern and Northern analysis were performed re- demonstrated crystals and granular vealing: a 1 kb insertion in one mildly affected homozygote, an ab- osmophilic material of unknown composition normally large mRNA in two unrelated severely affected homozy- contained within abnormal lysosomes. These gotes, and 11 others with grossly normal gene structure and tran- crystals are similar in appearance and scripts of the expected size and amount. Since all homozygotes location to those seen in cholesterol ester must have residual URO-synthase activity to make heme, efforts storage disease. This family demostrates were directed to identify the presumed point mutations for struc- an autosomal dominant inheritance pattern, ture/function analysis. Poly(A)+ mRNA, isolated from cultured as well as other differences from classic lymphoblasts of six CEP patients, was reverse-transcribed and PCR Bietti's crystalline dystrophy. We, amplified. The PCR products were subcloned and sequenced. Anal- therefore, suggest that this new entity be ysis of the first two mutations identified a C to T transition (nt 197) named autosomal dominant crystalline that substituted a Val for Ala at position 66, and a T to C transition dystrophy. (nt 216) that replaced a Cys by an Arg at residue 73. The two mutations were never found on the same allele indicating that the patient is a genetic compound. Hybridization analysis of DNAs from the other 10 homozygotes revealed that only two had an allele with the Cys73 to Arg mutation. These and additional mutations, are being expressed in E. coli for physical and structural studies.

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Insulin-like growth factor 2 serum levels are normal in The pathogenesis of dermatoglyphic alterations and patients with Bedwith-Wieamn syndrome. R. &ii l & arthrogryposis. W. Wertelecki. University of South Alabama, Eeigembiuml, K. Australiel, C. Ptlvdiral2 o of Mobile. Genetics, Hospital for Sick Children, Tronto, Ontario N5G Swinyard (1982) proposed the "Law of the Connective 1X8, Department of Paediatrics2 MGill University, Montreal, Tissue" as an explanation for the pathogenesis of Quebec Arthrogryposis Multiplex (AM) by noting that prenatal Beckwith-Wiedmtann, sydrrae (BS) is a contigucus gene fixation of a given joint (whether due to abnormalities of defect which maps between 11p15.4 arnd llpter. This corditicn the central or peripheral nervous system, myopathy or is an overrt sy pr inantly manifested in the fetus mechanical- constraints on fetal motion) resulted in and yaw child (< 5 years). The clinical features incrlude collagenous proliferation which greatly thickened the joint somatic gigantism, overgrowth of specific tissues ane organs, capsule and replaced atrophied muscle fibers. Experimental Acenatal hypoglycemia arnd a predisposition to the devel-ep-ert denervation results in increased collagen muscle synthesis, of specific embryanal tunirs (Wilms tumor, rhabtkyosarccma, not so if immobilization is caused by casting (Savolainen et hepatdblasttoa). Linkage analysis of autosc dominant pedi- al, 1988). grees with BWS has slhon linkage to 11p15.5 and no reoccrbina- Arthrogryposis and microstomia (MS), facial rigidity ticon has been d nntrated with the locus for insulin-like (FR), skeletal muscle atrophy (MA) and cardiomyopathy (CM) growth factor 2 (IGF2) which maps to 11p15.5. are landmarks of Freeman-Sheldon Syndrome (FS), among other Since many of the functional attributes of IGF2 could syndromes. We found evidence of skeletal and cardiac account for the syts of BWS,this growth factor has been myopathy in such patients. prcpcse to be a candidate gene for INS by nrmerous inves- Skin pterygia (P) are commonly considered to represent a tigators. IGF2 functions as a growth factor which affects sequelae of fetal edema. Hlartwig et al (1989) noted the fetal growth in mice ame demestrstes differential expression concurrence of multiple pterygia with cardiac hypertrophy in various tissues. Although the expression of IGF2 is in- and connective tissue abnormalities, which led him to creased in Wilms tumor and other BNS-associated embrycnal propose that the pathogenesis of P requires both fixation in txmunrs, no evaluations of senum IGF2 in ENS patients have been a flexion position and a weakened connection between the reaprt to date. skin and its underlying structures. T1enty patients with at least 3 major criteria for BS were We noted dermatoglyphic abnormalities (D) in FS similar included in the sttey. Two patients had erbryonal tumors and 3 to those noted by others in AM and Camptodactyly. Volar of then were in the neonatal hypoglycemic pease (IGF2 is known skin evolves into fetal pads which give rise to to affect serum glucose). IGF2 serum levels were neasured by dermatoglyphic patterns. We propose that the above radioreceptor and radioifmsay rollowing acid gel filtra- observations are congruent with recent investigations tion (J.Clin.Endo.Metab. Ag:739,1979). indicating that dermatoglyphic patterns reflect the Normal senum levels of IGF2 were demonstrated for the first arrangements of Merkel cells in the epidermal-dermal 10 patients assayed (including 1 patient with severe hypogly- junction (Moore and Munger, 1989). Perhaps, an expanded cemia). The average IGF2 level was 575 q/hml ± 346 for the ENS hypothesis focused on the functional unit dendrite-myosite- patients and 556 ra/ml± 243 for the age-matched normal con- Merkel cells and relatedly the regulation of the synthesis trols. This suggests that IGF2 may not be the primary defect of collagen and other components of the inter-cellular in BWS or, alternatively, if IGF2 overexprion causes the IBS matrix should be tested further to account for the phenotype, it does not result in icrsed IGF2 serum levels. pathogenesis of AM, MS, FR, MA, CM, FS, P, D seen in various combinations in a large number of malformative syndromes. Do not type on or beyond blue lines (printer's cut lines). A84 Clinical Genetics (0325) 1.168 (0326)

Expanding the spectrum of Killian Teschler-Nicola (KTN) syndrome: Genomics of human dysmorphology: Comparison of mendelian ovarian teratoma, hypertension, mild cognitive impairment and and chromosomal phenotypes. G. N. Wilson. Univ. of Texas phenotype ontology .D.A.H.Whiteman, C. Sunderman & M. R. Southwestern Medical School, Dallas. Rasouljgour, Dept. of Pediatrics, Univ. of Connecticut School of The number, distribution, and mechanism of genes involved Medicine, Farmington. in human morphogenesis may be indirectly studied using conditions. A detailed survey Syndrome diagnosis has traditionally been accomplished from a catalogues of dysmorphologic in Man (VA McKusick, 1989, 8th Ed.) static approach, without emphasis on either dynamic developmental of Mendelian Inheritance syndromes and 752 single defects among 4344 changes in morphology, or on the significance of low frequency, but revealed 810 The majority of syndromes were autosomal distinctive, features in particular syndromes. In the KTN syndrome a inherited disorders. specific cranlofacial pattern, ectodermal pigmentary dysplasia, and recessive, while single defects were more often autosomal syndromes, 54%, and 38% were assoc- compromise of neurologic function with severe mental retardation is dominant. Of genetic 30%, premature death, mental retardation, distinctive and recognizable, and the hypothesis can be tested by iated, respectively, with cytogenetic examination for mosaic tetrasomy 12p. The diagnostic and growth delay as opposed to 23%, 3%, and 13% of single marker allows exploration of the expanded syndrome, and evaluatlon defects. When analyzed according to the number and type of dominant syn- of ontologic changes in phenotype, without misclassificatlon. organ systems frequently affected, autosomal We report here a girl with several unusual features, whose dromes involved an average 3.43 systems per disorder, autosomal evolving phenotype led to several erroneous diagnoses prior to recessive 3.89, and X-linked 3.53. These numbers were much confirmatlon of KTN Syndrome. She presented after an uneventful lower than the average number of 10.63 systems affected in pregnancy, with severe neonatal hypotonla, somnolence and poor each of 96 chromosomal syndromes listed in the Catalogue of feeding. A congenital abdominal mass proved to be an ovarian teratoma. Unbalanced Chromosomal Aberrations in Man (Schinzel, 1984). At age 6 years, refractory hypertension (BP160/110 mmHg) Most commonly affected systems in the Mendelian disorders were developed. Response to beta-blockers and negative results of renal and limbs (12% of all anomalies), epidermal derivatives (7.7%), endocrine Investigations, suggests a central neurological basis. Early facial contour (7.5%), eye (7.0%), diffuse skeletal (6.5%), motor and functlonal delays were associated with hypotonia, but more and mouth (5.4%). Less specificity for organ system was noted recent tests of cognitive function yielded developmental quotients of in the chromosomal disorders, where even specific anomalies are 85% or greater. often associated with 15-20 different chromosomal syndromes. Examination at 5 1/2 years revealed typical craniofacial Ring syndromes averaged fewer systems affected compared to features, with skin pigment variation. Chromosome analysis of duplication/deletion syndromes, yet the correlation of systems peripheral blood gave a 46,XX karyotype (100 cells), but cultured affected to aneuploid segment length was inconsistent. Of the skin fibroblasts showed 46.XX (18%)/47,XX,t(12p;12p) (82%). mendelian syndromes, 47 have been localized to specific auto- Review of her sequential photographs allows definition of age somes and 58 to the X chromosomes. For single defects, 24 related changes in the physical phenotype, including exaggeration of have been localized to autosomes and 63 to the X chromosome. the normal increase with age of the vertical face dimension with Although genomic understanding of inherited anomalies is hin- increase in calvarium and forehead height with age, persistence and dered by the requirements of postnatal survival, familial delayed filling in of the broad nasal bridge, and excessive fullness of transmission, and clinical detection, catalogues such as the the facial subcutaneous tissues leading to exaggerated lateral facial MIM can provide useful information as illustrated by these dimensions. These ontologic changes can be correlated with her differences between mendelian and chromosomal phenotypes. previous misdiagnoses, expanding the differential diagnosis and aiding the clinical recognition of KTN Syndrome.

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C4 and Bf phenotypes in Black and Caucasian patients with childhood-onset The Neurofibromatosis-Noonan syndrome: compound heterozygosity Insulin dependent diabetes Mellitus (IDDM). R.J. Wyaft. C. Wan, GA or isoallelism? lumbhen, AgMUL. Rivas. Univ. of Tennessee-Memphis, Department of S. P. Yang, H. T. Hutchison and C. J. R. Curry. Pediatrics, Memphis. Valley Children's Hospital/U.C.S.F., Fresno, California. Specific alleles at the C4 and Bf loi have been shown to be useful markers for We report a family transmitting neurofibromatosis type 1 Insulin-dependent diabetes mellitus (IDDM) in samples of different racial and (NF-1) and Noonan syndrome (NS) through 3 generations. There ethnic composition. The same markers, however, are not demonstrable in each are 2 children with both NF-1 and NS, 1 with only NF-1 and 1 group studied. The purpose of this study Is to examine whether previously In Caucasians with childhood-onset with neither condition from one father; 2 children with neither reported Bf and C4 associations observed and 1 child with NF-1 from IDDM are also found in the US Black population and to report any significant condition from another father; only positive or negative associations with IDDM which are unique to US Blacks. a third father. All the children have the same mother and C4 and Bf phenotypes were determined on 168 Caucasian and 49 Black IDDM grandmother who both have NF-1 and NS. Various hypotheses patients from the Mid-South (USA). All of the patients had onset of IDDM of 9 17 exist for the frequently observed association between NF-1 and years and were followed as outpatients in Memphis. The age of onset was 9.3 ± NS: (1) the Neurofibromatosis-Noonan syndrome (NF-NS) is a 3.9 years for the Black and 7.9 ± 4.3 years for the Caucasian patients. The unique genetic entity rather than a phenotypic variant of malelemale ratio in the patient group was 20 29 for Blacks and 82:86 for either NF-1 or NS, (2) the NF-1 and NS genes are closely linked Caucasians. Controls were healthy hospital personnel from the Mid-South; 43 so that a sub-microscopic 17q11.2 deletion could then result in were Black and 92 were Caucasian. NF-NS, (3) the NF-NS phenotype is an example of the variable in and control Comparisons of alleic frequencies the Caucasian IDDM patient of NF-1 or the coincidental occurence of NF-1 of the vs expressivity (4) subjects yielded a significantly higher frequency Bf*F1 allele (0.063 of 2 common 0.016, pe .03) in the Caucasian IDDM patient group. At the C4 locus, a significant and NS is due to the independent segregation increase In the phenotypic frequencies of C4AQO (rr-2.13, p < .02) and C4A4 (rr Mendelian disorders. Our pedigree argues against (1) and (2), NF-NS transmitted NF-1 without NS to 2 of - 2.91, p < .01) was observed among Caucasian IDDM patients. The frequency of since the mother with the homozygous null C4A phenotype, however, was not significantly increased. her children, and supports either (3) or (4), since there is no In addition, significant negative associations of IDDM with C4A3 (p < .01) and transmission of NS without NF-1. C4A6 (p < .05) phenotypes and no significant positive or negative association = NF-1 genotype with any C4B phenotype were observed in our Caucasian patient population. C Data for Mid-South Blacks with IDDM indicate a positive, but not statistically (a = NS phenotype significant, association of IDDM with the Bf F1 phenotype (rr-3.4), similar to that

seen In our Caucasian sample. However, there was no evidence among Black = IDDM patients of the C4AQO and C4A4 associations observed in our Caucasian [-] presumed sample. The data do support a possible association of IDDM with the C4A2 (rr isoallele(s) 5.86) and C4B2 (rr 5.26) phenotypes among Black IDDM patients; due to small sample size these results, however, are not statiscally significant. f(~ -S The hypothesis that racial admixture may account for the higher frequency of IDDM in US Blacks as compared with African Blacks has been forwarded by others. Our pedigree also demonstrates the effects of various paternal The similarities and differences of the observed Bf and C4 associations in US alleles on the clinical expression of the maternal NF-1 allele. Blacks and Caucasians in our samples are supportive of possible cinical and/or Molecular studies are pending in this family and eventually genetic heterogeneity of IDDM in southern Blacks. Clearly a larger sample of Black IDDM patients will be needed to further clarify this point. Additional Mid- might show that NF-NS is due to compound heterozygosity or South Black IDDM patients and controls are currently being phenotyped for Bf isoallelism. Such a model could explain the Increasing and C4 complement proteins. These additional data will be used to further test severity of NS features in each subsequent generation, assuming the similarities/differences and strengths of associations between IDDM and that isoallele (i) interacts with the NF-1 gene less than complement alleles reported above. isoallele (ii) in terms of producing the NF-NS phenotype. Clinical Genetics A85 (0329) 3.10 (0330) 1.171

Cloning of the human aI(IV) collagen gene and characterization of mutations Angelan Syidre: Further Characterization of the Clinical in Alport syndrome. Humotype, aMd Natural History. IRT ZoriL JE Hundriokson.S J. Zhou- S. L Hostikka* D.F Bark&e+. L.T Choe, S.C. Gerkf ICL Woolven. CI Wills. University of Florida, Gairaeville. Arqelmn Research Gzra serves as a registry of Atjij+ and K_ *Blocenter and Dept. of Biochemistry, me Tryggvson, syreIr& (AS) patients and as a parent suport University of Finland, aDepts. of Medical Informatics, Biochemistry Oulu, The Arqelman Suport Group in the U.K. serves and Medicine, University of Utah, Salt Lake City, #Dept. of Biochemistry, a similar purpose. Th furthe delineate the clinical and Univ. of Rochester School of Medicine, Rochester, NY. dsvelcrrental features of AS, we evaluated two sources of a disease X-linked Alport syndrome is progressive hereditary kidney inforation btained through the oaztis: physical characterized by hematurla and hearing loss with structural defects in the basement membrane (GBM). We have previously isolated cDNA exmination ard him rolution data of 23 glomerular affected pati and family etii data about clones for a novel type IV collagen aS chain and located its gene (COL4A5) to prenatal develcvjzbI , initial p nting prcblees, and the lous of Alport syndrome on chromosome Xq22 (Hostikka at as. PNAS, behavial profiles (36 U.S. anxl 39 U.K. eto ). 1606, 1990). Using antibodies against synthetic peptides we also 87, deli of showed that the a5(IV) chain is located in the kidney practically only in the Eleven of 23 (47.8%) patients had tr stly of deetin positive and GBM. In the present study, we describe the structure of the 3' half of the 15q11-13, ard any features human a5(IV) gene (50 kb) containing the 19 most 3' exons as determined deletion nrgative patients did not identify from eight genomic X clones. The exon-intron structure was elucidated by niqwe to either group. restriction-enzyme mapping, DNA sequencing and heteroduplex analysis. The q tiir data describes a generally nrmal The a5(IV) and al (IV) collagen genes were shown to have an identical exon prenatal and birth history. Eleven of 67 (16.4%) of the size pattern in the 3' half. In contrast, the exon profile of the closely families reported sm complication in pregnancy or related a2(IV) collagen gene is considerably different, although it shares perinatally. Muree of these reported fetal distress, but some common intron locations in the coding sequence. Two mutations were baby was reported as aglyxic at birth. No neonatal problems found in COL4A5 in two kindreds with Alport: In one kindred, Southern ware reporte in the U.S. cohort, bit renatal hypotonia was analysis of DNA from a male Alport patient showed the absence of several noted in 3/39 (7.7%) of the U.K. grcup. Of 64 infants, none genomic Eco Rl fragents. This was shown to be caused by a deletion of about ware born less than 35 wek and three ware born 15 kb in the COL4A5 including exons 5-10 as counted from the 3' end. This with a birth weight of less than the fifth pexentile. mutation would result in the synthesis of an a5(IV) chain shortened by 240 The U.S. dtestiir da identified the first amino acid residues. Such a chain would not be able to assemble into a presentig clinical problems in 22 infants. All presented functional triple-helical type IV collagen molecule. In another kindred, a with either feeding difficulties, delpental delay, or point mutation was identified in exon 3, resulting in the conversion of a seizures. Seven of 22 (31.8%) presented by 3 unothe, 16/22 highly conserved cysteine residue to serine. This mutation could interfere (72.7%) presented by 6 mt, and all by one year. bTe with the folding of the C-terminal globular domain essential for triple helix diagnis of AS, howseer, was not -d in any infant prior to formation and irtermolecular cross-linking. The consequences of both 12 manths of ag. Several behavioral patterns were mutations can be considered sufficient to cause the structural and functional identified in infancy using both U.S. and U.K. data: 38/66 defects In the GBM in these Alport patients. The present results constitute (57.6%) had sleeping difficulties, 42/66 (63.6%) had feeding the first examples of a genetic basement membrane and kidney disease. difficulties in 2/27 (7.4%) hbd overeating for the two mutations infancy, Furthermore, this work has defined diagnostic probes behaviors, ard hyperactivity was reported in 42/56 (75%). and the potential for developing new probes for other mutations in Alport AS infants exhibit rcnepecific syndrome. Them firdings indicate that prior to the diagnosis.

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Lethal Melnick-Needles syndrome in a new mutation male. J. Zunich. Indiana Univ. School of Medicine Northwest Center for Medical Education, Gary. Melnick-Meedles syndrome (MNS) is an X-linked dominant bone dysplasia, lethal in males. Reports of affected males identi- fy severe skeletal involvement in association with multiple malformations. All reported males with lethal MNS were born to affected mothers. However, at least 3 cases of new muta- tion males with nonlethal MNS have been reported. It has been suggested that the lethality of MNS in male offspring of af- fected females may have an analogy in myotonic dystrophy (AJMG 27:153, 1987). This case represents the first reported in- stance of a new mutation male with lethal MNS. This male infant was born at 35 weeks gestation to a 31 yo G2P2 white female by vaginal delivery following a pregnancy complicated by oligohydramnios. Birth weight was 2430 g and length 47 cm. Meconium-stained fluid was present on delivery of a severely depressed infant and resuscitation efforts were unsuccessful. Autopsy noted the following abnormalities: cranial aplasia; bluish-white corneae; downslanting palpebral fissures; lowset ears; broad nasal root; narrow alae; triangu- lar-shaped mouth; severe micrognathia; U-shaped palatal cleft; webbed neck; omphalocele; malrotation; intra-abdominal testes; rudimentary penis and scrotum; flexion contractures of elbows and knees; ulnar deviation of hands; camptodactyly and clino- dactyly of digits 2 and 5 bilaterally; partial syndactyly of all fingers and toes; absent thumbnails and hypoplasia of ter- minal phalanges of thumbs. X-rays revealed absence of ossifi- cation of upper calvarium; curving, thin, irregular ribs; cur- ving of all long bones with marked periosteal changes; broad phalanges; and absence of terminal phalanges of thumbs. Chro- mosomal analysis demonstrated a normal male karyotype. The infant's mother was born with a tracheoesophageal fistula. Her physical examination is unremarkable and radiographs of spine, pelvis and long bones are normal. The abnormalities present in this case are most consistent with the diagnosis of MNS. The fact that this infant repre- sents a new mutation for this disorder argues against the ex- istence of some maternal humoral factor being responsible for lethal expression of MNS in sons of affected mothers. A86 Cytogenetics (0332) 1.172 (0333) 1.173

The effects of thymidylate stress and the fragile site at Xq27 The murine Rb(6,16) translocation: Evidence for sperm on chromosome aberrations in lymphoblastoid cell lines. M.A. selection and a modulating effect of aging. I. P. Aranha and Abruzzo, C.J. Ott, D.E. Ollendorff, V.L. Singer, and J.A. P. A. Martin-DeLeon. Univ. of Delaware, Newark Miller. California State University, Chico. Robertsonian translocations, common in man and occurring in A number of studies have found that the locations of other mammals, lead to an increased risk of chromosomally folate-sensitive fragile sites correlate with chromosome abnormal offspring. Estimation of this risk is dependent upon breakpoints in structural rearrangements, both those that are meiotic segregation, and pre- and post-zygotic loss. The congenital and those found in cancer cells. This has led to question of prezygotic loss is difficult to study in man but the hypothesis that individuals with rare heritable fragile can best be determined in first cleavage metaphases of mouse sites are predisposed to specific and/or increased rates of zygotes obtained under physiological conditions. structural chromosome aberrations and possibly to certain We have studied sperm chromosomes after sequential G- and C- types of cancer. As a test of this hypothesis, we banding in one-cell zygotes resulting from 6 male mice bearing cytogenetically examined five lymphoblastoid cell lines (LCL): the Rb(6,16) translocation. Since mating frequency has been two were established from control individuals, two were from shown to influence segregant distribution (Zackowski and individuals who expressed the rare folate-sensitive fragile Martin-DeLeon, 1989), the males were mated to superovulated site at Xq27, and one was from a female carrier of the fragile females at 3- and 14-day intervals. In 183 and 126 zygotes X gene mutation who did not express the fragile site at Xq27. analyzed in the 3- and 14-day populations, respectively, Replicate flasks of each cell line were treated as follows: segregants fertilizing oocytes were predominantly balanced (1) RPMI 1640 medium, (2) medium 199, (3) medium 199 wigh with alternate segregation accounting for 97% and 93% of the 10 M 5-fluorodeoxyuridine, and (4) medium 199 with 10 M zygotes. No unbalanced segregants of the translocation were was a excess sperm aphidicolin. After one week of treatment, all cultures were observed. There significant of with normal transferred to RPMI 1b40. At the end of weeks two and four, chromosomes compared to those with the translocation in both aliquots of each culture were removed, harvested by standard populations. In the 3-day group, the deviation from the cytogenetic methods, and whenever possible, 20 cells counted expected 1:1 ratio was highly significant (P

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Studies of the mechanism of restriction enzyme banding using Efforts toward chromosome mapping of the Roberts syndrome alphoid DNA and structural alterations at human centromeric defect. M. Askari and R. A. Schultz. Div. of Human Genetics regions. P. H. Arnt and E. W. Jabs2. Univ. of Florida and The and Medical Biotechnology Center, University of Maryland, Nemours Children's Clinic, Jacksonvillel and The Johns Hopkins Baltimore, MD. Univ., Baltimore, Maryland2. Roberts syndrome (RS) is a rare, autosomal recessive Banding of centromeric heterochomatin can be produced by CBG disorder clinically manifested by mental and growth staining or restriction endonuclease digestion followed by Giemsa retardation, tetraphocomelia, and a variety of craniofacial staining of human metaphase chromosomes. The mechanisms abnormalities. Cultured RS fibroblasts exhibit cytogenetic responsible for producing banding patterns are unknown. To abnormalities which can include random chromosome loss, determine if the presence or absence of C-like bands following pseudodiploidy, trisomy 7, and the premature separation of digestion by restriction endonucleases may be related to frequency heterochromatic regions of metaphase chromosomes. The high of recognition sites among sequences at a given centromere, we frequency of premature centromere separation (PCS) seen in examined whether or not the sizes of chromosome-specific alphoid Roberts syndrome cells offers a diagnostic tool and supports DNA fragments created by digestion with various restriction enzymes the notion that the primary defect in RS involves chromatid relate to the presence or absence of C-like bands produced by these pairing and disjunction. Analysis of proteins active in same enzymes. We sized alphoid DNA fragments from five different normal contromere structure and metaphase alignment has not chromosomes (6, 13, 21, X and Y), digested with each of six led to the identification of a defect in RS cells. Previous different restriction enzymes (Alul, DdeI, HaeIII, Hinfl, MboI, somatic cell hybridization experiments demonstrated RsaI). Generally, AluI produces larger restriction fragments than complementation of the PCS phenotype. Therefore, we have Hinfl; and Alul restriction banding produces C-like bands and Hinfl initiated a strategy to map the gene(s) which corrects the PCS does not. Otherwise, there was no direct correlation between the phenotype in RS cells following microcell mediated chromosome length of alphoid restriction fragments at specific human transfer (MMCT) of individual normal human chromoomes. To centromeric regions and the production of C-like bands. Because facilitate this effort, RS fibroblasts were immortalized by this suggested that DNA accessibility may be a factor in C-like transfection with plasmid pSV3dhfr which encodes SV40 T- banding, we compared AluI restriction and CBG banding of antigen. Independent foci of RS cells were isolated and found centromeric regions with conformational alterations. These to exhibit both T-antigen expression and indefinite growth in included dicentric chromosomes and Roberts syndrome chromosomes culture. More importantly, these transformed cells exhibit with 'splayed' centromeric regions and 5-azacytidine treated the PCS phenotype at significantly elevated frequencies prometaphase chromosomes with decondensed centromeric regions. In compared to both primary and transformed control cells, all cases bands produced by Alul resembled CBG banding. Markedly including cell lines representative of other chromosome decondensed portions of centromeric regions induced by instability syndromes. SV40 transformation, per so, was not 5-azacytidine did not band with either AluI and CBG banding found to affect the frequency of PCS in control calls. techniques identifying regions of euchromatin interspersed within Despite the RS tendency for random chromosome loss, the centromeric heterochromatin. Our studies demonstrate that transformed RS cells were found to serve as recipients for restriction endonuclease C-like banding is not strictly related to chromosome transfer. Thus, the PCS phenotype should provide the presence of restriction sites in alphoid DNA, and the condensed a means to screen for RS complementation. Mapping and chromatin conformation at the centromeric region plays a role in ultimate cloning of a gene which corrects PCS should lead to banding. These results underscore the structural complexity of the identification of the genetic defect in RS and offer insights human centromeric region. into mechanisms which influence chromosome stability. Cytogenetics A87

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Inversion of the Y chromosome [inv(Y)(pll.lql2)] High resolution Alu-banding of human chromosomes with biotinylated Alu- in a male fetus derived from a mosaic father, PCR products: use for in situ hybridization mapping. 45,X/46,X,inv(Y)(pll.1q12). A. Babu, F. A. Baldini. J. Menninger and D.C. Ward. Yale University School of Konstantinovsky, D. Punales-Morejon and V. B. Medicine, Department of Human Genetics, New Haven, CI 06510 Penchaszadeh. Division of Medical Genetics, The Alu sequences have been shown to be highly represented in the Beth Israel Medical Center and Mount Sinai positive reverse bands of human chromosomes (Manuelidis and Ward, School of Medicine, New York. Chromosoma 91:28-38). In situ hybridization of cloned Alu sequences produce an R-banding pattern whose quality is not fully satisfactory for An amniotic fluid sample was obtained from a general mapping purposes. We obtained a high quality R-banding using Alu- pregnancy of a 36 year old black female and a 42 PCR products (produced from primer #517 essentially as described by year old black male for prenatal diagnosis. Nelson et al., PNAS 86:6686-6690, 1989). The probe mix was biotinylated Chromosome analysis revealed a male 46,XY by nick translation, hybridized in situ to chromosomes, and detected with complement in all the cells examined. The Y fluorescein-labeled avidin. A possible explanation of these results is that Alu- chromosome, however, was of the size of the PCR products are highly enriched in diverged Alu sequences and when used G-group and showed an unusual morphology. The as probe, they hybridize to a larger number of target sequences than a cloned Y chromosome, by G-banding, had a darkly stained Alu repeat. short arm and a less darkly Stained long arm with Using multiple fluorescent labeling, we have combined the in situ R- a discrete banding pattern. QFQ and distamycin banding with in situ hybridization of probes either repetitive or single copy. A/DAPI banding revealed an inversion with an Results indicate that this method permisarapid--assignment-of DNAprobes------apparently heterochromatic short arm and an to specific chromosomal bands. entirely euchromatic long arm. Subsequent chromosome analysis of the father using peripheral blood lymphocytes revealed a mosaic chromosome composition with a 45,X karyotype in 7 cells and a 46,X,inv(Y)(pll.lql2) karyotype in the remaining 23 cells. No other tissue could be examined in the father. Since a retrospective evaluation of the father's and his family history indicated no fertility problems, the breakpoints involved in the inversion are considered to be located in the non-critical regions and the fetus was predicted to be a phenotypic male. This case is remarkable since the father is fertile despite his mosaic chromosome composition 45,X/46,X,inv(Y)(pll.lql2) in blood lymphocytes.

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Cytogenetic analysis of non transfered human abnormal embryos Isolation of a chromosome specific family of mouse minor after in vitro fertilization. satellite DNA. D. Broccoli. K.T. Trevor. O.J. Miller_.and M. BENKHALIFA (1), A.GENEIX (1), L. JANNY (2), D. BOUCHER (2) D.A. Miller. Wayne State University, Detroit, Michigan. and P. MALET (1). Human alpha satellite DNA is organized as chromosome (1) Laboratoire de Cytogdndtique Mddicale, Facult6 de specific families, based on higher order repeats of diverged Medecine, BP 38 63001 CLERMONT FERRAND CEDEX FRANCE. 170 bp monomers. The laboratory mouse, Mus musculus, has two (2) Laboratoire de Biologie de la Reproduction et du Develop- satellite DNAs. Both the more abundant major satellite and pement, C.H.R.U. BP 69, 63003 CLERMONT FERRAND CEDEX FRANCE. the less abundant minor satellite are associated with the centromeric region of every chromosome except the Y. The The chromosome aberrations are one of the main factors minor satellite is located closer to the centromere than is of in vitro fertilization failures. In our study we undertake the major satellite. We have cloned two minor satellite DNA to establish the karyotypes of non tranferable, non freezable sequences, HRS-1 and HRS-2. Sequence data shows a higher embryos. 47 embryos from 34 patients are analysed. 48 hours order arrangement based on a trimer of a 60 bp repeat unit. after insemination normal embryos contain 4 to 8 similar As in the human, such an organization is associated with blastomeres without cytoplasmic inclusions. In our sample chromosome specificity. Both HRS-1 and HRS-2 hybridized embryos with less than four blastomeres or presenting morpho- primarily to chromosome 4 in the SJL-derived RCS-5 mouse cell logical or cytological abnormalities are selected. A culture line. MspI digests mouse minor satellite DNA into two ladders, is performed during 8 hours according Tarkovsky (hypotonic one based on multimers of 180 bp (a trimer of the 60 bp mono- choc with 0,7 per cent of natrium citrate during mer) and the other based on multimers of 300 bp (a pentamer). 10-15 minutes, the fixatives being ethanol and glacial acetic HRS-1 and HRS-2 hybridize preferentially to a subset of the acid 2 : 1). The bad spreadings and the overlapping of two fragments in the trimeric ladder, one of 360 bp and the other blastomeres metaphases are not taken into account. 109 of 720 bp. The role of satellite DNAs in centromere structure metaphases are suitable for our study with at least 2 per or function is unclear. Recently, Masumoto et al. (J. Cell sample. Biol. 109:1963, 1989) identified a 17 bp site in human alphoid We observe 2 haploid embryos (23 chromosomes) with two DNA that binds to CENP-B, an evolutionarily conserved 80 kD blastomeres (probably related with a parthenogenesis centromeric protein. The same 17 bp sequence, with a single phenomenon), 29 diploid, 6 hyperdiploid, 2 hypodiploid and base change (the initial cytosine is changed to an adenosine), 5 polyploid embryos. is present in 2/23 monomers and another 3 monomers have 15 The chromosome aberrations ratio is 1/3. In vivo 33% is identical bases. The conservation of this binding site in the estimated figure. We don't find any correlation between these two rapidly changing tandemly repetitive sequence the type of cytological damage and the chromosome aberration families suggests that these repetitive satellite DNA ratio. It should be of definite interest to detect the sequences play an important role in centromere structure or aberrations before implantation. However ethical problems function. still remain regarding the damages due to sampling at an early stage of embryogenesis. A88 Cytogenetics (0340) 1.179 (0341)

The correlation between bleomycin sensitivity and functional Complex Translocation, Variant Translocation and Additional decline in adult patients with Down syndrome. L.M. Cherry, Chromosome Abnormalities in the Acute Myclogenous Leukemia K.A. Loveland, & D.B. Burt. The University of Texas Mental with 8; 21 Translocation. J. -Y. Chu and A. -D. Institute of Sciences Institute, Houston, Texas. Yang A breakdown in the cellular mechanisms for neutralizing Hematology, Tongji Medical University, Wuhan, China. the effects of superoxide radicals may be an important mech- anism in the development of functional declines in adult patients with Down syndrome (OS). To test this hypothesis, Chromosome analysis was performed in 16 patients with acute white blood cells growing in culture were treated with bleo- nonlymphocytic leukemia (ANLL) by using synchronization culture mycin, a radiomimetic antibiotic which induces chromosome G -banding and R -banding technique, all of these cases were M2 breakage through the generation of superoxide radicals in type. Loss of a sex chromosome was observed in 12 cases. Among the cell. Although bleomycin-induced chromosome damage has been measured before in patients with DS, no one has examined the 16 cases, twelve had standard translocation, 8; 21 )( q22; q22). this phenomenon in adult patients, nor has anyone examined Chromosome abnormalities additional to t(8; 21) were found in 3 functional differences between high and low responders to cases, the karyotypes were t(8; 21)(q22; q22), t(3; 11)(p22; q24); bleomycin. We measured bleomycin-induced chromosome breakage +8 in adult DS patients ranging in age from 20-70, and in a group t(8; 21) (q22; q22), del(9) (pl3) and t(8; 21) (q22Z q22), of age-matched patients with non-DS mental retardation respectively. In one case with a complex translocation involving (NDSMR). At baseline, eight patients with DS, but no patients chromosome No. 7,21, and 8, the karyotype was 45, X, -Y, t(7; were identified as having declines in functioning with NDSMR, 21; 8) (pl3; q22). And the remaining one patients had vari- relative to earlier levels as ascertained by caregiver report, q22; medical records, written reports of earlier psychological ant translocation t (8; 10) (q22; p15), without involving testing, and school records. Current functioning was docu- chromosome 21. Since all the cases had similar cell morphology and mented by neuropsychological testing and caregiver reports clinic characteristics, our results provided a furture evidence for a in the areas of intellectual functioning, adaptive func- tioning, and appropriateness of behavior. Cytological data greater weight of breakpoint 8q22 in the relative importance of the were available from six of these patients, and it was found two chromosomes in the genesis of ANLL. In addition, 4 cases in that the range of bleomycin-induced breakage rates in the this group were chloroma patients. Our rinding suggested that the group with functional decline was significantly higher (mean= to 1.37 breaks/cell, S.D.=0.21) than in the DS group as a whole pathogenesis of chloroma might be closely related the activation (mean=1.05, S.D.=0.33), or in the NDSMR group (mean=0.72, of the oncogenes involved in ( 8; 21). S.D.=0.23). These findings support the hypothesis that reduced ability to neutralize superoxide-induced cell damage may contribute to neuronal cell injury, resulting in declines in functioning.

(0342) (0343) Reciprocal translocations: A computerized and statistical Efficacy of coating culture surface with amniotic fluid super- natant in monolayer cultures. J. Davies, N. Villanueva, N. analysis system Hitter and S. Mate. SmithKline Beecham Clinical Laboratories, 0. Cohen. C. Cans. M. A. Mermet. J. ongeot. P. Jalbert Van Nuys, CA. Genetic Laboratory and Statistical Department, Medical A recent study by Chang and Jones (Am J Hum Genet, 45:A255, School of Grenoble, Grenoble, France 1989 supplement) suggested a coating of the culture surfaces with separate or pooled amniotic fluid supernatants as a means to enhance cell attachment and rapid cell growth in amniocyte cultures. We have attempted a control-matched study to inves- Reciprocal translocations (RCP) occur frequently in human tigate the efficacy of this coating process for both in-situ populations.They result from an exchange of segments and flask harvests for amniocyte and solid tissue cultures. between two chromosomes, any point of any chromosome Our results show that while this coating does promote a may be involved and thus an infinite number of different RCP slightly earlier cell attachment, the advantage may be limited is possible. to primary harvests without a change of medium, by the flask method, at the most. The cell attachment on the coated surfa- A given RCP produce unbalances nearly always by the ces is loose, and the cells come off easily during a medium change or a change of fluids during an in-situ harvest. This same could segregation type. Pachytene diagram reliably leads to a loss of many cells. With collagenase treated solid a predict the mode of unbalanced segregation, but is time tissues also, we did not observe any distinct advantage of consuming and a tedious method. The programm showes, growing the cells on the coated surfaces over the non-coated for a given RCP, the pachytene diagram, the different modes surfaces. Resuspension of the centrifuged amniocytes in auto- of segregation and their viability according to length criteria logus amniotic fluid supernatant, instead of a synthetic tissue ofDaniel. culture medium during initial inoculum of cells on the cover- slips, on the other hand, does help in early cell attachment With a sample of 1420 families carrying RCP ( about 5100 and a rapid growth of amniocytes. persons), we realise a discriminant analysis in order to define a predictive equation for the risks of unbalanced progeny in inherited RCP. In this prediction, both risk factors for segregation type and risk factors for viability have been analysed. We include parameters of chromosomes involved: quantitatives as well as qualitatives, and also some characteristics of RCP carriers. In the future, the knowledge of those risks of unbalance will be helpfull in prenatal diagnosis for each genetician, by using an European server. Cytogenetics A89 (0344) 1.508 (0345) 1.181

Comstutdon of a YAC pael of chromosomes lIp, 17p, 17q and 19q and The study of the miotic segregation pattern of a recipocal translocation in 2 mle cousins its rapid mappi by fuorescent In situ hybridization. M. S. Driesen. t(114)(p36.2Iq31.3) bsterozygous for the trmlocation. A. M. Zatop, K. Cieply, M. C. Wapennar. J. G. Dauwerse. T. Kievits E. J. Meershoek K H. V. Van Kirk, F. Levinson, and K. Garver. Dept. of Medical Fischbeck. B. Wierin'a and G. J. B. van Ommen. Department of Genetics, West Penn Hospital and Dept. of Human Genetics, Human Genetics, State Univ. of Leiden, The Netherlands;* Department Univ. of Pittsburgh, Pittsburgh, PA. of Neurology, Hospital of the Univ. of Pennsylvania, Philadelphia, USA,- The msiotic segregation pattern of a reciprocal Department of Human Genetics, Radboudziekenhuis, Catholic Univerity translocation t(1;4)(p36.2;q31.3) was studied in two male Nijmegen, The Netherlands. cousins heterozygous for the translocation. This was accomplished by using in vitro fertilization of human sperm The yeast artificial chromosome (YAC) cloning system has proven with zona-fre hamster oocytes. The two men were both to be a powerful technique for cloning complex genomes because of its ascertained after repeated pregnancy losses. However, donor potentially large insert capacity (50.1000 kb). YAC libraries of the total 1 has fathered two phenotypically normal daughters who both human genome are now available in several laboratories, but their carry a balanced form of the translocation, whereas donor 2 analysis remains an enormous task because of their vast sequence has not been able to produce any live born offspring. A total of 115 chromosom spreads were analyzed from donor 1 genome yields complexity. Targeted cloning of subregions of the human and 69 chromosome spreads were analyzed from donor 2. The a reduced compladty YAC panel, which facilitates contig mapping and following table depicts the results: probe isolation of the targeted regions. We have isolated YAC clones Donor 1 Donor 2 from the somatic cell hybrid 1040A6, containing as human complement % Total C Abnormal I Total C Abnormal a t(lp:19q) chromosome and chromosome 17. This hybrid is particularly Alternate (Norm) 31.3 2.6 19.0 2.9 Alternate (Bal) 14.7 0.8 22.0 4.3 interesting for directed cloning, since several human malignancy-related Adjacent-1 38.2 7.0 49.0 13.0 structural chromosomal alterations (deletions and translocations) map to Adjacent-2 7.0 1.7 7.0 1.4 chromosomes ip, 17p and 17q, as well as do several disease loci, e.g. 3:1 8.7 2.6 3.0 1.4 Miller-Dieker syndrome and Charcot-Marie-Tooth disease type 2 (17p) and Myotonic dystrophy and malignant hyperthermia (19q). Human The overall imbalance rate was 54% for donor 1 and 59% YACs ranging from 50 450 kb were efficiently isolated from the total for donor 2. This difference does not seem significant, however, donor 2 has an increased rats of structural hybrid cell library, after it had been-amplified, by high density colony abnormalities. Cells with structural abnormalities were screening with human Alu probe. Subsequently, Alu-fingerprinting and found in all types of segregation gametes. For each donor, Alu-PCR have been used to obtain independent, unique banding the frequency of cells that carry abnormalities, either patterns for individual YACs, thus providing identity information, structural and/or numerical, was 1S% in donor 1 and 23% in including possible overlap or reiteration. The chromosomal localization donor 2. Since 3% of those in donor 1 and 8 % of those in donor 2 were found in otherwise normal or balanced cells, of the isolated clones could be rapidly established by fluorescent in situ the adjusted frequency of normal/balanced cells then become hybridization (FISH), using biotinylated total DNA from YAC-containing 43% in donor 1 and 33% in donor 2. These results suggest yeast clones as a probe on metaphase spreads of human lymphocytes. that when the frequency of chromosomally abnormal sperm Thus, 51 YAC clones were mapped in under two months to sub- reaches a certain threshold, fertility may be completely chromosomal regions of chromosomes 1p, 17p, 17q or 19q. impaired.

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Recombinant Chromosome 6 Resulting in Trisomy 15p. L.G.S. Tetrasomy 12p mosaicism (Pallister-Killian Gannutz, M.P. McCurdy, P.N. Mowrey, M.J. Mascari, J.C. Ramer, Syndrome): report of two additional female cases. R.J. Hoover, R.L. Ladda. PA State Univ., Col. of Med., A.D.Garnica, R. S.Muneer, G.B.Schaefer, D.B.Domek, Hershey, PA. and D.Hopcus. Routine chromosome analysis of amniotic fluid fibroblast Since the initial report of Pallister et al., cultures established for AMA revealed an unusual non-staining in 1977, a number of tetrasomy 12p have been "gap" area on the proximal long arm of chromosome 6 in published. The majority of these cases demonstrated every cell. Parental blood chromosome studies revealed rcp chromosomal mosaicism in lymphocytes and/or skin t(6;15)(ql3gpl3)pat. This rec event resulted in the apparent fibroblasts. rejoining of 6p and 6q with retention of the chromosome 15 We have seen two patients with pigmentary stalk region which could potentially yield a duplication/ anomalies with dissimilar phenotype expression and deficiency of material of chromosome 6 and/or 15 in karyotypes. offspring. Chromosome analysis of a phenotypically and Case #1 is a 10 year old black female with developmentally normal 9 year-old half-brother to the fetus severe mental retardation, microcephaly, sparse revealed the identical rec(6) found in the fetus indicating hair, coarse facies, joint contractures, seizure little associated risk for abnormal fetal development. disorder and linear hypopigmented streaks on the Review of the literature identified numerous cases of extremities. Her lymphocytes demonstrated a 46,XX trisomy (15)(pter.qll or q13) defined as a bisatellited karyotype, while the skin fibroblasts showed marker. All had larger duplicated segments than in our case. 47,XX+i(12p) karyotype. Analysis of lactate Presumably the abnormal phenotype associated with marker dehydrogenase activity showed a dose effect chromosomes is a result of duplicated long arm material. consistent with the chromosome constitution. Band (6)(ql3) is a comon BrdU induced fragile site. Type IX Case #2 is a 4 year old white female with alpha I collagen gene has been provisionally localized to profound developmental delay, small pinpoint pupils, (6)(ql2ql4). Our cell line has been submitted to Coriell prominent forehead, broad nasal bridge, Institute for transformation and banking. These unusual hypertelorism, synophris, cornea eliptics and recombinant events furnish valuable tools to aid in streaky hypopigmentation on the upper lateral developing strategies for gene mapping. aspects of both legs, extending anteriorly and medially. Her lymphocytes demonstrated a normal 46,XX karyotype while the skin fibroblasts showed F&fUS 46,XX/47,XX,+i(12p) mosaicism. Pwr In each case the parents karyotypes are normal. We are now evaluating the karyotypes from the non- % SD. pigmented fibroblast cultures established from II k different sides of the midlines for both patients.

6 ro 6 t(6.;i5) 6 A90 Cytogenetics (0348) 1.516 (0349) 1.182

Construction of irradiation-reduced hybrids for human chromosome 3 Nucleolar transcriptional activity in Sertoli cells is dependant on centromere and characterization by IRS-PCR (interspersed repetitive sequence arrangement. T. Haaf LI gndM. ShMjd a 1Stanford University, PCR) analysis. T.W. Glover. B. K. Hall and E. Lesius. Univ. of Stanford, CA; 2University of Wurzburg, FRG. Michigan, Ann Arbor. The interphase nucleus is a highly organized structure in which individ- Irradiation-reduced hybrids have proven to be powerful resources ual chromosomes occupy compact territories. The higher-order spatial or- for mapping and obtaining region specific probes. We have utilized ganization may be related to gene expression, DNA replication, and/or nu- this approach to generate irradiation-reduced hybrids for human clear division. Indirect immunofluorescence with human autoantibodies to chromosome 3 as part of our study of loci in band 3p14. A somatic centromeres and RNA polymerase I (RPI) and mouse monoclonal anti- cell hybrid, UCTP-2a, containing human chromosome 3 as the only bodies to fibrillarin was used to analyse the relationship between nucleolar human material was used as donor. Prior to irradiation, some cells transcriptional activity and centromere distribution in mouse Sertoli cells. were transfected with the neor gene during a period of fragile site The CENP proteins specifically recognized by human CREST antibodies expression in an attempt to promote integration of the gene into the induce positive cytochemical staining of centromeres (kinetochores) during 3p14 fragile site during successive periods of breakage and repair. interphase. RPI molecules occur in association with transcriptionally active Cells were irradiated with 5000 to 20,000 rads of gamma irradiation rRNA genes, but are absent from nontranscribed nucleolar chromatin. and fused with hprt- Chinese hamster cells. Hybrids were selected Fibrillarin, a 34-kD protein of the U3-RNP particle, interacts with the free with either G418 or HAS. These fusions resulted in hundreds of ends of nascent ribosomal transcripts, being involved in the early stages of hybrids, some of which were isolated for characterization. Hybrids rRNA processing. Two different types of Sertoli cells were consistently were initially characterized by IRS-PCR using LINES (LIHs) and Alu found in five animals analysed: A type I pattern consisted of nuclei, in primers to determine the extent of human chromosomal material which the centromeres were arranged into two or three clusters, each con- present. The location of these IRS-PCR fragments was determined by taining a bunch of individual centromeres. The nucleolus did not show any prior analysis of a panel of chromosome 3 hybrids deleted for various cytochemically detectable amounts of RNA polymerase L. This is direct portions of chromosme 3. We were thus able to quickly determine evidence for the fact that the underlying rRNA genes are transcriptionally in- both the extent and general location of origin of the human material in active. As an internal control, RPI was immunolocalized to the nucleoli of all the irradiation-reduced hybrids with this approach. Furthermore, spermatocytes and round spermatids of the same preparations. In contrast, isolated gel fragments serve as a direct source of probes. Promising type II nuclei contained one large central nucleolus strongly fluorescing hybrids were also screened with PCR primers constructed for probe with antibodies to RPI and fibrillarin. This is correlated with rDNA tran- D3S3, in 3pl4, and ITIH3, in 3p21, to obtain those with limited scription and pre-RNA processing. Use of nonimmune serum instead of the human material around band 3pl4. We have thus obtained a number of antinucleolar antisera as well as omisson of either the primary or secondary hybrids positive for one or both of these markers and containing antibody resulted in negative immunofluorescence. The centromeres of varying portions of chromosome 3. Fluorescence in situ hybridization type II nuclei were fused in an invariant way to form several centromeric using these hybrids as probes back to human metaphase chromosomes bodies, in which individual centromeres were not discernible. A total of is being used to verify the localization and size of human sequences in some 1000 cells (from 5 different animals) was analysed; 10-20% of nuclei the hybrids. In addition, in situ hybridization with the neor gene is belonged to type I and 80-90% to type II. Centromere fusion appears to be being performed to determine if any neo sequences have recombined a prerequisite for nucleolar activity in Sertoli cells. In mouse chromosomes, at the fragile site. the centromeres and rRNA gene clusters lie closely adjacent and are embed- ded in pericentromeric heterochromatin. Specific chromosome arrangements during interphase as visualized by centromere movements are likely to play an important role in the regulation of gene expression.

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Chloramphenicol modulates the level of expression Heritable NOR deficiency as a possible factor in human of the fra(X) site associated with the fragile X spontaneous abortion. J.R. Hartv. J.A. Brown. C. Jackson= J.B. B. D. Cook, Medical College of Virginia, Richmond, VA. syndrome. P.J. Hagerman, Mills, Lunt, Heritable nucleolus organizer region (NOR) deficiency has and L. McGavran. University of Colorado School of been described in other organisms, leading to delayed or Medicine and Childrens' Hospital, Denver. abnormal development, and/or embryonic failure. To see if During the course of our studies of the possible heritable NOR deficiency is a factor in human pregnancy role of mitochondria in the clinical expression of failure a cytogenetic examination of NOR activity has been the fragile X syndrome, we have obtained evidence performed on a series of spontaneous abortions (SAB) using suggesting that mitochondrial function may ammoniacal silver staining, which is thought to deposit silver on active NORs in proportion to their level of transcriptional influence the level of expression of the fragile activity. Samples were also counterstained with GTG for site. In particular, following induction of unequivocal chromosome identification. To date, our study fragility with 5-fluorodeoxyuridine (FdU), we have sample consists of 57 first trimester SAB classified as either observed that (i) addition of chloramphenicol (CAM) blighted ova or missed abortions. Of these, 25/57 (44%) were to cultures of fragile X lymphoblastoid cells karyotypically normal, while 32/57 (56%) demonstrated a (GM4025) or to primary (phytohemagglutinin- variety of chromosomal abnormalities including trisomy (22/57, stimulated) lymphocyte cultures from a fragile X 38%), sex chromosome monosomy (3/57, 5.2%), triploidy 4/57, 7.0%), and tetraploidy (3/57, 3.5%). Since NOR activity has male partially blocks deoxythimidine (dT)- or 5- not been extensively examined in early pregnancy tissue, a bromodeoxyuridine (BrdU)-reversal of fragile site control cohort (n-15) of comparable gestational age was expression; (ii) in the absence of dT, addition of obtained from diagnostic CVS. Parental blood samples were also CAM results in a small, but significant increase in obtained for the control CVS and normal SAB. the level of expression in GM4025; and (iii) in the Among the 25 karyotypically normal abortuses both the dT-reversal system, deletions at the Xq27.3 fragile total level of cellular NOR activity (19.19 + 3.40) and the site were frequent in contrast to the total number of active NORs per cell (8.77 ± 1.01) were (>5%), significantly lower in the SAB than in the control pregnancies absence of such deletions in control cultures (activity: 20.71 + 3.22; active NORs: 8.77 ± 1.01). containing FdU. The provisional conclusion from Furthermore, the normal SAB demonstrated a significantly these observations is that CAM is blocking some higher frequency of lower NOR scores (little or no silver) aspect of mitochondrial function that is required than the controls (30.0% vs 22.4%, p

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Phorbol esters as additional mitogens for the Kinetochore analysis of micronuclet allows insight into the actions of preparation of karyotypes from human neonatal cord Colcemid and Mitomycin C. D.I. Hoar, N.L. Rudd, S. Williams, I. blood. W.G. Heim, F.A. Brunnhoelzl and C.E. Kirk. Evans and U.G.G. Hennig. Univ. of Calgary and Alberta Children's The Colorado College, Colorado Springs. Hospital Research Centre, Calgary, Alberta, Canada. With the increasing numbers of chemicals in our environment comes Human neonatal umbilical cord blood often a need for reliable assays for the detection of potential aneugens and yields only a few mitotic spreads suitable for clastogens. The fibroblast micronucleus assay recognizes whole karyotyping and analysis. To increase the number chromosomes (aneuploidy) and chromosomal fragments (breakage) of mitotic spreads, phorbol myristate acetate (PMA) excluded from the main nucleus during mitosis. Kinetochore (K) was added at various concentrations to standard immunofluorescence of micronuclei allows insight into the mechanisms culture medium based on RPMI 1640 and containing of their formation. In this study, two diploid fibroblast strains were the usual mitogen phytohaemagglutinin (PHA). treated with a known aneugen, Colcemid, and a known clastogen, Preparation of karyotypes was carried out in the Mitomycin C (MMC) to examine the effectiveness of this assay in usual manner with particular attention to initial distinguishing between the two mechanisms of micronucleus formation. and final volumes. At the optimal concentration of These results demonstrated the predicted increase in K-positive 1 ug/ml, an average of 4.26 (S.E.=0.72) times as micronuclei in Colcemid-treated cells. The aneuploid index (Al)1 many mitotic spreads were obtained as from control increased an average of 26.8 fold (3.9 to 153.4 and 6.1 to 98.7) in these cultures without PKA. When PMA was used as the strains. However, the chromosome breakage index (CBI)1 also rose sole mitogen, very few spreads were seen. Thus, significantly. In cells completely arrested with Colcemid, elevated a synergistic relationship exists between the breakage did not appear until after the release of the block. These actions of PMA and PHA. Phorbol dibutyrate (PDBu) findings suggest that the increase in K-negative micronuclet was was also tested as a mitogen in the same manner as related to chromosome breakage rather than to Colcemid-induced K PMA. Neither a significant difference in mito- damage, an interpretation supported by previous reports.2'3 genicity nor a significant synergistic relationship The CBI in MMC-treated cells rose an average of 7.9 fold (11.5 to between the two esters was found. No damage to the 105.2 and 14.0 to 95.5) in the two test strains, a clear reflection of chromosomes or degradation of morphological quality its clastogenic action. However, a 4-fold increase in the Al was seen was detected with either ester. The mitotic (5.5 to 22.1 and 4.7 to 22.5). The magnitude of the increase is similar spreads were suitable for routine karyotyping. to that observed in ataxia telangietasia, suggesting that the K-containing micronuclei are secondary to elevated breakage (eg dicentrics and rings) rather than to an MMC-induced lag of whole chromosomes. We conclude that the fibroblast micronucleus assay, coupled with K immunofluorescence provides a useful approach for the screening genotoxic agents. The delineation of the precise mechanism by which an agent perturbs the CBI or Al or both, may require more detailed analysis. 1Hennig et al. (1988), Mutat Res 203:405; 2Woods et al (1985), Biochem et Biophys Acta 824:177; 3Lafi & Parry (1987) Mutagenesis 2:23.

(0354) 1.185 (0355) Analysis of YACs containing DNA from the human centromeric region. Dialyzed Fetal Bovine Serum (DFBS) Doubles FUdR-Induced fra(X) E.W. Jabs and Xians Li. The Johns Hopkins School of Medicine, Frequencies in Whole Blood Cultures. E.C. Jenkins. CJ. Duncan. H. Baltimore, Maryland. Gu. M. Genovese. M.S. Krawczun. NYS Institute for Basic Research The library created with total genomic DNA partially digested in Developmental Disabilities, Staten Island. with EcoRI and cloned into yeast artificial chromosomes (Burke et A with without DFBS showed al. 1989; Brownstein et al. 1989), was screened with the polymerase comparison between cultures and chain reaction using primers from the consensus sequence of alphoid consistently higher fra(X) frequencies in DFBS cultures. Whole blood 170 bp monomers under conditions of low stringency. Over four from 5 unrelated males with the fragile X syndrome was exposed to hundred clones were obtained. We obtained some of these clones for 0.1 pM FUdR for the last 24 hours of a 4 day incubation period. Half analysis from Ian Dunham of Maynard Olson's laboratory at of the cultures contained 15% DFBS (GIBCO) in their complete Washington University. The location of some of these sequences was medium (including RPMI-1640) and the other half contained regular, determined to be chromosome specific by in situ hybridization. non-dialyzed FBS. At least 100 cells were analyzed per variable. As The library was also screened using primers that generated DNA shown (Table I), fra(X)frequencies ofall individuals increased in DFBS fragments that were confirmed by hybrids and in situ hybridization cultures. to be specific to the centromeric region of chromsomes 6, 7, and Y under conditions of high stringency. Less than 10 clones were obtained for chromosome 6. There were three classes of YACs: 1) YACs containing alphoid, LINE, Alu sequences; 2) YACs with alphoid TABLE I. Fra(X) % in FBS TABLE II. Fra(X) % Versus Folic and Line sequences; 3) YACs with only alphoid DNA. These YACs may and DFBS Cultures Acid Concentration provide a means of looking at the macromolecular organization of Case No. FBS DFBS yM Folae FUdR Case 6 Case 7 the human centromeric region and identifying sequences or sequence 1 12 33 2 0 4 2 organization responsible for centromeric function. 2 9 28 40 0.05 43 40 3 18 24 400 0.05 41 30 4 15 29 4000 0.05 40 37 5 6 21 400 0 0 6

In most cases, the fra(X) % doubled and in one case, it more than tripled. Addition of increasing concentrations of folic acid to whole blood cultures from two other fra(X) individuals did not reduce their fra(X) frequencies (Table II). Therefore, increasing doses of folic acid alone do not appear to interefere with the FUdR fra(X) induction system. These results have reinforced our use of DFBS in one of the protocols that we employ routinely for fra(X) identification in whole blood cultures. Application of our findings should reduce the possibility of false negative results thereby improving the reliability of fra(X) detection in whole blood cultures. A92 Cytogenetics (0356) 1.186 (0357)

Eigenanalysis of digital images in the Fourier Origin of a small extra ring chroaosoae. Z. B. domain: A study of high resolution human chromo- Johnson, K. J. Thompson, and G. S. Sekhon. somes. Z. Jericevic. L.C. Smith and L. McGavran Univ. of Wisconsin, Madison. Baylor College of Medicine, Houston, Texas, and The Recent developments of in-situ chromosome Children's Hospital, Denver, Colorado labelling with centromere-speclfic DNA probes Cell synchronization techniques and high resolu- and chromosoae painting techniques have tion analysis have enabled cytogeneticist to detect increased the scope of cytogenetic diagnosis. increasingly small chromosome abnormalities that We describe a 30 year old male with a s6all have a profound clinical implications. Subband extra ring chromosome in 20X of his cells. changes in chromosomes at > 1000 bands level ap- Earlier studies had suggested that the ring proach the limits of technical ability to observe chromosome was derived from chromosoae 21. with any degree of confidence. We developed the Repeat studies with G, Q, C, and DAPI/Dist A eigenanalysis based procedure to analyse images of staining suggested that the ring was mor -likely high resolution chromosomes and have found excel- to have originated from chromosome 1, 9, or 16. lent concordance with the high resolution ideograms In-situ hybridization with a chromosome 1 from ISCN 1985. Eigenanalysis is a mathematical derived highly repetitive sequence probe showed approach used to obtain characteristic roots and the ring chromosome to be no. 1. vectors from a matrix and is an important method for extracting information from digital images in microscopy. To use multiple images as input data, the images must be correctly aligned and the pat- tern common for each input image must not be geometrically distorted. Otherwise, the different geometries produce blurring of features. Analysis of the common pattern in 40 digital images of ran- dom examples of high resolution preparations of hu- man chromosomes as a data set has been achieved us- ing the phase synchronization approach in which eigenanalysis is performed in the Fourier spectral domain on the phase and the amplitude of the Fourier transform. Our results show that eigenanalysis in the frequency domain provides a better resolution of features than does eigenanalysis in the spatial domain. After the eigenanalysis further improvement of images is achieved by the iterative Fourier synthesis using a phase retrieval approach similar to that used in crystallography. Statistically based prototypes for each high resolution Giemsa banded human chromosome are constructed and their potential use in detect- ing the abnormalities is discussed.

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Evolution of alphoid DNA on homologous pairs of NOR-bearing De novo monocentric Robertsonian translocations associated chromosomes in man and chimpanzee. with a bisatellited microdhromosome in two infants with A. Lund Jrgensen?* Carol Jonesl, and A. Leth Bak. Institute Down syndrome: mechanisms which can produce this double of Medical Microbiology, The Bartholin Building, University cytogenetic error. L. A. Kaepernick, J. V. Higgins and of Aarhus, Aarhus, Denmark. *) Present address: Dept. of G. S. Sekhon. Department of Pediatrics/Human Development, Medical Genetics, University of Washington, Seattle WA. 1) College of Human Medicine, Michigan State University, East Eleanor Roosevelt Institute for Cancer Research, Inc. Denver, Lansing and Department of Pediatrics/Medical Genetics, CO. University of Wisconsin, Madison. An understanding of the evolution of the human genome will rely on analyses of sequence organisation of the various The finding of de novo monocentric Robertsonian trans- classes of DNA in man, chimpanzee (Pan troglodytes), and go- locations with associated bisatellited microchromosomes in rilla (Gorilla gorilla). We studied the non-coding chromosome two infants with Down syndrome is described. The micro- specific and centromere located alphoid DNA. X-chromosome chromosomes are thought to consist of the short arms of are alphoid DNA has diverged 5% between man and chimpanzee, and the acrocentrics involved. Suggested karyotypes both species show 12% divergence from gorilla, indicating 47,XY,-21,+t(21q;21q),+t(21p;21p) in patient 1 and that man and chimpanzee are more closely related than either 47,XY,-14,+t(14q;21q),+t(14p;21p) in patient 2. Possible is to gorilla. The alphoid DNA of the acrocentric chromosomes mechanisms giving rise to this rare chromosomal constitution has diverged 261 between man and chimpanzee. However, a spe- include abnormal exchange between chromatids in the bivalent cific pairwise exchange (cross-homogenisation) of alphoid DNA stage of meiosis I, centric fusion prior to or during that operates among human non-homologue acrocentric chromoso- meiosis, and centric fusion prior to the first mitotic mes, between 13 and 21 and between 14 and 22, is also found division of a fertilized egg with trisomy 21. An isochromosome among the human homologous chimpanzee chromosomes. Thus, cannot be ruled out in patient 1, therefore misdivision cross-homogenisation has occured between chimpanzee chromoso- of the centromere during gametogenesis could also account l mes 14 and 22 and between chimpanzee chromosomes 15 and 23. for this karyotype. The ikel ihood of each mechanism The results indicate that increased recombination frequency resulting in the long and short arm products presenting operates within the alphoid DNA of the human chromosomes. together is discussed. The rarity of this event is compared to estimated frequencies of monocentric Robertsonian trans- locations and the incidence of bisatel Iited microchromososes in otherwise normal karotypes. Cytogenetics A93 (0360) (0361)

Occurrence of 9p tetrasomy. Mary K. Kukolich, S.M. Jalal, C-BANDING TECHNIQUE: A RELIABLE AND SIMPLIFIED Mary Garcia, Donald W. Day, Toni R. Benjamin and Franciel MODIFICATION. Lacassiz . M.V. Carva&l, and Morgan. Genetic Screening and Counseling Services, Denton, M.A. Alliende. Louisiana State University Medical Texas. Center and Children's Hospital, New Orleans; INTA, University of Chile, Santiago, Chile. Whole arm autosomal tetrasomy is extremely rare in abort- The C-banding techniques produce selective uses or live births, with the possible exception of Pallister- staining of constitutive heterochromatin. Since the Killian syndrome for 12p mosaicism. Ten cases of 9p tetra- original technique reported by Arrighi and Hsu in somy, including the present (6,9p tetrasomy and 4,9p tetra- 1971, several techniques and variations have been somy including a portion of 9qh region), have been reported published. Although C-banding is a well-established as live births. Characteristic features have been severe procedure in most cytogenetics laboratories, the developmental delay, psychomotor retardation and failure to techniques thus far reported have some limitations. thrive. General features include brachy- or micro-cephaly The necessity of aging the slides, and the varia- with sutures or fontanelle, skeletal anomalies with severe tion in the duration of alkali treatment depending malformations of the extremeties, ambiguous genetalia, heart on the age of the slide and the tissue of origin, defect and characteristic facies. Facial features of our are the most significant. Most techniques require case included hypertelorism, bilaterally short palpebral aging the slides 1 or 2 weeks. If one day old fissures, apparent microphthalmia, beaked nose, bilateral slides are to be used, pre-treatment with heat has cleft lip and palate, cupped, rotated and very dysplastic been recommended. Timing of alkali treatment is ears and short webbed neck. The infant survived for two crucial to avoid under or excessive denaturation of months. The extra 9p isochromosome was confirmed by multiple the chromosomes. bandings (G and RF) from lymphocyte and skin fibroblast This is a report on a modification of the Sumner cultures. Karyotypes of both parents were normal. technique used for the last four years. This tech- A case of 9p tetrasomy, confirmed by G and RF bandings, nique produces consistently good quality C-banding was also observed in an abortus, estimated to be at 15 weeks on fresh slides, even those prepared the same day of gestation with possible cranial defect. The extra 9p of the harvesting. We use Ba(OH)', at 60- C for 30 isochromosome was also Ag-NOR negative and had just the peri- seconds. This time is constant, independent of the centric C-positive region. Cytogenetically, 9p isochromosome duration the pellet has been kept in the freezer. may occur at a relatively high proportion because of the known The detailed step by step technique will be pre- predisposition of the 9qh area to breaks. Though 9p dupli- sented. cation could occur as an isochromosome from misdivision of centromere, it could arise also as a translocation product, (pter-q12: :p11-pter).

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Aphidicolin-sensitive common fragile sites are Karytypic and moleculsr anslysis is the gemus Msstacus as a model for strongly induced by treatment during early S phase chromosome evolution. HP Loy. MM Cohen. and RA Schultz. Div. of but not treatments during late S phase and G2 Human Genetics ad Medical Biotechnology Center, Univ. of Maryland, alone. J.C. Leonard. Wilson Genetics Unit, George Baltimore. Washington University, Washington, D.C., U.S.A. Cytogenetically visible alterations of the human genome sre associated with Type 4 fragile sites, the most numerous and a spectrum of clinical outcomes including spontaneous abortion, comgenital ubiquitous group of coinon fragile sites, are malformation, neoplasia, and biochemical defects. Elucidation of processes induced by exposure tat aphidicolin for the final 24 involved in karyotype evolution may provide insights into mechanisms hours of culture, presumably due to the inhibitory underlying chromosomal andlor gone rearrangements. An ecellent exmple effects of aphidicolin upon both replicative and of extreme karyotypic variation occurs within the door genus Msntacus. The DNA repair synthesis. Because of the preferential Indian muntiac (Mf. sUxac) has the lowest diploid number among mammals distribtion of type 4 fragile sites among light 0 (fmale 2N-6; male 2N-7), while the Chinoe muntjac (M.rewsi) has 46 bands, it can be hypothesized that they are located chromosomes. Thes two species are phenotypically similar and are capable in euchromatin and that the effective induction of of producing viable (but sterile) hybrids. We are developing comparative type 4 fragile sites is dependent upon exposure to karyotypic and molecular genetic maps between human and each of thes on aphidicolin during early S phase but not late S muntjac species, focusing initial efforts the Indian muntqac. A G-banded phase or G2 phase. To test this hypothesis, idiogram of the Indian muntjac has been generated in accordance with accepted nomenclature (ISCN, 1985). Flow cytometric quantification of cultured lymphocytes were exposed to aphidicolin propidium iodide (PI)-stained nuclei revealed the cellular DNA content ofthe (0.2 x 10-i M) for varying intervals between the Indian muntjac to be 91% of the human value, confirming expected similarity final three and 24 hours of culture. Whole blood in genome size. Hybridizations with 7 independent cDNAs representing cultures were established using RPMI 1640, human housekeeping' genes haw identified specific bends in Indian muntjac harvested, and prepared for analysis as previously genomic DNA, suggestive of significant sequence conservation in singlebcopy described (Leonard et al., 1988, Hum. Genet. gene. Indian muntjac chromosomes, stained with PI (for DNA content) and 79:157-162.) A 3-hour treatment immediately prior fluorescoinated anti knetochore antibody (for contromere siz), were flow- to harvest includes only cells exposed during sorted to greater then 90% purity for gOneration of chromosome-specific G2 Southern blots and hybridization with human cDNA probes. Two human and the very end of S phase. The level of type 4 chromosome 19-liked DNA repair genes, BRCC2 and XRCCI, both sites seen in this treatment group was the same as hybridized oexlsively to DNA from muntjac chromosome 2. Specificity of in the DMSO solvent control. For treatment ERCC2 hybridization was confirmed by PCR amplification of muntjac DNA intervals up to 12 hours prior to harvest, the with primers flanking an evolutionarily conserved sequence. The resulting level of type 4 sites Is approximately 10 per 100 product contained the expected internal sequence and yielded an appropriate cells with no individual site showing consistent signal on Southern blot analysis. Is asu hybridization to sublocalizo this expression. For treatments ranging between 15 and assignment on chromosome 2 is in progress. To explore potential 24 hours, approximately 100 sites per 100 cells are relationships among repetitive DNA sequences, a set of phap clones expressed. These data indicate that the presence containing -l8kb inserts wes generated from Hindli digested Indian muntjac of aphidicolin during early S phase is crucial to DNA. One such cloue, LU103, beas a repetitive squence which hybridizos to all non-contromeric regions of Indian chromosomes is slY. This the efficient of 4 sites mun*c expression type fragile repetitive sequence wes not detected in human DNA and conservation in the and support the hypothesis that type 4 fragile Chinoe mustjac is under investition. To date, our efforts demonstrate the sites are early replicating and euchromatic. amenability of the Indian muntc as a model to assess chromosomal evolution. A94 Cytogenetics (0364) 1.189 (0365) 4.10

Sister chromatid exchange (SCE) frequencies are different in (direct) Sex chromosome alpha satellite arrays: size estimates and nondisjunction. cylotrophoblasts and (cultured) mesenchymal core cells. L U. LP Shulman, AT M. M. Mahtani. R. Wevrick. and H. F. Willard. Stanford University, Tharapel. B Tucker, JL Simpson. S Elias. Univ. of Tennessee, Memphis; Stanford, CA and University of Toronto, Toronto, Canada. Memphis, Tennessee. The X and Y chromosomes have a relatively high frequency of aneuploidy Determining the frequency of sister chromatid exchange (SCE) in chorionic and are unusual in that, at least in males, pairing of centromeric DNA cannot villus cells may prove useful if chorionic villus sampling (CVS) is to be used for occur prior to chromosome disjunction at meiosis I. By sizing and mapping evaluating fetuses exposed to clastogens or at increased risk for chromosome the centromeric a satellite arrays on the sex chromosomes, we are breakage syndromes. However, there is little information concerning of cells obtained from chorionic villus specimens. examining the possibility that anomalous a satellite array length or background frequency SCE in In errors. Although several groups have reported SCE frequency in chorionic villi, with organization may play a role segregation Using pulsed-field gel values ranging from 5.2 SCE/cell (Zahed et al, 1988) to 14.5 SCE/cell (Markovic electrophoresis (PFGE) and a Y chromosome-specific a satellite probe, we et al, 1987), none distinguished between cytotrophoblasts and mesenchymal generated long-range mapping data and size estimates of the array at the Y core cells. We have therefore determined SCE frequency in both centromere (DYZ3) In 20 normal males. The array lengths appear to fall cytotrophoblasts and cultured mesenchymal core cells. Chorionic villus into a bimodal distribution: 11 DYZ3 arrays between 250-430 kb (mean . specimens were obtained by elther transcervical or transabdominal CVS or at 330), 7 arrays between 900-1200 kb (mean = 1060), and 2 arrays of first trimester pregnancy termination. After cleaning and incubating villi in intermediate size at 570 and 600 kb. To test the hypothesis that DYZ3 complete medium (Alpha-MEM supplemented with 15% Chang medium, 15% arrays at one or the other end of the distribution might participate more fetal bovine serum, 100 lU/ml penicillin, 100 pgml streptomycin and 1% L- frequently in paternal melosis 11 errors, we examined the length of a were to deoxyuridine glutamine) at 37° C for 15 hours, villi exposed 5-Bromo-2 satellite arrays in 47, XYY males. In four cases, centromeric DYZ3 array (Brdu) for 72 hours, at a final concentration of 30 ug/ml. Chorionic vill were then treated with trypsin-EDTA to assist in separating cytotrophoblasts from lengths were 310, 310, 540, and 1100 kb. These data Indicate that errors mesenchymal core. Cytotrophoblasts were detached from their chorionic villi by in the melotic separation of Y sister chromatids can occur in chromosomes vigorously flushing vill in a Pasteur pipette. Remaining pieces of villi represent with a satellite arrays at either end of the array size distribution found in portions of mesenchymal core, and were used to initiate cultures. normal males. In contrast, a satellite array sizes at the X chromosome Cytotrophoblasts were then harvested and chromosome preparations were centromere (DXZ1) comprise a much more homogeneous population. In a made in the usual manner by treating the cytotrophoblasts with colcemid for 30 group of 36 X's from normal individuals, the frequency distribution of array minutes. Mesenchymal core cells derived from the same villi remained in culture sizes approximated a normal curve. DXZ1 arrays from 35 X chromosomes for approximately 12 days. These cells were then subcultured and 5 to 6 hours ranged in size between 2260-3730 kb (mean 2997; SD 442). The later exposed to Brdu (final concentration 30pg/ml) for 48 hours. Harvesting average array size on the X Is comparable to that found on several autosomes, were as with direct preparations. and chromosome preparations then performed Is DXZ1 array In a normal male, Staining of all slides was done using the technique of Wolff and Perry while that on the Y notably smaller. One (Chromosoma 48: 341, 1974). RESULTS: Eight first-trimester chorionic villus however, was significantly smaller than the rest, at 1490 kb. We have specimens were evaluated; five were obtained by CVS (10.4 to 12.6 weeks generated detailed long-range restriction maps both of this array and an gestation) and 3 were obtained following pregnancy termination (9.8 to 11.7 array of 2530 kb, using one- and two-dimensional PFGE. Other than their weeks gestation). SCE were observed in all 8 specimens from both size, the general features of the two maps were basically identical. Based on cytotrophoblast (direct) and mesenchymal core cell (culture) preparations. From the fact that (i) restriction sites for several frequent cutting enzymes were direct preparations, 80 cells were scored with a mean frequency of 6.6 SCE/cell consistently found at the array edges and (ii) similar total array lengths ± preparations, 160 cells were 1.4 SD (range 3-11 SCE/cell). From culture were obtained with each of several enzymes in both cases, it Is likely that scored with a mean frequency of 10.4 SCE/cell ± 0.4 SD (range 7-17 SCE/cell). single-copy DNA flanks the satellite sequences on each side. Further, it Is CONCLUSIONS: Baseline SCE rates differed between directly-prepared DNA Is within each cytotrophoblasts and cultured mesenchymal core cells, using our technique unlikely that abundant single-copy present array, (Tharapel et al, 1989) in which cytotrophoblasts and mesenchymal core cells are although small amounts (<100 kb) would have gone undetected. Measure- derived from the same villi. We conclude that SCE analyses involving chorionic ment of DXZ1 arrays in 47,XXY males should allow evaluation of a potential villi must take into account cell type and preparation. relationship between array size and nondisjunction.

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The distribution of aneuploidy in human gametes: comparisons SI oRAL AtI:ERATIMS OBSERVED IN 10 CASES OF HEUN RING between spermatozoa and oocytes. R.H. Martin, E. Ko and A. adICOSH 21. M. J. g:inslH }amziaa. .1, M. B. Rademaker. Alberta Children's Hospital and Univ. of Calgary, Peterseni18I E. E , . i , J. M. etZ Calgary, Alberta, Canada. LAQP I S. R3 EU1 l, A. SdizlL. nraren3 and S. E. The frequency and distribution of aneuploidy was compared in A__ _L. The Johns Hcpkirs Uhiv. Schl. of Med., 11,615 karyotyped human sperm and 772 karotyped human oocytes Balboa, MD, 2 Univ. of Geneva, Switzerland, 3 Balor Coll. to determine if all chromosomes are equally likely to be involved in of Med., Holston, TX, 4 Univ. of Aarhus, Demrki, DInst. for aneuploid events or if some chromosomes are particularly susceptible Mental Health, Belgrade, Yugoslavia, 6 nve Univ. of Iowa to nondisjunction. The results are based on 7958 karyotyped sperm Hospitals aad Clinics, Iowa City, 7 Inst. for Medical Genet., and 44 karyotyped oocytes from our laboratory and a collation of data Zurich, Switzerland, 8 e J. F. Inedy Inst., Glostnip, published from other labs. In general, hypohaploid chromosome Decmark. complements were more frequent than hyperhaploid complements, Previous work in this laboratory with one de noyo case of in both human sperm and oocytes. The distribution of chromosome human ring doianeana 21 (r(21)) (Proc. Natl. Acad. Sci. 86: an isorocn loss in the hypohaploid complements indicated that significantly 1914,1989) irndicated that intezirate of fewer of the large chromosomes and significantly more of the small one maternal dchrcoez 21 preceded ring formaticon. We tested a or an chromosomes were lost, suggesting that technical loss predominantly the hypothesis that Rbertsonria translocation affects small chromosomes. A conservative estimate of aneuploidy isochromossme is a frequent intermediate of r(21) formation (2 X hyperhaploidy) was approximately 3-4% in the human sperm and in 10 additional cases (5 de novo, 5 familial) of r(21). 18-19% in human oocytes. All chromosome groups were represented Phematypic variation was noted among the ten patients. Four among hyperhaploid human sperm and oocytes. For human sperm, the patients were phenotypically normal and two showed mild observed frequency of hyperhaploidy equaled the expected frequency mental retardation with few or no dysmorphic features. The exhibited mental retardation, growth based on the assumption that the frequency of nondisjunction is equal remaining four patients aad features. A panel of for all chromosome groups, with two exceptions: group G and the sex deficiency dysmorphic single-opy Aseqeces that map to chrosace 21q ( D21S13, D21S110, chromosomes. Among individual chromosomes in human sperm, D21S82, HMG14, M21542, OL6A1) was used to asoertain copy chromosome 1, 21 and the sex chromosomes had a significant excess number by polymorphim ard/or dosage analysis in CMA saples of hyperhaploidy. For human oocytes, there were fewer hyperhaploid of these 10 cas. Six cases showed evidence of loss of DM oocytes than expected for chromosome groups C and F and more than sequeces in the rirs at ()L6Al, the most distal polymorphic expected for chromosome groups E and G. Among individual marker on 21q. No evidence was seen for duplication of DM chromosomes there was a significant excess for chromosome 21. to sequenes in the rixs at the two most proximal long arm loci These results indicate that all chromosomes are susceptible tested (D21S13, D2lSllO). A r(21) macrorestriction nondisjunction but that chromosome 21 is particularly prone to breakpoint fragment has not yet been identified with plsed aneuploidy in both human sperm and oocytes. Sex chromosomal field gel electohoresis studies on [M fry 3 is common in human but not in human this aneuploidy sperm oocytes; digested with several rare-cuttinr restriction enzymes usirn corroborates new information from spontaneous abortions and probes frym 21q22.3 (ErS2, D21S3, MN, D21S113, CD18, PMK). much of sex chromosomal liveborns which demonstrates that Results frma these 10 r(21) patients were more consistent Overall these results aneuploidy is paternal in etiology. suggest with breakage and reunion of p and q arms than with heterogeneity in the etiology of aneuploidy: a common mechanism asymmetric breaks in the q anns of an iso or all chromosomes and mechanisms specific affecting targeting Pobertsonian translocation intermediate. Work with other [M chromosomes. markers will localize the breakpoints in these r(21) patients. Cytogenetics A95 (0368) 1.191 (0369) 1.192

Are chromosome-specific alphoid sequences evolutionarily A chromosome specific alpha satellite DNA subset on conserved? D.A. Miller. M. Rocchi. A. Baldini, L. Singh, human chromosome 2. 0. J. Miller. M. Rocchi .2. A. and 0.J. Miller. Wayne State University, Detroit, Michigan Baldini1.3. N. Archidiacono'. S. Lainwala'. and D. Human alphoid satellite DNA sequences are organized in A. Miller'. 1) Wayne State University, Detroit, MI, families that are localized on one or a few chromosomes. USA. 2) Istituto G. Gaslini, Genova, Italy. 3) Each family is marked by variation in number and arrangement Yale University School of Medicine, New Haven, of slightly diverged 170 bp monomers. In order to find out CT, USA. whether this specificity originated before the divergence of We have cloned a 1.4 kb HAE III fragment of human and great apes, we have hybridized cloned human probes human alphoid DNA in a plasmid, pBS4D. This to great ape chromosomes. A human chromosome 12-specific fragment was from the DNA of a human-hamster probe, pBR12 (Baldini et al., Am. J. Hum. Genet. 46:784,1990) hybrid cell line containing chromosome 2 as its hybridized specifically to its purported homeolog gorilla no. only cytologically detectable human component. 10. Similarly, Jorgensen et al. (EMBO J 6:1691, 1987) Under high stringency conditions, pBS4D hybridized reported that a cloned human X alphoid sequence hybridized in situ mostly to chromosome 2 and to a lesser to the chimpanzee X. We have isolated a cloned alphoid extent to chromosomes 18 and 20. Restriction sequence from a pygmy chimpanzee, PAN-3, that hybridized analysis using the DNA from 17 selected somatic specifically to chimpanzee chromosomes 1, 9 and 19 and to the hybrid cell lines confirmed these localizations homeologous human chromosomes 1, 11 and 17, as well as the and revealed that the genomic organization of this human X. These results indicate that the chromosome specific alphoid DNA differs on each of these three organization of these families has been conserved among chromosomes. The alpha satellite DNA subset human, chimpanzee and gorilla. identified by pBS4D on chromosome 2 is Such conservation is not always found. A cloned human characterized by a tetrameric (about 680 bp) sequence, pBS4D, that hybridized to human nos. 2, 18 and 20 reiteration detectible after digestion by (O.J. Miller, this conference) was hybridized to great ape restriction enzymes Hind III, HinfI and XbaI. metaphase chromosomes under standard conditions. In both pBS4D hybridized to Southern blots of DNA of chimpanzee and pygmy chimpanzee, pBS4D hybridized specifically chimpanzee, gorilla and orangutan only under low (13% and 22% of the total grains, respectively) to the stringency conditions. centromere of chromosome 11 (homeologous to human no. 9). A very small concentration of grains (6%) was at the centromere of chimpanzee 12 (homeologous to human 2q). No other grain concentrations were observed. In the gorilla, pBS4D hybri- dized specifically (12% of the total grains) to the centromere of no. 19 (homeologous to human no. 17). No grain concentra- tions were found in either orangutan or gibbon. Thus, pBS4D detects chromosome specific sequences in human, chimpanzee and gorilla, but surprisingly on non-homeologous chromosomes.

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Deletion 3p25: Another Contiguous Gene Syndrome. P.N. Detection of human X chromosomes in micronucleated Mowrey, M.J. Chorney, J.C. Ramer, M.J. Mascari and R.L. lymphocytes by fluorescence in situ hybridization. Ladda. PA State Univ., Col. of Med.; Hershey, PA. N.Z. Parsa and A.B. Mukherjee. Department of Deletion of 3p25 is associated with a complex and Biological Sciences,Fordham University, Bronx, NY. consistent set of phenotypic abnormalities. We previously Micronuclei are small cellular entities which reported two such children (Am J Med Genet 33:108, 1989) and result from the subfraction of the complete di- have more recently followed another child with an identical ploid genome. Colchicine induces micronucleation phenotype characterized by pre- and post-natal growth in cells by interfering with the assembly of deficiency with failure to thrive, swallowing disorder with microtubules and the mitotic spindle. It is also severe gastroesophageal ref lux and severe developmental known that chromosome integrity is preserved dysfunction. The craniofacial features of the 3 children during colchicine-induced micronucleation. The aim included microcephaly, with small C-shaped ears, midfacial of this study was to determine the X chromosomal hypoplasia with prominent blepharophimosis and ptosis, distribution pattern in micronucleated human characteristic puffiness about the eyes, carp-like mouth and lymphocytes induced by colchicine. Lymphocytes micrognathia. Edema of the hands and feet was noted in 2 of derived from 2 females were cultured for 72 hrs. 3 patients. This typical phenotype has been reported in 15 and the cells were exposed to 2 dosages of colchi- other patients reported in the literature. In our previously cine (2.5 pg/ml and 5 pg/ml) for 24 hrs. before unreported patient, failure to thrive was associated with being fixed. The cells were then processed for extraordinarily excessive oral secretions leading to fluorescence in situ hybridization with X-chromo- recurrent pulmonary aspiration in spite of gastrostomy and some-specific biotinylated DNA probe and were Nissan fundoplication procedures. The child died at 6 months screened under a fluorescent microscope. Each X of age of respiratory complications. Each of our 3 patients showed its clear fluorescent spot in the nuclei. had identical chromosomal breakpoints at the haploid band An analysis of a total of 2385 cells indicates level of 550. that micronucleation is dose-dependent and the 3p25-3pter is estimated to represent about 8.33% of distribution patterns of the Xs in various nuclei chromosome 3 (1.8 x 107 bp). This large segment would be alter with increased colchicine dosage. The expected to contain a large number of contiguous genes. highest % of micronucleated cells (87-89%) had Monoallelic gene expression in 3p25 region appears to both the X chromosomes located in a single large produce a complex phenotype as a result of abnormal cell nucleus/cell. The proportion of cells (8%) with proliferation (small tissue mass) and aberrant cell migration one X in a large nucleus and the other X in a (abnormal organogenesis). These patients serve as another micronucleus/cell at 2.5 yg/ml colchicine in- model to investigate the parental origin of the chromosomal creased to 13% at 5 jag/ml dosage. There was no deletion and the role of monoallelic gene expression on the increase in the proportion of cells (2%) with the evolution of the phenotype. two Xs in two separate micronuclei/cell as colchi- cine dosage increased. This indicates that only one X in a diploid female cell is more prone to be housed in a micronucleus with higher micronuclea- tion, presumably suggesting differential topology of the two Xs. (Supported by a Whitehall grant to A.B.M.) A96 Cytogenetics (0372) 1.193 (0373)

Identification of bisatellited marker chromosomes by in situ A rapid C-banding protocol with consistently good results. hybridization with alpha-satellite DNA probes. R. Plattner, S. Sahakian, N. Mitter and N. Villanueva. SmithKline Beecham N. A. Heerema and C. G. Palmer. Indiana Univ. School of Clinical Laboratories, Van Nuys, CA. Medicine, Indianapolis. Selective staining of constitutive heterochromatin by C- Small supernumerary marker chromosomes cannot be banding is believed to involve successive depurination and de- identified cytogenetically because of their small size and naturation of chromosomal DNA, followed by a loss of DNA from lack of distinct banding pattern. Some are associated with non-C-banded regions during incubation in a hot salt (SSC) mental retardation or infertility and others are carried solution. The process of depurination is generally achieved without phenotypic effect. Clinical outcomes, therefore, through treatment of DNA with HC1, while denaturation is perfo- cannot be predicted for markers diagnosed at amniocentesis. rmed through exposure of DNA to Barium Hydroxide. A loss of Some markers are bisatellited and others resemble small DNA from C-banded regions during the salt treatment may result rings or dots. One type of bisatellited marker stains from a modification of the constitutive heterochromatin regions intensely with distamycin/DAPI, (DA/DAPI). Original reports by certain non-histone proteins during depurination and denatu- describing the DA/DAPI procedure stated that the centromeres ration processes. The most rapid C-banding procedure, publish- of 1,9,15,16 and Yq were the only regions to fluoresce ed so far, as known to us, is by Kwong (Karyogram, 14:55,1988), brightly. Recent literature has disputed these findings by and may take 136 minutes as the minimum time. For some years, demonstrating that other acrocentric centromeres may also we have been using a modification of the basic C-banding tech- stain with DA/DAPI. Based on the early DA/DAPI procedures, nique, which takes less than 10 minutes to perform, and is good bisatellited markers that were DAPI(+) were identified as for all kinds of specimen tissues and for both fresh and aged inverted duplicated 15s but the recent literature has shed slides. The technique works for both in-situ and flask harvest doubt on their origin. protocols for non-suspension cultures. Essentially, it invol- We report here on the identification of six DAPI(+) ves replacing a 30 minute or longer treatment in 0.2N HC1 by a bisatellited markers by radioactive and nonradioactive in short rinse in acetic acid, and is followed by a 5 minute or situ hybridization with pDl5Zl, an alpha-satellite probe less immersion in Barium Hydroxide at room temperature. These specific for chromosome 15. All six markers demonstrated modifications result in requiring only a one minute rinse in probe hybridization on both ends of the dicentric markers, the hot, 2 X SSC, and a quick giemsa staining to achieve C-ban- confirming chromosome 15 derivation. Two DAPI(-) ding. We have also tried other alternatives to using acetic bisatellited markers were also tested for chromosome 15 acid with varying success. Use of this modified C-banding origin by IX Al" hybridization with pDl5Zl. Neither marker technique can obviously save a lot of time and rapidly provide demonstrated 15-specific hybridization. Further studies are the critical information needed in certain decision making underway to identify both bisatellited markers by the use of situations. alpha-satellite probes for the other acrocentric chromosomes. Ten additional ring markers are being studied by fluorescent nonradioactive in situ hybridization with other alpha-satellite probes.

(0374) (0375) 1.195

Mosaic 45,X/47,XY,+8 in a pheotypic male infant. fragmmntation detected in human lymphocytes exposed to different B. Schofield. D. Punlse-nveion. A. B1bu. S. Paveecu. J.R. anti-psychotic medications la vi=. D.A. Shafer, L.JB V.G. Naba and A. Fal k, Emory Univ. and Mental Health Inst., GA. Ebatt ai V. B. BO- Iph. Divisiln of Medical Genetics, Georgia Atlanta, The phenothiazine anti-psychotic drugs commonly used for Bath Isrel Medical Canter and Aunt Sinai School of schizophrenia therapy have a chemical structure similar to chromatin Medicine, Ne York. dyes that can intercalate DNA. In a previous study of schizophrenic and Me report a mle infant roted to hve multiple normal womn in a hospital prenatal care program, found that mothers features at birth. FNriiaral blood kzytypes who took a variety of anti-psychotic drugs (principally phenothiazines) 45,X-16/47,XY,+8m14. me karyotp of cultured ddn during pregnancy and their exposed newborns had elevated levels of fixablbets 47,XY,+8, with no 46,X cells detected chromosome damage. We have adapted the Single Cell Gel (SOC) 50 cells counted. At 13 wnths of a heigst m 70 ca(<5), Electrophoresis method (Singh et al. 1988) into an effective and might 8.2 kg(<5N), H.C. 43.5 ce(<5X). 71t followsing consistent procedure for direct detection of DNM fragmentation. Individual calls are Iiotypic Abrx ulitiis nar noted: icnoc nual non-dividing sandwiched between thin agarose gals on a hair udrl an the central an anterior regixm, broed microscope slide, the nuclear INK is lysad and unwound with alkali, nsl root and prominnt naxze, btroinMm, high-arched detergent and other agents and then subjected to brief electrophoresis (10 min). This causes DNA with single or doule strand bra to palate, maaligad teeth, large promizunt aur with thick separate and migrate out of the nucleus to form a comat tail of small helices, abort reck with nabbing, limited elboa inaticn, DMA fragments that can be visualized by ethidium bromide fluorescence. creesee eqira, delp in both soles, bilaterl pae Whole blood lymphocyte samples from normal donors were treated for 90 partial sydactyly of 2nd a1 3rd toes bilaterally od sin with four cmon phenothiazine anti-psychotics: fluphenazine hypwlestic nails. Genitalia wsre mal, with overriding (Prolixin), trifluoperazine (Stelazine), thioridazine (Nellaril) or scrotum, mild caorcal hypoepediesma iniml chorde, chlorpromazine (Thorazine), as wall as with two non-phanothiazine retzwtile left teetis in irgUnal canal, ad noalpable anti-psychotics: haloperidol (Haldol) or clozapine, which lack the right testis. Ultrsm %sy failed to visualize the right tricyclic structural component of the phenothiazines. The SOG treated cells were evaluated for the testis. famil intezial orwa wera dltected. A voiding frequency, length, and density of the comet tails of fragmented DMA. Untreated controls had few cells with comet cystogzu_ d an from the lmaer right side of tails and thAse wmre usually short thin tails with low DNM density. the bl Poasible for the origin of this Long and very dense tails occurred with very high frequency in cells rwvionsly Lcsicimara: a) tno irdanit unreported exposed to Prolixin or Stelazine at 25 - 50 ugml dose levels. p~usna of poet zyg1tic nom-disjunctimn .a/or anOhses Thorazine and Wllail also produced DA frntation, hoawver, higher or b) a chimea, with a trismy 8 fetus conized by doses (50 - 100 ugiml) more required to achieve comparable effects. In cells from a zabedI45, fetus. In ditian to features contrast with the phenothiazines tested, Haldol and clozapine produced typical of trimmy 8, the praobul had dabrt nobed rack, nail no tailing at the 50 ug/sl dose level at which all phenothiazines mare laypte Ad urognital Omliss dngstin that the 45,X strongly positive. Some tailing did occur with Haldol in 10 - 20 cell line my be presant in aditional tiast besides percent of cells when 100 - 200 ugl was used, while clozapine produced perizinral blood. no tailing at any dose up to 200 ugo/m. The preliminary results suggest a liner dose response effect for the phenothiazines. Thes in vitr studies provide clear evidec that certain anti-psychotic drugs have direct genotasic effects on cellular DNA and that these drugs my differ significantly from one another in their relative genotoxic potency. These results also suggest that genetic toxicology should now be considered in the pharmacological evaluation of anti-psychotic drugs. Cytogenetics A97

(0376) 4.3 (0377) 2.6

Identification of isochromosomes 21 through molecular genetic Trisomy 21: Association between reduced recombination and non-disjunction. S.L. Sherman*, N. Takaesu*, S. Freeman*, analysis and maximum likelihood estimation. LG. Shaffer, LE C. Phillips*, R.D. Blackston*, B.J. Keats+, P.A. Jacobs#. Spence. J.M. Meyer. JA Brown and C. Jackson-Cook Medical A.E. Cockwell#, D. Kurnit@, I. Uchida%, T.J. Hassold*. College of Virginia, Richmond. *Emory University, Atlanta, GA; +Louisiana State University, The largest class of de novo rearrangements (rea) in Down New Orleans, LA; #Wessex Regional Genetics Laboratory, syndrome are rea(21q21q). Classically, these rea have been termed Salisbury, Wiltshire, England; tUniversity of Michigan, Ann Robertsonian translocations (rob) implying an attachment of two Arbor, MI; %McMaster University, Hamilton, Ontario, Canada. different chromosome 21 homologues. However, we provide To assess the association between recombination and non- disjunction of chromosome 21, we have analyzed cytogenetic molecular evidence that the majority of rea(21q21q) may be and DNA markers in 100 cases of trisomy 21 and their parents. a isochromosomes (i) derived from single parental chromosome 21. Our initial DNA studies of 56 cases demonstrate that the Blood samples were obtained from 14 probands with Down overwhelming majority, 52/56 (93%) are due to errors in syndrome and homologous rearrangements of chromosome 21 [13 maternal meiosis. This figure is significantly higher than rea(21q21q) and 1 rea(21;21)(q22;q22)] and their parents. In order those based on cytogenetic polymorphisms (70 - 75%) and to distinguish between rob(21q21q) and i(21q) and to determine illustrates the increased accuracy obtained from the use of the parental origins of the rea, we used 24 RFLPs spanning the DNA markers. Using the maternally derived cases and probing with over length of chromosome 21. The parental origins were also assigned 20 chromosome 21 DNA markers, we have created a centromere independently and blinded to the RRLPs using QFQ and NOR map that spans most of 21q, including the most proximal-known cytogenetic variants. Using all RFLPs, the method of maximum markers, D21S16/D21S13, and the distal marker COL6A1. We likelihood was used to estimate the parental origin and have detected recombinants in 57% of those cases having at rearrangement definition in each family. The maximum likelihood least three evenly spaced, informative markers; this value estimates indicated that the majority of rea were isochromosomes is significantly lower than expected if we assume interference and a normal female map of approximately 90 cM (X21 4.76, (11/14), with 8/11 paternally derived. Three of the rea were ppter). (X29 22.41, p

(0378) (0379)

Neither age nor sex influence the expression of Medium 199 + FUdR is superior to three other systems for folate sensitive common fragile sites on human site at in NI chromosomes. induction of the fragile Xq27.3 lymphocytes. D.F.C.M. Smeets. Department of Human Genetics, j11gj4U, B Finucane(2), BS Emanumli31 and EJ Ramos(na3). (1) Albert University of Nijmegen, Radboud Hospital, Institute, Nijmegen, The Netherlands. Einstein Medical Center, Philadelphia, PA; (2) Elwyn Elwyn, PA; Common fragile sites are present in the (3) Children's Hospital of Philadelphia, Philadelphia, PA. chromosomes of every individual. Their expression Cytogenetic diagnosis of the Fragile-X syndrome is based on in vitro strongly depends on the culture at in cultured underconditions conditions. However, when the same culture demonstration of the fragile site Xq27.3 cells conditions are applied, the number of fragile which limit DNA synthesis. Protocols currently being used clinically sites often varies significantly between include: folate deficient medium (e.g. Medium 199); inhibitors of individuals, suggesting that also other factors, influence the propensity of common fragile sites thymidylate synthase (e.g. 5'-fluorodeoxyuridine -FUdR-), dihydrofolate to be expressed. To study a possible effect of reductase (e.g. Methotrexate -MTX-), or ribonucleotide reductase (e.g. sex and age we therefore investigated the excess thymidine -THY-). Recent recommendations for laboratory expression of folate sensitive common fragile sites in 42 females and 40 males of various ages. diagnosis of the Fragile-X syndrome suggest using at least two different They were normal healthy volunteers or induction systems, in light of the variable expression of the fragile site in individuals whose chromosomes were examined for individuals known to carry the fragile-X mutation (0 to 50% of cells the presence of heritable translocations. Peripheral blood lymphocytes were cultured for 72 analyzed). hours in medium 199 supplemented with 5% fetal We studied expression of the fragile site at Xq27.3 in 6 individuals calf serum. Colcemid was added 90 min before in the different induction harvesting started. After chromosome spreads had known to have the Fragile-X syndrome following been made according to routine procedures, from systems: A) Medium 199, B) 199 + 10-7M FUdR, C) 199 + 600 ggml every culture 100 unbanded Giemsa stained + FUdR. One hundred were metaphases were studied and all aberrations THY and D) RPMI 1640 10-7M metaphases recorded and photographed. Subsequently, the blindly scored from each experiment and we determined: 1) the number of slides were destained and GTG-banded for slides necessary to score 100 metaphases (as a measure of mitotic index) localization of the lesions. A total of exactly site at In all 1500 aberrations were scored. The expression of and 2) the percentage of cells carrying the fragile Xq27.3. lesions in every individual culture was quite cases cells cultured in Medium 199 + FUdR expressed the highest variable, ranging from 0 and 56 aberrations per percentage of fragile sites. In one case the patient was found to have 0% 100 analysed mitoses (mean 18.3 + 10.3 SD). The 1640+FUdR mean number of aberrations per 100 cells in males expression in Medium 199, 1% in 199+THY, 4% in RPMI (17.0 + 11.0 SD) did not vary significantly from and 8% in 199+FUdR. Mitotic index was found to be adequate for clinical that in females (19.5 + 9.5 SD), nor did it show studies slides used per experiment) using all of the cultures tested. any correlation with age (Pearson correlation (1-2 coefficient = 0.06). Two "new" common fragile Based on these results, we suggest that Medium 199+107M FUdR be used sites were discovered, located at 6p21 and 17q21. as one two induction in studies. Their fragile site status, however, needs to be of the (or more) systems Fragile-X confirmed. A98 Cytogenetics (0380) 1.196 (0381) 1.197

Three fragile sites on the canine X chromosome are Different expanded chromosome regions in a single cell lineage selected homologous to recognized common fragile sites on the human for amplification and deamplification ofthe UMP synthase gene. X chromosome. D. M. Stone, P. B. Jacky,' and D. J. D. Parker Suttle and Mazin B. Oumsiyeh. Veterans Administration Prieur. Department of Veterinary Microbiology and Medical Center and Dept. of Pharmacology, St. Jude Children's Pathology, Washington State University, Pullman, and Research Hospital, Memphis, TN 38101. Kaiser Permanente Medical Program,' Portland, Oregon. The UMP synthase gene is stably amplified within expanded Investigations of fragile site expression in domestic chromosome regions (ECRs) in Chinese hamster lung cells selected for dogs were undertaken. Peripheral blood lymphocytes from resistance to pyrazofurin (PF and 6-azauridine (6AUR), inhibitors of 17 dogs were cultured for folate sensitive fragile site the decarboxylase activity of the bifunctional UMP synthase enzyme. expression and cells from 3 of these dogs were also The orotate phosphoribosyltransferase activity initiates the metabolism cultured for expression of aphidicolin inducible fragile of 5-fluorouracil (EU) to its cytotoxic form. Growth of the PF+6AUR- sites. Whole blood cultures were established in RPMI 1640 resistant cells in EU results m selection of cells that have undergone medium and lymphocytes were stimulated with pokeweed deamplification, rapid loss of the amplified UMP synthase gene copies, mitogen. Bactrime (Roche), containing trimethoprim and and consequently loss of resistance to PF+6AUR. (Suttle, Som. Cell sulfamethoxizole, or aphidicolin, was added for the last Mol. Genet. 15:435,1989). Acquisition and deletion of ECRs correlated 24 hours of culture and 100 cells from each culture were with the selection for amplification or deamplification of the UMP examined. Canine chromosomes expressed 3 specific folate synthase gene (Qumsiyeh and Suttle, Som. Cell Mol. Genet. sensitive and aphidicolin inducible fragile sites on the X 15:503,1989). Detailed cogenetic analyses was conducted on a single chromosome; one on the short arm at the band corresponding cell lineage taken through three consecutive cycles of amplification and to human Xp22 (13 dogs, folate sensitive), and two on the deamplification. In each case there was a unique structure and/or long arm at bands corresponding to human Xq2l (16 dogs) position for the ECR that contained the amplified UMP synthase and Xq27.2 (12 dogs). The Xq2l site was the most genes. In situ hybridization studies confirmed the cytogenetic data frequently expressed site and also was expressed in the suggesting a correlation between amplification as an ECR and greatest percentage of cells from all dogs (2% to 12% of chromosome rearrangements. The chromosomal site of both the cells) and was expressed on both homologs in metaphases primary and secondary amplification was the same, a small acrocentric from two female dogs. No other fragile sites were marker chromosome that has resulted from a rearrangement of expressed on the X chromosome by either method in any of chromosome 4, the endogenous site of the UMP synthase gene. The these 17 dogs. Silver staining disclosed no nucleolar third cycle of amplification resulted in an HSR of varying size located activity on the canine X chromosome. on a marker chromosome containing the 4p region where UMP These findings document 3 folate sensitive and synthase maps in the wild type cells. The variation in position, size and aphidicolin inducible fragile sites on the canine X structure of the consecutive ECRs can be explained by differences in chromosome which show G-band homology to 3 recognized coamplified DNA sequences or by complex chromosome common fragile sites on the human X chromosome. This rearrangements. The ability to cycle cells of a single lineage through suggests that not only is the mammalian X chromosome states of amplification and deamplification will facilitate study of the highly conserved evolutionarily in its morphology, banding gene amplification process and the factors that effect the composition pattern, and linkage groups, but that common fragile sites and stability of amplified regions. on the mammalian X are also conserved. (Supported by NIH Supported by VA Merit Review Award and in part by CA-21765 from grant RR00515 and grants from the American Cancer Society NCl and by American Lebanese Syrian Associated Charities. and Morris Animal Foundation).

(0382) 1.198 (0383) 1.200

Fluorescent inaIlU hybridization with chromosome 18 alpha satellite DNA probe for Telomeric attachment of isodicentric Barr bodies revealed by in-situ the verification of a derivative chromosome and the quantification of mosaicism. hybridization with biotinylated and DNP labeled probes. C. L. Walker. A T Tharapel. M. B. Oumsiyeh. P R. Martens, J. C. Ward and R. S. Wilroy. C. B. Carnile. K.M. Flov and B.R. Miseon. The Johns Hopkins Cytogenetics Laboratory, University of Tennessee, Memphis, Tennessee. University, Baltimore, Maryland. A biotin-labelled alpha satellite DNA probe detecting locus D18Z1 (commercially One feature of the inactive X chromosome is that it forms a unique obtained) was used to study two abnormalities involving chromosome 18. A known chromatin structure in the interphase nuclease, the Barr body. As complete trisomy 18 patient was used as a positive control. In the first case, GTG- isodicentric X chromosomes are always the inactive X and form banding analysis revealed an unbalanced chromosome complement with a modal bipartite Barr bodies, they are ideally suited for studies of the count of 45 chromosomes. Each cell showed only one normal 18 and one 21, inactive X during interphase. We first examined the structure of along with a translocation chromosome consisting of the long arms of chromosomes isodicentric X chromosomes in interphase skin fibroblasts, using 18 and 21. Parental chromosome analysis showed the mother to have a balanced biotinylated probes for either centromeric or subtelomeric DNA translocation between a chromosome 18 and a 21, involving the pericentromeric sequences. Then we carried out similar studies in metaphase and in region in both. Thus, the origin of the centromere of the two resultant derivative mouse-human hybrids derived from these cells (having only the chromosomes could not be determined. jnoluhybridization with D18Z1 probe on inactive X). Comparisons of the distance between the centromeres of the mothers cells showed bright fluorescence on the centromeres of both the an isodicentric joined at the p-arms with two isodicentrics joined at normal 18 and the translocatlon chromosome, i.e., der(18). The infant received the the q-arms surprisingly reveal that the distance between centromeres der(18) subsequent to a 3:1 disjunction and therefore has monosomy for both the is greater when separated by p-arms than by q-arms. For the q-arm short arm of 18, and the short arm and the centromere of a 21. attached isochromosomes, these distances do not change in metaphase nuclei (obtained by mitotic shakeoff), suggesting that the Barr body CARRIER MOTHER UNBALANCED INFANT structure is the same at both stages of the cell cycle. For the p-arm isodicentric, the distance between centromeres is less in metaphase than interphase, perhaps reflecting condensation of transcriptionally active chromatin on the p-arm. Also, we found that the distance between centromeres in hybrids did not differ significantly from that in the human cell suggesting that Barr body structure is maintained in chromosomes 18 der(18) der(21) 21 18 der(18) 21 the mouse-human hybrid. In all of the isodicentric analyzed, the distance between telomeres was not greater (and was often less) than the distance between centromeres. This along with The utility of inkU hybridization, using the D18Z1 probe for the quantification of confocal imagery of chromosomes (labeled with both probes, using trisomy 18 cells in a mosaic patient (46,XY/47,XYV+18) was assessed in the second biotin for one and DNP for the other) to analyze three dimensional case. Based on routine cylogenetic scoring of metaphase spreads with nuclear structure, suggests that the telomeres are close to one distinguishable 18's, a 2:3 ratio of normaLtrisomy 18 cells was detected. Analysis by another, attached to the nuclear membrane, forming a loop-like inslU hybridization yielded a more accurate ratio of 1:4, because even in structure. underspread metaphases the centromere fluorescence could be clearly visualized and cells counted consecutively. We also tested the feasibility and accuracy of scoring interphase cells for rapid screening of trisomy 18. Fluorescent inaiju hybridization with alpha satellite DNA probes has multiple applications in clinical cytogenetics including determination of the centromere in a derivative chromosome, rapid assessment of mosaic trisomy 13, 18, and 21, and potential direct screening of amniocytes and trophoblast cells for the common autosomal and sex chromosomal aneuploidies. Cytogenetics A99

(0384) 13.3 (0385) 1.201

Uniparental heterodisomy for chromosome 14 in a phenotypically Discrimination between symmetrical and asymmetrical trans- abnormal familial 13/14 Robertsonian translocation carrier. locations by combined hybridization of chromosome-specific J.-C. Wang, M.B. Passage, P. Yen, L.J. Shapiro.and T.K. and alpha satellite probe DNA. Heinz-Ulrich G. Weier. oe N. Mohandas. Howard Hughes Medical Institute, UCLA and Harbor- Lucas. Richard Segraves, Daniel Pinkel and Toe W. Gray UCLA Medical Center. Torrance, CA. Biomedical Sciences Division, L-452, Lawrence Livermore Although the majority of individuals with familial balanced National. Laboratory, Livermore, CA 94550, USA. translocations are phenotypically normal, a small number of We describe here, a procedure based on fluorescence in situ such individuals have varying clinical abnormalities. These hybridization that allows rapid determination of the frequency are generally attributed to unknown causes independent of the and type of reciprocal translocations in metaphase spreads. This translocation. A nine-year-old mentally retarded girl (GR) was procedure is particularly well suited to assessment of the initially evaluated during the newborn period because of frequency of stable aberrations such as translocations as a multiple congenital anomalies including a small thoracic cage. measure of the degree of exposure to ionizing irradiation at long marked angulation of the ribs, bilateral subdural hygromas times after exposure. In this procedure, selected "target' requiring a VP shunt, and facial dysmorphism. By age 9 she had chromosomes are fluorescently stained from pter to qter by in severe kyphoscoliosis. a seizure disorder. and coarse facial situ hybridization with chromosome-specific composite probes. features with frontal bossing, prominent maxilla and mandible. Detection of the biotinylated composite probe is accomplished Chromosome studies showed GR to be a carrier of a balanced with fluorescent avidin-FITC so a the stained chromosomes 13/14 Robertsonian translocation [45.XXt(13ql4q)]. which was fluoresce green under blue light excitation. The chromosomes also present in her father. The mother was a carrier of a are additionally stained by hybridization with an in vitro balanced reciprocal translocation between chromosomes 1 and 14 synthesized pan-centromeric probe DNA specific for alpha [46.XX.t(1;14)(q32;q32)]. Both parents were phenotypically satellite repeat. Hybridization of this probes is detected with normal. Molecular studies were carried out to determine the AMCA-conjugated antibodies so that the centromeres show blue parental origin of chromosomes 1. 13, and 14 in the patient. fluorescence under UV excitation. Thus, structural aberrations Using probes for D14S13 and D14S22, we could show that the involving target and non-target chromosomes appear both red patient inherited both 14's from her father and none from her and green under blue excitation and can be scored rapidly. mother. The most likely origin of the karyotype in GR is the Discrimination between symmetrical and asymmetrical fertilization of an ovum missing chromosome 14 resulting from a translocations is accomplished rapidly by observing the number 3:1 segregation in the mother, by a sperm with the 13q/14q and of blue-fluorescing centromeres along the aberrant a normal 14 resulting from a 2:1 segregation in the father. chromosome(s) during UV excitation. This procedure appears Recent studies indicate that both maternal and paternal applicable both to human and to mouse chromosomes. homologs of chromosomes are required for normal development and Work performed under the auspices of the U.S. Department of that uniparental disomy can lead to congenital anomalies Energy by the Lawrence Livermore National Laboratory under resulting from genomic imprinting. Our findings show that contract number W-7405-ENG-48 with support from USPHS uniparental heterodisomy for 14 most likely accounts for the grant HD17665. Additional support was provided by the phenotypic abnormalities observed in GR. It is suggested that Program for Analytical Cytology. uniparental disomy may be the basis for abnormal development in at least some phenotypically abnormal familial balanced translocation carriers.

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Characterization of Robertsoniam translocations using in situ hybridization An X/Y translocation resulting from recombination between with biotinylated DNA probes. DJ. Wolff. A.R. Brothman'. and S. Schwartz. homologous sequences on Xp and Yq. P.H. Yen, S.P. Tsai, S.L. Division of Human Gemetics, Univ of Maryland School of Medicine, Wenger, M. Steele, T.K. Mohandas, and L.J. Shapiro. Howard Baltimore and 'Cytogenetics Division, Eastern Virginia Med School, Norfolk. Hughes Medical Institute, University of California. Los Angeles and Children's Hospital of Pittsburgh. PA. Molecular studies utilizing probes for specific DNA sequences in the The mammalian sex chromosomes are thought to have evolved centromeric and short arm regions of the acrocentric chromosomes are useful from a common ancestral homologous chromosome pair and for studying Robertsoniam transocatioms. Fluorescent in situ hybridization significant sequence similarities remain between the present chromosomes: two was used to characterize six Robertsonian translocation day X and Y chromosomes. In addition to the pseudoautosomal t(14;22), three t(13;14), and one t(14;21). The probe alphaXT(680),22-94, region which is essential for proper pairing of X and Y during specific for alphoid sequences of both chromosome 14 and 22, was labeled with bio-16-dUTP during PCR amplification utilizing pUC primers. male meiosis, extensive homology has been found between Xp22 Subsequent hybridization in itu to metaphase spreads was visualized with and Yqll in humans. These are the regions where the break- fluorescence microscopy. Double signals were detected on each t(14;22), points in the majority of X/Y translocations occur. It has indicating that both centromeres were present. No NOR-positive material been suggested that these translocations could be caused by was present in the translocated chromosomes. Thus, the most likely homologous recombination between Xp and Yq. However, to date chromosomal breakpoints were in the proximal p arms. Single signals were none of the breakpoints in X/Y translocations have been detected in each of the t(13;14) and the t(14;21) translocation, demonstrating studied. We now report the isolation and characterization of that, at least, centromeric material from chromosome 14 was present in each. the breakpoints in one patient with an X/Y translocation and However, C-banding indicated that the t(13;14) and the t(14,21) were show that the breakpoints indeed fall within homologous elements. Two additional Li.26 for alphoid repeats dicentric probes, (specific sequences shared by X and Y. of chromosomes 13 and 21), and SB (a clone containing lSS ribosomal has a 46.t(X;Y),Y karyotype with breakpoints at Xp22.33 sequences), were labeled with biotin via nick-translation and hybridized to R.P. that and Yqll.1. The translocation chromosome was inherited from four t(14,21) Robertsonian translocations. Preliminary results revealed a chromosome 21 alphoid material was present in at least two of the his mother. Both R.P. and his mother are Xg negative while translocations. These three probes are being used to further characterize both maternal grandparents are Xga positive and have normal these, as well as other Robertsonian translocations. Our results suggest that karyotypes. At 13 months of age he had severe developmental most Robertsonian translocations contain alphoid sequences from the delay, seizures, ichthyosis. depressed nasal bridge. anteverted centromeres ofboth chromosomes involved and that the breakpoints lie in the nares and bilateral simian creases. Genomic DNA was hybridized p arm regions of DNA repeats proximal to the centromeres. with various probes from Xp22.3. The results show that R.P. has deleted the terminal portion of Xp including the STS, DXS237, S232-A and S232-B loci, but not the more proximal DXS143. A probe CS19A from the S232-C locus on the X detects a normal Y fragment (JRPl) and an altered X fragment (JRP3). Both fragments were isolated and characterized. Hybridization between JRP1 and its X homolog CSl9A showed that they share over 9 kb of sequence homology. Comparison of the restriction maps of CS19A, JRP1 and the junction fragment JRP3 showed that JRP3 is the product of homologous recombination between CS19A and JRP1. Therefore the X/Y translocation in R.P. is the result of an aberrant homologous recombination event between Xp and Yq during spermatogenesis in the maternal grandfather. Al 00 Cytogenetics/Differentiation and Development (0388) 1.203 (0389) 1.204

The possible use of in-situ hybridiyatoo1i for t h Simultaneous expression of the rare fragile X site (FRAXA) and the detection of mosaicism common site (FRAXD). Zaslav and W.T. B Dept. of Pediatrics, North Shore University Hospital-Cornell University Zahed. L.*+. Murer-Orlando, M.* and Vehematis. M.s medical College, Manhasset, New YorL The Fragile X (fra(X)) syndrome is the most common inherited form *Paediatric Research Unit. Guy s Hospital, London of X linked mental retardation. It is diagnosed by demonstrating a Bridge. SEi 9RT. UK rare folate sensitive fragile site on the long arm of the X chromosome at band Xq273 (locus FRAXA). Low level expression (1-2%) +Department of Pathology, Montreal Children Hospital, of an apparent fra(X) chromosome has been observed in many seemingly 2300 Tupper Str., Montreal. Quebec, H3H 1P3. Canada normTindividuas and in amniotic fluid cell cultures, leading to diagnostic dilemmas. Recently, a common fragile site located at In situ hybridization can complement conventional chromosome band Xq27.2 (locus FRAXD) was identified near FRAXA cytogenetic techniques in mosaic cases or cases (Sutherland and Baker, Clin Genet 37:167, 1990). suspected of mosaicism, where a wide number ot The ability to distinguish cytogenetically between the two metaphases need to be scanned, a tedious and sometimes fragile sites may resolve questionable fra(X) diagnoses. In order to impossible task. With the use of probes specific for establish the conditions required for induction of both types of particular chromosomes, this could be accomplished by fragile sites, samples from a control male, two fra(X) males and a scoring interphase nuclei. carrier female were studied using various protocols. Rare and In order to establish the sensitivity and common fragile sites were induced in cells cultured in RPMI 1640, specificity of this technology for the possible with 10%6 FBS and 2 mM L-glut by stressing the cells with different detection of mosaicise. we applied biotinylated agents for the final 24 h. The 4 Protocols used consisted of centromeric probes specific for chromosomes X, Y. 18 and exposure to (1) 1 uM aphidicolin (APC) and 0.025% ETOH; (2) 1 uM 21 to normal and fully aneuploid samples and scored, in APC,0.025% ETOH, and 300 nmaL thymidine; (3) 0.1 uM FUdR; (4) 1 uM each case. 500 nuclei, for the number of spots of label APC, 0.025% ETOH, and 0.1 uM FUdR. Cultures were harvested 72 h present. With the exception of the centromeric probe for after initiation and fragile sites were identified in a minimum of chromosome 21, which cross-hybridizes with the 100 trypsin Giemsa-banded cells from each protocol. In the control centromere of chromosome 13. we tound that the probes sample using Protocol 1, 3% of the cells exhibited the common fragile provided a high degree of sensitivity (84%.-96%) and site while no cells exhibited the the rare fragile site using specificity (97%). Protocol 3. Both common and rare fragile sites were present in the We then applied the probes on a few mosaic or samples from the fra(X) subjects after treatment with Protocols 2 and suspected mosaic cases which had been studied by looking 4. 1-2% of cells showed both common and rare fragile sites on the at metaphase chromosomes. Results from in-situ same X chromosome at the 500 band level. Breakage at the common and hybridization confirmed and supplemented the cytogenetic rare fragile sites could be distinguished as follows: when the rare findings, and the proportion of aneuploid cells in fragile slte was expressed the major portion of band Xq27 remained mosaic cases, with both techniques, were comparable. attached to the broken chromosome end, whereas the common fragile These preliminary results suggest that in-situ site showed darkly stained material equally divided between the hybridization can be a valuable and reliable technique terminal end of the X chromosome and the displaced fragment. for the detection of mosaicism. particularly in CVS In conclusion, using these protocols and criteria, the rare and cases, where mosaicise is common. common fragile sites on the X chromosome can be distinguished. This approach should be of assistance in resolving questionable fra(X) syndrome diagnoses. Differentiation and Development (0390) 1.205 (0391)

Pregnancy outcomes in epileptic women followed prospectively. Lipid abnormalities of the XX = mouse. T. G. Atkinson and E. Andermann, M. Oguni, L. Dansky, A.L. Sherwin. Montreal S. R. Blecher. Univ. of Guelph, Guelph, Ontario, Canada. Neurological Hosp. and Inst., Dept. of Neurology and NXu- rosurgery, McGill Univ., Montreal, Quebec, Canada XX= ("sex-reversed") mice have several abnormalities and our observations indicate that abnormal testicular lipid Over 250 pregnancies of epileptic women have been fol- levels are also characteristic of these mice. lowed prospectively with monthly monitoring of serum anti- §= is a duplication of the testis-determining region of convulsant drug levels, serum (SF) and red cell folate the Y chromosome, transposed to the distal end of the (RCF) levels, and maternal serum alphafetoprotein (MS-AFP) chromosome. This region of the Y chromosome undergoes pairing levels. The offspring were assessed with respect to growth and crossing over with the X chromosome during meiosis, and developmental parameters and presence or absence of resulting in translocation of = to the X. Such An-carrying major congenital malformations. Major malformations X chromosomes produce, on fertilization, chromosomally XX occurred most frequently in offspring of epileptic women individuals of apparent male phenotype. These mice are taking primidone (PRM) (40%) alone or in combination, fol- sterile and are appropriately described as pseudomales. lowed by phenobarbital (PB), ethosutmide (ESM), valproic Previous microscopic examination demonstrated a greater acid (VPA) and phenytoin (PHT) - all around 20%. The low- number of lipid droplets within the Sertoli cells of est frequency of major malformations was seen with cdrbdma- pseudomale testis compared with wild-type testis. Also, zepine (9%). Developmental anomalies (omitting postural abnormally large peritoneal fat pads have been observed in deformities and hernias) were most frequent with PRM and XX pseudomales. These observations and the fact that VPA and least frequent with CBZ. Head circumference was pseudomale testes have a dark, yellowish pigmentation suggest significantly lower in children exposed to polytherapy vs. that there may be a lipid metabolism abnormality in the XX§= monotherapy, particularly for CBZ and VPA. mice. Testicular lipids of normal (XY) males and XX)a Plasma VPA levels during pregnancy were significantly littermates have been examined by thin layer chromatography. higher in mothers of malformed babies than in mothers of Levels of both cholesterol ester and triglyceride appear to normal babies (P< 0.01). No consistent relationship was be higher in the pseudomale than in the wild-type. found between serum anticonvulsant drug levels and AFP Quantification of lipid levels by elution from the levels or between SF levels and AFP levels. However, ab- chromatography plates and calorimetric assay has been normal fluctuations of MS-AFP levels were associated with initiated. Examination of lipids in other tissues including abnormal pregnancy outcomes (malformations and spontaneous liver, kidney and serum is also in progress. ascribed to the abortions). The ratio of CBZ-epoxide to CBZ levels was The only role usually testis-determining chromosome is of a male was significantly higher in pregnant than non-pregnant region of the Y development gonad. an of epileptic women, and when CBZ was taken in combination Our preliminary results suggest interesting abnormality with VPA. lipid metabolism in the XX = genotype. In conclusion, the type and number of medications and phadrmacokinetic parameters play an important role in preg- nancy outcomes of epileptic women. Differentiation and Development Al 01

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Molecular and ultrastructural characterization of the products of the human Perinatal Lethality (pie): A mutation caused by integration of a retinopathy candidate genes ROMI and RDS. R.A. Bascom. G.Connell. J. transgene into distalimouse chromosome 15. Garcia-Hems. LCollins. D.Ledbetter. R.S. Molday. V.Kalnins and R.R. D.R. Beier and P. Leder. Department of Genetics. Harvard Medical McInnes. Hospital for Sick Children and University of Toronto, Toronto, School.Howard Hughes Medical Institute. Boston. MA. 02115 Ont and University of British Columbia, Vancouver, Canada; Baylor College of Medicine, Houston, Texas. We have identified a transgenic mouse line in which it appears that romn- (formerly termed rosp-1) is a membrane protein located in the outer a recessive insertional mutation has occurred as a result of transgene segment of the rod photoreceptors that, together with a homologous but integration into a locus required for post-natal viability. This line seperately-encoded polypeptide, rds, constitutes a new mammalian cames an MMTV-LTR/c-mvc fusion gene which was shown to be photoreceptor-specific protein family. The membrane orientation and cellular associated with the development of a variety of tumors. In a non- location of the bovine rds polypeptide have been recently established inbred background. homozygous transgenic animals survive. but have (Connell et al., ARVO 1990), allowing us to further compare the rds and reduced fertility and viability. When the transgene was introduced rom-) proteins. To begin defining the respective roles of these proteins in the into the inbred C57B1/6J background by serial backcrosses. a more biogenesis, structure and function of the rods, we have examined the dramatic recessive phenotype was noted. Newbom mice homozygous membrane topology and subcellular localization of the rom-1 polypeptide. for the transgene are smaller than their sibs. feed poorly. and We have used electron microscopy, and a polyclonal antiserum that detects generally die within one day after birth. Pathological analysis of not to to the rims of the rod disks, a location these mice reveals that their livers have variable. but often quite rom-I but rds, localize rom-1 and to In vitro and translation of rom-) in the severe. vacuolization of hepatocytes. The histochemical identical that of rds. transcription, of presence and absence of canine pancreatic microsomes, demonstrates that, ultrastructural appearance of the affected livers is not suggestive like rds, rom-) does not have a cleaved NH2-terminal signal peptide. known carbohydrate or lipid storage disorders. or of a respiratory appears similar to that seen in toxic Protease peon analysis shows th rom-) is cotranslationally inserted into disorder. The cellular damage membranes NH2 and COOH liver inJury. the and has 4 transmembrane domains, with the the ends being cytoplasmic, a topology similar to that of rds. Preliminary We have used recombination analysis to map the position of linked to a cluster evidence suggests that rom-1 and rds, each of which migrates at -33 kDa as transgene in the mouse genome. It is very tightly caracul and anemia monomers, a disulfide bonds: a) in the absence of loci that include the mutations (Ca) microcytic form heterodimer in vivo by the of E-mercaptoethanol, rds migrates at -68 kDa, consistent with its existing in (mk). This region is also linked to the int-Vproto-oncogene. gene family. and the 11 gene family: this vivo as either a homodimer, or heterodimer, b) the central hydrophilic region lfo-x-3 cytokerati61type synteny is conserved with the homologous genes found on human of rom-1 has 7 cysteines, all of which are conserved with rds; c) rom-1 12. appears to beimmunoprecipitatedwith rds as a complex of-68 kDa from a chromosome We call the mutated locus in the transgenic line pie. for perinatal rod outer segment fraction. We conclude that: (i) both rom-1 and rds are the sequences which flank the membrane topologies, and no lethality. Cloning of genomic localised to the rod disk rims, have similiar the identification of the (ii) the sequence similarity of these two transgene insertion site should facilitate cleavage of the NH2-terminus; molecular basis of the proteins, their co-localization to the same region of the photoreceptor, and disrupted gene and the characterization of the The conservation of further their possible association in a rom-lfrds heteodimer, all suggest they share a presumptive metabolic defect. synteny a for this locus on human role in the biogenesis and structure of the photoreceptor; (iii) since a mutation suggests that there may be homolog in rds (retinal degeneration slow) causes a murine retinopathy characterized by chromosome 12. defective morphogenesis of the disks in the rod outer segments, ROMI and RDS are both candidate genes for human retinopathies. To facilitate disease association in man, we have sublocalized ROMI to chr. 1Iq13 by in situ hybridization.

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A Conserved X-linked Transcript IdentIfied by a Sequence Mitochondrial DNA deletions in normal adult humans detected and N Arnheim Molecular Biology from the Sex Determining Region of the Mouse Y by PCR. G.A Cortonassi Chromosome. Section, Univ. of Southern California, Los Angeles, 90089- C.E. 'Mltchell M. and Qumlyeh M.D 1340 USA *BIshoR High levels of a number of large mitochondrial DNA Univ. of Tennessee, Memphls,*Dept of Ob/Gyn, Molecular (mtDNA) deletions are associated with some human diseases Genetics Laboratory; #Dept. of Pediatrics. tKearns-Sayres syndrome, progressive external In an effort to Isolate sequences transcribed from the mouse Y opthalmoplegia). The frequency at which these same deletions occur in normal individuals has been largely chromosome a flow sorted Y library was differentially screened unknown. Using the polymerase chain reaction (PCR), we detect the presumed products of deleted mitochondrial mouse testis using labelled cDNA probes reversed transcribed from genomes in a number of DNA samples from selected normal RNA and male and female liver RNA. Clone pY8 was identified as adult tissues. In addition, we find that samples of placental and fetal origin produce much less PCR product per hybridizing to testis RNA but not male or female liver RNA. Y8 maps normal mitochondrial genome. to flank two 13-bp direct repeats mouse is Primers were designed to the sex determining region of the Y (short arm) and which bound the most common mtDNA deletion associated with translocated to the Sxr (sex reversed) region In sex reversed mutant mitochondrial myopathy. Under conditions of rapid temperature cycling, the PCR product of a specific deleted mice. It detects three discreet Sxr specIfic lad, two of which have mtDNA is expected to out-replicate the non-deleted product, been localized 3' to the Zfy-1 and Zfy-2 genes. Y8 detects a 3.5kb because the replication of the shorter product is completed in a much shorter time. testis specific mRNA transcript In adult tissues. Extensive screening Several observations support the hypothesis that the amplification products are a result of preexisting, in vivo of a cDNA library Identified a 2.0kb cDNA, cR5 which shares mtDNA deletions rather than an artifact of the PCR.(l)DNA approximately 200bp of homology with Y8. Unlike Y8, cR5 strongly samples of different origin show very different frequencies of deletion product, even when equivalent amounts of normal hybridIzes to the X chromosome and only weakly to the Y. It detects mtDNA are used as template. (2)Under amplification conditions which detect single DNA molecules, samples can be diluted to 3.5kb testis In numerous other adult tissues both the transcript a concentration sufficient to produce 50% strong positives male and female. The cDNA has been ocalzed by backcross analysis and 50% negatives, a result consistent with Poisson expectations of a discrete deleted molecule. (3) A to the centromerlc region of the mouse X chromosome and by In situ statistical 'concordance" test using an experimental design analysis to human Xpl 1.21. The relationship of the X and Y loci and of Luria and Delbruck indicates that detection of deletions is independent of the number of amplification cycles. the origin and function of the transcript Is currently under Our data are consistent with an ±nviv origin of the mtDNA deletions amplified by PCR. Also, our results suggest Investigation. that deletions of the mitochondrial genome accumulate with age in normal individuals. Experiments to test this second hypothesis rigorously are underway. Al 02 Differentiation and Development (0396) 1.213 (0397) 7.10

Chromosomal sublocalization and cellular expression of the retinal homeobox Gene silencing and gene competition: Independent mechanisms involved gene HOX10. LDe Chen, L. Plodr, L. Collins. P. Thorner, V. Kalnins, A, in human hemoglobin switching. T. Enver. N. Raich. B. Nakamoto. B. Duncan, B. Taylor, and R.R. McInnes. Hospital for Sick Children and Joseohson. Th. Palavannooouiou and G. Stamatovannoooulos. Division Univ. of Toronto, Toronto, Queen's University, Kingston, Ontario, Canada, of Medical Genetics, Univ. of Washington, Seattle WA. and The Jackson Laboratory, Bar Harbor, Maine, USA. The activation and high level transcription of the 8 giobin locus is HOX1O encodes a developmentally regulated homeobox gene originally thought to be dependent on the locus activation region (LAR); a region identified on the basis of its relatively-abundant and retina-specific expression of DNA located 6-20 kb upstream of the e gene and characterised by a (De Chen et al, Amer. J. Hum. Genet. 45: AlIl, 1989, where HOX10 was series of erythroid specific superhypersensitives sites. To analyse the termed RETI, and shown to map to chr. 14). As an initial step in evaluating role of the LAR in the temporal control of human embryonic (e) globin the role of HOX1O as a candidate gene in human and murine retinopathies, we expression we linked a 3.7 kb fragment containing the entire human e have determined its precise chromosomal location in both species. To globin gene to a 2.5kb LAR cassette and analysed its stage specificity sublocalize HOX1O on human chromosome 14, a tritium-labelled HOX10 in transgenic mice. During human ontogeny the e globin gene is first probe was hybridized at high stringency to human metaphase chromosomes, expressed in primitive erythroid cells derived from the yolk sac blood and the distribution of silver grains analyzed over well G-banded metaphases islands and later silenced when definitive erythropoiesis begins in fetal at the 500-550 level of resolution. A clear peak of hybridization was liver. The LAR-e construct showed a similar developmental pattern observed at 14q24.3, placing HOX1O close to FOS, and proximal to arl- of expression; being expressed in yolk sac derived primitive erythroid antitrypsin. In addition, minor but significant hybridization was also seen at cells but not in fetal liver or bone marrow derived definitive erythroid 14q21, 9q33-34, and 1 lq22-23. Together with preliminary low-stringency cells of transgenic mice. The absence of e expression in definitive Southern blotting and cDNA cloning data, these findings suggest the presence erythroid cells suggest that the developmental regulation of the e of genes homologous to HOX10 elsewhere in the human genome, on globin gene is mediated by an autonomous negative control mechanism chromosomes where no homeobox genes have been previously mapped. The which cannot be overcome by the LAR. By deletion mutagenesis we murine homolog (Hox-10) was mapped to the distal part of mouse have identified the cis active element in the c globin gene that chromosome 12 by linkage analysis. TaqI-digested DNA from the testcross mediates this process. The autonomy of e globin stage specificity with CAST/Ei x MEV crossed to strain BXD-32 or SWR/J was scored for a 1.7 kb regard to the LAR distinguishes it from the y and 8 globin genes band seen in CAST/Ei. Hox-10 is strongly linked to 2 markers on distal which lose developmental stage specificity when linked individually to chromosome 12, a-l-antitrypsin (Aat) and an HMG CoA synthetase (Hmgs) the LAR. In this case stage specificity is restored in LAR variant, giving the apparent gene order and distances (cM) of Hmgs 19.5 + constructs containing both -y and i genes suggesting that fetal to adult 4.4 - Hox-10 - 12.2 + 3.5 - Aat, thus placing Hox-10 close to Fos. These globin gene switch is mediated by a reciprocal mechanism in which data document further the conserved syntenic regions between distal human the genes compete for the influence of the LAR. These results chr. 14q and mouse chr. 12, and provide a chromosomal focus for HOX10 suggest that at least two independent mechanisms, autonomous and linkage analysis in retinopathies. Northern blot analysis indicates that competitive, are involved in hemoglobin switching in Man. HOX1O expression may be retina-specific in the adult. In situ immunofluorescence is being used to determine which retinal cells express this protein, using an affinity-purified antiserum raised to a trpE fusion protein. Initial results show that staining is confined to the nuclei, and that all 7 retinal cell types appear to express the protein. We speculate either that HOXIO plays a general role in the differentiation of the retina, or alternatively, that the polyclonal antiserum is detecting related homeoproteins expressed in different retinal cell types.

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Maternal origin of familial aneuploidy for chromosome 21. Analysis of collagen in the "sex-reversed" (XX=) mouse. S. A. Gerwehr and J. R. Korenberg. Cedars-Sinai Medical JK. Griffin and S.R. Blecher. University of Guelph, Guelph, Center, University of CA, Los Angeles. Ontario, Canada. Recent york has shown that the parental origin of a gene or chromosome region can profoundly affect the phenotype of Sxr is a duplicated portion of the testis-determining offspring. To evaluate the possible contribution of this region of the mouse Y chromosome which is translocated to the phenomenon to the variability of the Down Syndrome phenotype X chromosome during meiotic cross-over. When an XSxr-bearing and of the monosomy 21 phenotype, the parental origin vas sperm fertilizes an ovum, the resulting chromosomal female investigated for a panel of cell lines derived from patients (XX~xr) develops as an apparent male. The "sex-reversed" carrying partial aneuploidy for chromosome 21. A total of 38 individual (pseudomale) shows a spectrum of abnormalities, informative cases with partial trisomy or partial monosomy of including small, aspermatogenic testes and abnormal chromosome 21 were examined. Of these, 10 were found to be of epididymides de novo occurrence, 27 were maternally and 1 was paternally Electron microscopic studies on the epididymis and testis derived. Previous work has shown largely preferential of the pseudomale mouse previously indicated an maternal transmission of aneuploid gametes in balanced ultrastructural abnormality in collagen. Instead of long, carriers of Robertsonian fusions. To assess whether the low organized fibrils there are thin, wispy structures which level of paternal transmission observed in our study was a appear to be immature or poorly assembled. This study examines more general property of balanced translocation carriers or the biochemical nature of the abnormality. more specific to chromosome 21 aneuploidy, the parent of Two approaches have been undertaken: hydroxyproline assay origin was investigated in 165 informative partially aneuploid and SDS-PAGE-Western blot analysis. Hydroxyproline, a post- cell lines maintained by the NIGMS mutant cell repository. The translationally modified amino acid unique to collagen, is analysis revealed that 133 (81%) of the cell lines' used to estimate collagen quantity. Preliminary data indicate aberrations were maternally derived, and similar to the that there appears to be significantly more hydroxyproline in chromosome 21, the results were consistent for partial trisomy the pseudomale testis (p<p>0.1) than in the normal organs. The the sex of the proband. Several possible mechanisms which hydroxyproline content of the epididymis is greater than that could account for these findings, including abnormalities of of the testis in both males and pseudomales. SDS-PAGE and meiotic pairing, abnormalities of spermatogenesis, decreased Western blots using anti-collagen IV antibodies seem to competitiveness of aneuploid sperm during fertilization, or support these observations. Studies examining whether the differential survival of embryos produced from aneuploid male hydroxyproline is free or bound in collagen are in progress. to gametes. Our results suggest a significant effect of These data suggest that = , previously thought chromosome 21 on these processes due to the high level of influence only sex-determination, may be involved somehow with maternal transmission of that chromosome in aneuploidies. collagen in not only the gonad but also in the epididymis, an Further, most of the de novo occurring partial aneuploidies accessory sex organ. This possibility is of interest because stem largely from intra-chromosomal events (deletions and of the ubiquity of collagen and its important role in insertions), while the familial cases derive mainly from development. inter-chromosomal events (translocations). Finally, the molecular determination of the parent of origin for the de novo aneuploidies may further elucidate the mechanisms of chromosomal mutation. Differentiation and Development Al 03 (0400) 1.215 (0401) 1.216

OURA:A new association of Qmphalocele and Effect of phenytoin and an inhibitor of epoxde hydrolase on Unilateral Sadial aplasia. B. D. Hall, Univ. of phalangeal ossification in the C57B146J mose fetus. UJ.K Kentucky, Lexington. Hartsfield. Jr.. J.G. Morel and L.B. Holeg. Univ. of South In 1987 Robinson et al (Proc Greenwood Genetic Florida Colleges of Medicine and Public Health, Taspa; Mass. Center 6:130, 87) reported 3 unrelated males with General Hospital arid Harvard Medical School, Boston. supraumbilical omphaloceles and unilateral left The teratogenicity of phenytoin (PHr) has been attributed upper limb deficiencies. No etiology was determined to the formation of the arene oxide metabolite. Irsed and all 3 had normal chromosomes. Two of the 3 had teratogenicity has been observed in rodents when the cardiac anomalies and 1 of the 3 had thoracic metabolism of the arene oxide by epaxide hydrolase has been vertebral defects. Doctor Dian Donnai (personal inhibited by trichlorrcene oxide (TCPO). We tested this communication) has identified a similarly affected hypothesis in C57BW6J mouse fetuses and evaluated the effect male who also had complex heart defects. I report of PrH alone versus TCPO + Pir on the ossification of the the 5th known case of this unusual association. forepaw in the cleared skeleton, an effect of Pir on the This is a male whose mother smoked 1 1/2 packs of souse which is analogous to an effect cbserved in RI-exposed tobacco a day and drank 6-7 cups caffeinated coffee humans. No interactive effect of PHr and TCPO was found which a day. He was appropriate for gestation for all raises doubt as to the relationship of the activity of epoxide prenatal growth parameters. He had a left absent hydrolase on the teratogenic effect of PHr on digit radius and thumb and a large supraumbilical ossification in the msose fetus. omphalocele. No other defects were found. He had The mice in this inbred strain were assigned rardcmly to successful surgical treatment of the omphalocele one of seven groups: Par (r=133), TCFO (rv=127), PEfr + TCPO and was discharged at 6 weeks. Follow-up at 9 (n=71), PSr + TCRO vehicle (ri=46), TCPO vehicle (r=100), sham months showed him doing well, developmentally, but control (rr125), and untreated controls (n=161). below the 3rd percentile for weight and length. PHEr was administe daily by gastric gavage (6QOg/jkg) Chromosome analysis was normal and family history prior to breeding and throughout pregnancy. The maternal serum negative for similar problems. levels of PHr were ararable to human therapeutic levels. Thus far, the OURA association has been very TCPO (100mgJkg) was administered on days 9, 10 and 11 of consistent. All 5 cases were sporadic, males, with gestation. Fetuses were recovered on day 18, stained with supraumbilical omphaloceles, and unilateral left alizarin red-S and cleared with 1% NOH to visualize directly upper limb defects. The only one with documented the ossification centers in the digits in the left forepaw. follow up is developing normally, but growth has Statistical analysis was performed using logistic regression, been poor. The lack of affected siblings, absence correcting for litter effect, where the et variable was independent of consanguinity, normal chromosomes, and no the absence or presence of ossification and the common teratogenic exposures leaves the etiology variables were the groups. There was no interaction (p=0. 51) undetermined. between PH1 and TCPO, although both groups had a lower proportion of centers ossified than the control group. This is consistent with the observations of Strickler et al. (Lasxet 2:746-749;1985) in humans that the deficiency of epoxide hydrolase activity that predisposes to Oenytoin astbrycpathy does not correlate with the presence of digit hypoplasia. Supported by NIH grants DE 07008-10, HD 15241 and DE 00243.

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Demethylation in CpG Islands During Fetal Development M. M. DEFECTIVE TYPE II COLLAGEN BIOSYNTHESIS IN HUMAN Holland. D. J. Driscoll. J. C. Robinson and B.R. Miseon. The Johns HYPOCHONDROGENESIS. Horton WA, Machado MA, Bartley J, Lee Hopkins University, Baltimore, Maryland B, Ramirez F. Department of Pediatrics, University of Texas Medical Little is known about how the DNA methylation patterns observed School, Houston; Loma Linda University; Mount Sinai Medical School, in adult tissues are established. Enzymes have been found with NY. maintenance methylation activity as well as de-novo methylation Hypochondrogenesis, a member of the spondyloepiphyseal activity, but enzymes that specifically demethylate DNA have not been dysplasia (SED) family of human chondrodysplasias, is characterized by identified. There is evidence to suggest that demethylating activity distinctive clinical and radiographic findings, structural abnormalities of exists during spermiogenesis in the mouse (Trasler et al. MCB 10:1828, growth plate cartilage and electrophoretic abnormalities of type I collagen. 1990), and in transgenes and virally transformed cells in culture. A single base mutation in COL2AI resulting in a gly->ser substitution at Observations of demethylation events in endogenous genes during fetal position 943 (exon 46) has been detected from analysis of genomic DNA in development have not been previously reported. CpG islands are one case. To better define the molecular pathogenesis of the condition, we clusters of CpG dinucleotides (most often found in the promoter investigated chondrogenesis in cultured chondrocytes from an affected regions of housekeeping genes) that are uniquely unmethylated in infant. mammalian genomes, except when on the inactive X (in eutherian Chondrocytes released from epiphyseal cartilage were allowed to mammals) where they have been shown to have a major role in dedifferentiate in monolayer culture and subsequently "redifferentiated" by maintaining the silence of the chromosome. The CpG island in the culture over agarose. Tissue-like structures were produced which HPRT locus on the murine inactive X becomes methylated de-novo resembled cartilage grossly and by light and electron microscopy. several days after X inactivation occurs (Lock et al, Cell 48:39, 1987). Compared to similarly prepared control cultures, the differentiated As these islands in active genes (autosomal as well as X-linked) are chondrocytes contained extensive dilated RER. The relative amount of unmethylated in human sperm and oocytes (Driscoll and Migeon, Som matrix produced by the mutant cells was less, the ratio of collagen fibrils to Cell Mol Genet 16:267, 1990), it seemed likely that they were never proteoglycan granules was less and the fibrils were thicker and irregularly methylated and that de-novo events affecting methylation status in the organized. These morphologic changes were identical to those seen in intact CpG islands would be limited to those on the inactive X. However, epiphyseal cartilage from the case. Electrophoretic analysis of 3H-proline our analysis of the methylation status of clustered CpGs in genes labeled collagens from the culture-derived structures revealed a "doublet" (PGK, G6PD, P3, HPRT) on the active X chromosome during fetal al(II) band and extensive proal(II) and incompletely processed proal(II) development reveals that specific demethylation events affecting sites chains. Analysis of COL2AI genomic DNA fragments prepared by EcoRI within the islands are occurring in the 6-8 week human fetus. We digestion and by PCR amplification of exons 44-52 was unremarkable and have observed unique methylation patterns present in many fetal COL2AI mRNA transcripts were normal size (5kb). A cDNA was reverse tissues at this time as well as subsequent changes which produce the transcribed from total cellular RNA and target sequences corresponding to adult patterns. Such changes within the same tissues indicate specific exons 38-50 of the COL2AI cDNA were amplified by PCR. The resultant sites have been demethylated. Our results suggest that the process of fragment was the expected size (1.5kb), and direct sequencing revealed no de-novo methylation, that occurs in the fetus, methylates at least abnormalities. These observations demonstrate defective biosynthesis of some sites within CpG islands in active genes, and that site-specific type II collagen and formation of abnormal cartilage collagen fibrils in demethylations occur subsequently. Further studies should reveal if hypochondrogenesis. They implicate a mutation in a different region of demethylation is attributable to a demethylase, or reflect changes in COL2AI from that reported previously in hypochondrogenesis or in other proteins binding at these sites which inhibit remethylation. SED cases and suggest that the phenotype may result from severely deficient or defective cartilage collagen fibrils regardless of the specific mutation. Al 04 Differentiation and Development (0404) 1.220 (0405)

CONFIRMATION OF A POLYMORPHISM IN THE 5' The neurofibranatosea paracrioathies ors UPSTREAM REGION OF THE LIVER ARGINASE GENE syrdrcss? B. G. Kiusseff. M.D. and W. Fro0m. Ph.D. Division ASSOCIATED WITH EXPRESSION IN RED BLOOD CELLS. D. of Medical Geneic, University of Scoth Florida, Taopa, Deartment of iman Genetica, Ulm University, Geramny. Klein. W.W. Grody. 1LM. Kernm A. Dodson, and S.D.Cederbaum. In 1931, Yakovlev and Guthrie coined the term Dept. of Psychiatry, UCLA School of Medicine, Los Angeles, CA. rar ae synrIrc (NCS) for Von ckln disease Persistence of arginase (Al) activity throughout the erythrocyte (NF-1) tuberus clerosis and Sture-Weber syrore. Since lineage has been demonstrated to occur generally in higher primates, then, NCS has been used for many chronic xorditicns with but not in lower mammels. Absence of red blood cell (RBC) arginase cutaneous and 0aS signs (Gomz, 1987). expression with attendant normal liver arginase activity has been shown In 1987, Busseff proposed a hypothesis that all pakcnatoses are pacrities. Acrdig to the to segregate as an autosomal co-dominant trait in wild and captive hypothesis a lifelorn dynamic predi ition to dysregulation populations of Macaca fascicularis. These data suggested that the of growth factors (GFs) caused by the genes of the Muruselian expression of arginase activity in RBCs is the result of a regulatory phkcmatoses arn by the unknown causes of the sporadic alteration. entities, leads to pre ard postnatal hamartc=a. Agirn, Southern analyses of the DNAs of many homozygous expressers and hormcnal and envirormantal influences play a role by non-expressers revealed a specific deletion within the 5' non-coding disnrptirn the precaricus innate grath equilibrium amors the GFs, their receptors, extracellular matrix and the region of the Al gene of non-expressing individuals. Using the respective cells/organs (Kousseff, 1990). Analysis of 285 polymerase chain reaction (PCR) with genomic DNAs as templates, prcpositi with 24 different E t_, 175 having NF, three reproducible amplification products of 685, 540, and 420 bp were su~orted the hypothesis. obtained from an expressing individual only. In contrast, PCR of the We used a radioiassay to determine the ta in-C genomic DNA from a non-expressing animal, using the same primers, (Sm-C) levels within NF-1 neurofibrcis arcd cpare then to was completely negative, suggesting a structural difference in this DNA the levels in the adjacent. "normal" skin of NF-1 patients. In all neurofibranas, there was at least two fold increase of region between the two populations. DNA sequence analysis of the Sm-C. This implicated increased synthesis/actmulation of largest PCR fragment demonstrated the presence of regulatory Sm-C as a pat etic factor for the hamartomatous consensus sequences, though bearing no overall resemblence in overgrowth. EM studies of melawmytes fr-ia "rmeal skin of sequence order to the homologous 5' region of the human AI gene. NF-1 patients arc frym their cafe au lait (CAL) macules ware A hexanucleotide core DNA sequence specific to genes expressed in cFmpared to controls; the melarncytes of the "nobrml" NF-1 several erythroid cell types resides at the appropriate position in this skin showed a five fold increase of melanin mcobluli. In CAL macules, the increase was eleven fold (1aufrann et al., 5' regulatory region. These data collectively support our original 1989). This was interped as "primirg", predisposition of hypothesis of a regulatory polymorphism in this species, and suggest cells of NF-1 patients to paracrine dymzwulation. Taus, that several sets of DNA elements control arginase transcription and laboratory data sPPOrt the thesis that NF ard the respond in a cell specific manner. hakaatlces are paracrinapathies. This in turn voids the current usage of NCS as a meanirgless term applied to different conditions sharing sane tanecs and nairologic signs and prepares its exodus frm dymnerdology, meical genetics and medicine in general.

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Detection of transcripts homologous to guy in the testis and Towards the Cloning of the Murine Y-Located Minor Histocompatibility epididymis of mice of XY and XX =r genotype. P. Hastrangelo. Gene (Hya) and Spermatogenesis Gene (Spy). S.R. Blecher and R.P. Erickson. University of Guelph, Guelph, M.J. Mitchell and C.E. Bishop. Univ. of Tennessee, Memphis, Dept. of Ontario, Canada and University of Michigan, Ann Arbor, Ob/Gyn, Molecular Genetics Laboratory. Michigan. In the mouse Hya was defined by skin graft studies in inbred mouse strains. Grafts from males were, rejected by females while grafts from (sex reversed) is a duplication of the mouse sex- females were accepted by males. In-vitro cell mediated assays have now determining region translocated to the pseudoautosomal region been developed, which are MHC restricted, using either cloned killer T- of the Y chromosome. It is transmitted to 50% of X chromatids cells or proliferative T helper cells and a human homoiogue (HYA) has by the obligatory crossover that occurs between the X and Y been defined. The Hya locus has been mapped to the Sxr region of the chromosomes during meiosis. Two homologues of uy (Zfy-l and mouse Y chromosome and the human HYA to Yp1 .2-Yqter. The Zfy-2) are present in the Sxr region. ZL. has been implicated precocious male specific expression and Y-linkage of Hya and HYA argues in sex determination. XX mice carrying I= on one X chromosome a role in male development. We have previously shown that an Sxr have an abnormal male phenotype and are called 'pseudomales". variant, Sxrb, that lacks expression of H-Y antigen (the product These pseudomales have small, abnormally coloured testes and controlled by Hya), was deleted for an Sxr locus defined by DNA probe lack the initial of the Interest in the segment epididymides. pY291. This locus was termed Sx 1. This mouse also had highly cause of these abnormalities in the XX= pseudomale have lead compromised spermatogenesis which mapped a spermatogenesis factor us to study the transcription of Zfy in the testes and (Spy) to the DNA deleted from the Sxr region. A further deleted locus epididymides of these mice. was defined by the gene Zfy-2, which Is one of the Y homologues of the Northern blots of normal and pseudomale testis and human zinc finger containing gene (ZFY). We have now cloned a third epididymis RNA probed with the Zty-2 genomic clone pDP1171 locus that is deleted from the Sxr region. This new region was detected (courtesy D. Page) has revealed homologous transcripts in using a subclone T5.1.1PH from a 17kb phage recombinant isolated with these organs in adult mice (at medium stringency). The our Sxr probe Y8. T5.1.1 PH defines four Sxr reglons, two of which are hybridization patterns differ between normal and pseudomale absent in Sxtb. One of those regions is the same as that defined by Zfy-2 organs and between testes and epididymides. but the other region Is novel. This region is termed T5. We have Reverse transcription PCR of Zfy-l has also amplified initiated *chromosome walking' in all these regions and have isolated fragments of the predicted size from prepubertal normal testis 270kb of DNA, which Is, therefore, the minimum size of the deletion. and epididymis RNA as well as pseudomale epididymis RNA. The DNA from the Zfy-2 locus is present in multiple copies (2-4 copies The basic paradigm of mammalian sex determination states within the Sxr region) while the DNA from Sxl and T5 is not duplicated that sex determining genes control testis determination but on the Y chromosome. We are currently testing the cloned DNA for not secondary sex organ differentiation. Our preliminary sequences that are conserved on the Y chromosome across species, as an finding of putative Zfy transcripts in the mouse epididymis indication of the presence of a gene. Our system detects the X and Y as well as the testis raises other possibilities. The presence homologues of Zfy-2 in human, horse, rabbit and pig. Zfy-2 has of Z&f in the aspermatic pseudomale argues against sperm being diverged considerably from these homologues and, therefore, if exonic the only source of Zy transcripts in testes (and sequences are present in our walk DNA, and they have no more diverged epididymides). Northern blot hybridization pattern differences than Zfy-2, we are confident that we will identify them. between normal and pseudomale testis and epididymis RNA may reflect the morphological differences previously described. Differentiation and Development Al 05 (0408) 1.223 (0409) 2.1

Genetics of Trophoblastic Disease: Differential effects of Homologous ribosomal protein genes on the human X and Y chromosomes Genomic Imprinting. P.A. Mowery. W. Leger. J. Davare. U. may underlay the haploinsufficiency in Turner Syndrome. D.C. Page. Surti. Department of Pathology, Magee-Womens Hospital, E.M.C. Fisher, P. Beer-Romero, A. Ridley. and LG. Brown. Whitehead University of Pittsburgh. Institute and Dept. of Biology, Massachusetts Institute of Technology, During 1972-1989 (18 years) 304 cases of complete Cambridge. hydatidiform moles (CHM) and 425 cases of partial 'Turner Syndrome" is a complex human phenotype with four hydatidiform moles (PHM) have been diagnosed at Magee-Womens cardinal components: 1) high embryonic mortality, 2) short stature, 3) Hospital, Pittsburgh. One hundred and ten complete moles gonadal dysgenesis, and 4) numerous specific anatomic abnormalities have been karyotyped 99 cases having 46,XX karyotype and including webbed neck, lymphedema of the dorsal surfaces of the 11 having 46,XY karyotype. Chromosomal heteromorphisms extremities at birth, and aortic coarctation. Turner Syndrome is one of and/or restriction fragment length polymorphisms in the the most common chromosomal (as opposed to Mendelian) disorders. molar tissue and the parental bloods were compared in order Though classically associated with a 45,X karyotype, the Turner to determine the parental origin in 41 complete moles and phenotype has been observed in some 46,XY females with deletlons of the all of them were of androgenetic origin. Penetrance of this sex-determining region of the Y chromosome. These observations led us imprinting is complete with all cases showing trophoblastic to pursue the hypothesis that Turner phenotype is the result of monosomy hyperplasia, cistern formation and no evidence of the for a gene or genes common to the X and Y chromosomes. By DNA- presence of the fetus. Two hundred and thirteen partial hybridizatlon-based deletion analysis of XY females with and without moles have been karyotyped, 181 of them being triploid Turner stigmata, we have mapped the 'Turner gene(s)' to a small portion (69,XXY, 69,XXX or 69,XYY). Similarly, parental origin of of the Y chromosome. The most likely location Is a 90-kb segment of the sixty-eight triploid PHM was determined. Origin of the distal short arm. Within this region we identified a gene that appears to extra set of chromosomes was found to be dispermy or encode one of the roughly 80 proteins which, together with four RNA consistent with dispermy. Morphology of these cases was species, comprise the ribosome. This Y-chromosomal gene, called much more gross variable, ranging from obvious molar changes RPS4Y, has several characteristics that one might predict for the Turner to very inconspicuous presence of rare cistern formation and gene. It has a homolog, RPS4X, on the X chromosome. As judged by trophoblastic inclusions. Morphology in some cases Northern both genes are ubiquitously transcribed in fetal overlapped with blotting, and the morphology found in double trisomies, adult tissues. Nucleotide sequence analysis of cDNAs suggests that the two trisomies, 45,X and normal karyotype. In addition several genes encode basic proteins that differ at only 19 of 263 amino acid cases with nonmolar triploids are being analyzed for the residues. The similarity of the predicted protein sequences to that of the origin of the extra haploid set of chromosomes. known rat RPS4 protein strongly suggests that the two human proteins function as RPS4 isoforms. As judged by transcriptional analysis of 1) human cell lines carrying 1, 2, or 4 X chromosomes and 2) human- rodent hybrids retaining 'inactive' human X chromosome, the human RPS4Xgene is not dosage compensated and is not subject to X inactivation. We hypothesize that much of the complex Turner phenotype is the result of having a single RPS4 gene. Having one rather than two RPS4 genes would likely reduce the number of ribosomes per cell, thereby reducing the cell's capacity for protein synthesis. Certain haploinsufficlencies in Drosophila (i.e., the Minute mutations) provide striking precedents for such dosage effects associated with ribosomal protein genes.

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Prenatal and genetic risk factors in neuronal migration dis- Skewed X-inactivation in normal monozygotic twin pairs. orders. A. Palmini, E. Andermann, F. Andermann. Montreal C.S. Richards. J.G. Hall. M.L. Falterman W.I. Nance. Neurological Hospital and Institute, Dept. of Neurology and GeneScreen, Dallas, Texas; Univ. British Columbia, Neurosurgery, McGill Univ., Montreal, Quebec, Canada. Vancouver; Medical College of Virginia. We reviewed the family and pregnancy histories of 45 Uneven Lyonization has been proposed as the underlying patients with the following neuronal migration disorders: mechanism for X-linked disease discordancy in some HZ female bi-rolandic symmetric macrogyria (8); asymmetric bilateral twin pairs (Richards *t al. Am J Hum Genet 46:672, 1990). macrogyria (2); generalized cortical dysplasia (3); hemi- Skewed X-inactivation in HZ female twins may be directly megalencephaly (3); megalencephaly (1); focal cortical dys- related to the twinning process. To test this hypothesis plasia (17) and forme fruste of tuberous (11). There was a DNA isolated from peripheral blood lymphocytes of 20 normal high incidence of prenatal risk factors in the pregnancies female twin pairs was examined for. methylation differences of the mothers, These included other congenital defects in using methylation-sensitive and methylation-insensitive the patients; anencephaly in a sibling; stillbirth in a twin; restriction enzymes and the the VNTR probe M27B located on elevated maternal age; viral infection of the mother in the Xp. The results demonstrate that 18/20 HZ female twin pairs first trimester; uterine pathology; and exposure to radiation, showed concordance for X-inactivation patterns, while drugs and trauma in early pregnancy. There was, however, also 2/20 twin pairs showed opposite (skewed) X-inactivation a high incidence of perinatal risk factors which might have patterns. Because 80% of HZ twin pairs are monochorionic, been misinterpreted as the main etiological factors. When their prenatal vascular anastomoses would lead to a brain imaging reveals architectonic abnormalities, detailed concordance and correlation in the X-inactivation profiles review of pregnancy and family histories may provide clues to of their red cells and/or lymphocytes, as has, in fact been the etiology of the dysplastic lesions. reported by Brewer et al. (Biochem Genet 1:41, 1967). However, the phenotypes of dichorionic (DC) HZ pairs would accurately reflect variation resulting from the original X- inactivation process in precursor cells along with any additional differences that might be attributable to the twinning process. While placentation and fetal membranes were not documented in this study, in both cases showing discordant inactivation phenotypes, the parents of the twins had been told at their daughters' birth that they were dizygotic, a mistake often seen with DC HZ pairs. Our results indicate that at least 10% of HZ female twins may show marked discordance in their X-inactivation profiles, and suggest that similar differences in other pairs may be obscured in the X-inactivation profiles of myeloid cells by prenatal placental anastomoses. Further observations on twins of known placentation will be required to confirm the contribution of the twinning process to skewed X- inactivation in HZ female twins and its relationship to the time at which twinning and X-inactivation occur. Al 06 Differentiation and Development/Gene Structure and Regulation (0412) 1.226 Developmental regulation of the human 4 globin gene in transgenic mice. E.A. Spangler. K.A. Andrews and E.M. Rubin. Lawrence Berkeley Laboratory, Berkeley, CA. Because the programs for human and murine a globin expression during development are essentially the same, the mouse should provide an appropriate model system in which to study the process of human a globin gene switching. We have characterized the expression of the human zeta (C) gene, which encodes an embryonic a-like globin, in transgenic mice. We find that a 777 base pair fragment spanning hypersensitive site II from the distal 5' region of the human p globin gene cluster potentiates expression of the C globin gene. In the absence of the HSII fragment, no C expression is observed. It has been reported that human fetal and adult p globin transgenes are not developmentally regulated in mice when the individual genes are linked to several of the major hypersensitive sites. We have examined the expression in transgenic mice of a human adult p globin gene linked to the HSII fragment. We find that this construct also lacks developmental regulation, and is expressed in both embryonic and adult murine hemopoetic tissues. In contrast, expression of the human C gene in mice parallels expression of a murine embryonic a-like globin gene (x), and appears to be limited to the primitive erythroid cells of the embryonic yolk sac. Our results indicate that the factors involved in a globin switching during development are evolutionarily conserved. We find that expression of the human C gene in mice requires linkage to an erythroid-specific enhancer sequence, but that the presence of the enhancer does not effect the developmental regulation of the transgene. Thus, the transgenic mouse is an in vivo system in which the requirements for the developmental switch in a globin gene expression can be analyzed in detail.

Gene Structure and Regulation (0413) 1.227 (0414) 1.228 Subtraction hybridization of control vs. Alzheimer's disease An Examination of Loci from Distal 4p for Genomic Imprinting via brain cDNA libraries. M.J. Alberts, P. Ioannou, J. Gilbert, Methylation that Might Contribute to Paternally Derived Juvenile H. Taylor. M. Mihovilovic. J. Lee. R.J. Bartlett. A.D. Onset in Huntington Disease. M.R. Altherr, S. Plum er, X, Roses. Duke University Medical Center, Durham, NC 27710. MacDonald and J.J. Wasmuth. Dept. Biol. Chem., Univ. Calif, Subtraction hybridization provides a technique for the Irvine. characterization and isolation of genes that are The juvenile onset of Huntington disease (HD) is often associated differentially expressed in various tissues. We have made with paternal transmission of the disease gene. Numerous human brain (temporal cortex) cDNA directionally cloned investigators have suggested that this bias in the mode of inheritance libraries from control and Alzheimer's disease (AD) patients in juvenile cases may be due to the differential "imprinting" of using lambda Gem 4. Asymmetric PCR of the amplified phage maternal and paternal alleles. Many of the arguments in favor of this DNA was used to produce complementary driver and tracer hypothesis are derived from the study of transgenes in the mouse. In strands. The driver strand was radiolabelled with HJ-GTP, an effort to determine whether differential methylation may be the tracer strand with P32-CTP, and the driver DNA was associated with imprinting in the vicinity of the UD gene we examined biotinylated for removal post-hybridization. Approximately the methylation state of six distinct loci that span the terminal 4 Mbp 40 ug. of driver was hybridized against 1 ug. of tracer for of chromosome 4p in 2-3 pedigrees per locus where the parental 24 hours at 420C in the presence of 50% formamide. Sub- origin of the alleles could be unequivocally assigned in at least 2 traction was performed using streptavidin precipitation. offspring. Two of the loci used in these experiments D4S95 and Analysis of counts post-subtraction-hybridization showed D4S98, have recently been shown to exhibit significant linkage >99% success for removal of driver and 75% removal of disequillibrium with the HD gene. Both HD and non-HD pedigrees tracer. Asymmetric PCR was used to amplify and clone the were employed in these analyses. The DNA derived from subtracted tracer DNA. A subtracted library of approxim- lymphoblasts of parents and offspring was digested with enzymes that ately 1 million clones (>80% with viable inserts) was reveal the polymorphism and subsequently, in separate reactions, with constructed. Most of the unsubtracted trace DNA was due to the methylation sensitive enzyme HpaII and its methylation rare species that were bidirectionally amplified during the insensitive isoschizomer Mspl. If the alleles of the offspring are asymmetric PCR due to internal priming sites. Differential differentially methylated depending on parental origin, i.e. imprinted, colony hybridization of 150,000 clones has Identified 12 than the prediction is that the offspring would reveal a consistent clones that appear to be subtracted. One clone, MA/N2, is difference in allelic methylation patterns, as determined by approximately 600 bp and Identifies a 13 kB mRNA that is Hpall/MspI digestion. Although, overall, the terminal 4 Mbp of 4p greatly diminished in temporal cortex from AD vs. control appears to be hypermethylated, we failed to observe any consistent brains (n=6). This clone appears to map to the peri- hereditable difference that would be consistent with differential centromeric region of chromosome 19. The sequence of MA/N2 methylation of maternal vs. paternal alleles. While this result does does not match any known gene in Genebank. This technique not rule out imprinting as a contributing factor in juvenile onset HD it is useful for identifying potential candidate genes in AD. does suggest that differential methylation may not be involved. Alternatively, it is possible that the region of imprinting is small, possibly including only the HD gene itself, and therefore not detected in this analysis; that only specific cell lineages (i.e. the neurons affected in HD) are imprinted; or that another unknown mechanism unrelated to DNA methylation is responsible for imprinting in this region.