Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5499-5503, September 1984 Genetics

Failure to demonstrate mutations affecting protein structure or function in children with congenital defects or born prematurely (electrophoretic mutations/genetic monitoring/etiology of congenital defect) JAMES V. NEEL AND HARVEY W. MOHRENWEISER Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI 48109 Contributed by James V. Neel, April 16, 1984

ABSTRACT An effort has been made to confirm the re- responsible for these 12 proteins on the basis of mutability, port [Dubinin, N. P. & Altukhov, Y. P. (1979) Proc. Natl. these loci were presumably neither more nor less mutable in Acad. Sci. USA 76, 5226-5229] that children born prematurely this setting than the loci responsible for the thousands of oth- or exhibiting congenital defects can be shown to exhibit rela- er proteins that make up the human body. To a first approxi- tively high frequencies of rare (nonpolymorphic) electropho- mation it follows that, if the battery of proteins studied could retic variants of proteins and that a large proportion of these be expanded to several thousand, then each defective child variants are due to mutation in either the father or the mother. would harbor several such mutations. Normal children, on In a series of 178 children who were comparable with those the face of these findings, would be very much less likely to described in the earlier report, we failed to encounter a high harbor such mutations. It would be difficult to escape the frequency of these variants in some 5341 determinations in- inference that mutations of this type played a primary role in volving 45 proteins, nor were any mutations observed. Data the genesis of congenital defect and/or prematurity, an infer- from 1583 determinations of enzyme activity on a subset of the ence with no less startling implications for human develop- panel of proteins were also unremarkable. We are thus unable ment and teratogenesis than for the subject of genetic moni- to confirm the earlier report. toring. Inasmuch as there was already in place in our labora- tory a program examining consecutive newborn infants for In 1979, following several similar reports in Russian lan- the occurrence of mutations influencing electrophoretic mo- guage journals, Dubinin and Altukhov (1) published in these bility or enzyme activity (2, 3), we deemed it worthwhile, as PROCEEDINGS a communication reporting a high frequency an extension of this study, to attempt to repeat the above- of mutations affecting proteins among a selected infant pop- described observations. ulatioh. The abstract reads as follows: PROTOCOL Twelve proteins of enzymic and nonenzymic nature in The subjects of this study were drawn from three sources. blood samples of infants that deviate from the average popu- The first source (the Pediatric Referral Series) was a group lation in physical development (50 premature and 177 full- of children referred to the University of Michigan Medical term infants with rough and multiple developmental defects) Center for diagnostic studies or surgery because of serious were studied by electrophoresis in polyacrylamide and starch to inherited gels. The control group consisted of 500 normal newborns. In congenital defects not corresponding any simply infants with developmental disorders, the frequency of rare genetic entity. The necessary blood samples from the chil- electrophoretic protein variants was found to be about one dren were obtained in connection with other diagnostic stud- order of magnitude higher than in the control. It has been ies. Samples were also routinely obtained from both parents shown for at least five cases that such variants are de novo at the time of the child's hospitalization, for reference in the mutations. According to these data the mutation rate is -2 x event an unusual finding was encountered in a child. All 10 3 per locus per generation for the group selected and -6 x samples were obtained with informed consent. 10-5 for the total population. Despite the fact that further The second source of samples (the Cord Blood Series) was specification of the estimations found is required, we consid- from the study of the occurrence of biochemical variants in er the results obtained as evidence in favor of the efficiency infants mentioned of the earlier substantiated monitoring model of gene muta- blood samples from consecutive newborn tions in the human population.. . This approach, which in- above. A description of this program will be found in refs. 2 fers electrophoretic screening of blood proteins in a specially and 4. Because the Obstetrical Service of the University of selected group of newborns, makes it possible to reduce the Michigan Medical Center is especially oriented towards the size of samples needed for statistically reliable estimations of needs of the high-risk pregnancy, a disproportional number the alteration of mutation rate. of the children delivered on this service are either premature (here defined as a birth weight less than 2000 g) or at risk of a The far-reaching implications ofthis report for the study of congenital malformation. In this program, for all deliveries mutation are clear. It implies, inter alia, as the authors point- for which informed consent can be obtained, venous blood ed out, that a search for biochemical mutations among the samples are obtained from both parents and a placental sam- predominantly normal children born to a cohort subjected to ple is obtained from the umbilical cord of the child after the some mutagenic exposure, such as the survivors ofthe atom- cord has been severed. The subset of children included in ic bombings, is a largely misplaced effort. In addition, there this program who were premature or exhibit significant con- is a second, no less far reaching, implication, not stressed by genital defects are the second source of material for the pres- the authors. Assume the report is correct, that there was a ent study. The deliveries of all premature infants included parental mutation rate of approximately 2 x 10-3 at the loci in the series were initiated spontaneously, although in some responsible for the 12 proteins sampled in a group of 227 instances the labor was for obstetrical reasons terminated by defective infants. Since there was no preselection of the loci caesarian section. Twins and triplets were not included in the premature series, given that low birth weight is a normal of the ace- The publication costs of this article were defrayed in part by page charge concomitant of such births. Congenital dysplasia payment. This article must therefore be hereby marked "advertisement" tabulum and talipes as isolated defects are not included as in accordance with 18 U.S.C. §1734 solely to indicate this fact. diagnoses in the newborn series because of the wide spec- 5499 Downloaded by guest on September 27, 2021 5500 Genetics: Neel and Mohrenweiser Proc. NatL Acad Sd USA 81 (1984)

Table 1. Electrophoretic variants detected in the Study Series and the Control Series Study Series Control Series No. of No. of Determi- nonpolymorphic Determi- nonpolymorphic Protein Symbol EC no. nations variants nations variants Albumin Alb 132 0 2,827 2 Ceruloplasmin CRPL 138 3 3,063 26 Haptoglobin HP 88 0 Transferrin TF 138 3 3,063 66 Acid phosphatase ACP1 3.1.3.2 169 0 3,162 6 Adenosine deaminase ADA 3.5.4.4 169 0 3,141 0 Adenylate kinase 1 AK1 2.7.4.3 169 0 3,132 3 Carbonic anhydrase I CAl 4.2.1.1 101 0 789 2 Carbonic anhydrase II CA2 4.2.1.1 111 0 1.036 1 Esterase A ESA 3.1.1.1 169 0 3,153 5 Esterase B ESB 3.1.1.1 145 0 1,804 0 Esterase D ESD 3.1.1.1 168 0 3,162 0 Galactose-1-phosphate uridlyltransferase GALT 2.7.7.10 157 0 3,097 1 Glucosephosphate isomerase GPI 5.3.1.9 167 1 3,141 13 Hemoglobin a HGBa 114 0 3,141 4 Hemoglobin f8 HGB/3 62 0 3,141 0 Hemoglobin 8 14GB8 62 0 Hemoglobin y HGBy 52 0 3,141 4 Isocitrate dehydrogenase IDH1 1.1.1.42 154 0 3,095 7 Lactate dehydrogenase A LDHA 1.1.1.27 169 1 3,141 6 Lactate dehydrogenase B LDHB 1.1.1.27 169 0 3,141 2 Malate dehydrogenase MDH 1.1.1.37 168 1 3,141 0 Nucleoside phosphorylase NP 2.4.2.1 168 0 3,155 3 Peptidase A PEPA 3.4.11.- 169 2 3,156 7 Peptidase B PEPB 3.4.11.- 169 0 3,162 7 Peptidase C PEPC 3.4.11.- 166 0 2,938 8 Peptidase D PEPD 3.4.13.9 165 1 2,942 11 Phosphoglucomutase 1 PGM1 2.7.5.1 169 0 3,162 3 Phosphoglucomutase 2 PGM2 2.4.5.1 169 0 3,162 6 Phosphogluconate dehydrogenase PGD 1.1.1.44 166 0 3,140 1 Triosephosphate isomerase TPI 5.3.1.1 166 0 3,141 2 Glucose-6-phosphate dehydrogenase G6PD 1.1.1.49 124 1 2,538 5 Glutamic-oxaloacetic transaminase (soluble) GOT1 2.6.1.1 168 2 3,159 16 Hexokinase 1 HK1 2.7.1.1 168 0 3,064 2 Hexokinase 2 HK2 2.7.1.1 168 0 3,141 0 Phosphoglycerate kinase PGK 2.7.2.3 34 0 2,095 0 Uroporphyrinogen I synthase UPS 4.3.1.8 35 0 2,076 7 Aldehyde dehydrogenase ALDHR 1.2.1.3 19 0 1,190 0 Enolase ENO1 4.2.1.11 19 0 1,192 0 Fumarase FUMi 4.2.1.2 13 0 919 1 Glutamic-pyruvic transaminase GPT1 2.6.1.2 19 0 1,192 S Inosine triphosphatase ITPASE 3.6.1.19 17 0 1,191 0 Inorganic pyrophosphatase PPASE 3.6.1.1 19 0 796 0 Apolipoprotein APOLIP 13 0 978 4 Glyoxalase GLO 4.4.1.5 47 0 963 0 5341 15 108,963 236

trum in severity and uncertainty of diagnosis in the newborn. series. Likewise, children in the first two series found in the Our original protocol was designed to exclude malformed course of their diagnostic work-up to have karyotypic abnor- children found to have karyotypic abnormalities, on the mality are presented with the children in the Karyotypic Se- grounds that a sufficient cause for their defect has been es- ries. These three groups form the Study Series of Table 3. tablished. When, however, an abstract appeared in 1982 (5) The control values for this study are based on studies of the that we interpreted as reporting that 38% of the children in normal infants in the previously described Cord Blood Se- the Russian series had demonstrable karyotypic abnormali- ries. These control frequencies were obtained concurrently ties (predominantly trisomy 21), a third group of children with the determinations on the children with various defects. (the Karyotypic Series) was added to the protocol. These The Russian investigators have never described in any de- were all children with a variety of physical abnormalities re- tail the composition of their sample, but we have the impres- ferred to the Pediatric Cytogenetic Unit for diagnostic stud- sion that the severity of the defect in the children was such ies. Those children among these referrals who were not that they required institutionalization. In an effort to obtain a found to have chromosomal abnormalities but who did have sample of children with a high degree of handicap, we have major physical abnormalities have been combined for pre- made a particular effort in our Congenital Defect Series to sentation with the defective children making up the first two obtain samples from children with major cardiac or central Downloaded by guest on September 27, 2021 Genetics: Neel and Mohrenweiser Proc. Natl. Acad Sci USA 81 (1984) 5501

Table 2. Enzyme deficiency variants detected in the Study Series and the Control Series Study Series Control Series No. of No. of rare Enzyme Symbol n variants* n variants Adenylate kinase AK 148 0 1,224 2 Diphosphoglyceromutase DPGM 107 0 445 0 Enolase ENO 107 0 440 1 Glyceraldehyde-3-phosphate dehydrogenase GAPD 93 0 443 0 Glutamic-oxaloacetic transaminase GOT 92 0 1,033 1 Glucose-6-phosphate dehydrogenase G6PD 148 0 (1) 1,233 3 Glucosephosphate isomerase GPI 148 0 1,234 2 Lactate dehydrogenase LDH 148 0 1,266 0 Malate dehydrogenase MDH 148 0 1,265 2 Phosphoglycerate kinase PGK 148 0 1,265 0 Pyruvate kinase PK 148 0 1,254 1 Triosephosphate isomerase TPI 148 0 (1) 1,194 5 1583 0 (2) 12,2% 17 *Numbers in parentheses indicate enzyme deficiency variants occurring in polymorphic frequency.

nervous system abnormalities and also, as noted above, add- duction of new systems into the laboratory battery over the 3 ed a group of children with karyotypic abnormalities. years that the data have been accumulated, as well as the fact that some proteins cannot be typed with accuracy in LABORATORY PROCEDURES newborn infants (e.g., haptoglobin and carbonic anhydrases Table 1 lists the plasma and erythrocyte proteins examined I and II). In a total of 5341 determinations in the Study Se- for electrophoretic variants, and Table 2 lists the subset of ries, there were 15 classified as exhibiting heterozygosity for these proteins that, as enzymes, were examined for activity a rare variant. All of these variants were present in one par- levels. For the purposes of these studies, heterozygosity for ent or the other and so were presumably inherited. an enzyme deficiency variant is defined by an activity level The findings with reference to activity variants are sum- -66% of normal. The rationale for this definition has been marized in Table 2. There were in the Study Series only two developed in detail elsewhere (6, 7). Variants detected by determinations characterized by approximately half-normal this criterion will for the most part be the result of alleles levels, among a total of 1583 determinations. These variants, whose gene product (if any) has no enzymatic activity (7, 8) oftriosephosphate isomerase and glucose-6-phosphate dehy- or is very unstable. Descriptions of the techniques employed drogenase were observed in the same Black child. As has in this laboratory for the detection of both kinds of variants been previously described (11), these variants occur as poly- will be found in publications of Neel and co-workers (4, 9), morphisms among the American Black populations (al- Fielek and Mohrenweiser (10), and Mohrenweiser and Fie- though they do not attain polymorphic proportions in this lek (11). ethnically admixed series). The triosephosphate isomerase Whenever a variant other than one corresponding to a variant was inherited from the father and the glucose-6-phos- common genetic polymorphism was encountered, the sam- phate dehydrogenase from the mother. ples obtained from the father and mother were examined, to determine whether the variant was inherited or had arisen DISCUSSION within the last generation in consequence of a mutation. For Dubinin and Altukhov (1) reported that rare (i.e., ndnpoly- convenience we term such variants "rare" variants. Failure morphic) electrophoretic variants were encountered in 5 of to do family studies when a common, polymorphic variant is 5085 determinations (10,170 locus tests) in their control-se- encountered carries the risk of missing some mutations that ries, from which they calculate a frequency of 0.00031 ± mimic common polymorphisms, but we have shown that this 0.00014 per locus per individual, and in 15 of 2357 determina- is a minor source of bias (9). The protocol allowed for high- tions (4714 locus tests) involving children with congenital de- resolution cytogenetic studies of any child exhibiting an ap- fects/prematurity, from which they calculate an average fre- parent mutation but, as we shall see, the need for such stud- quency of 0.00203 ± 0.00052 per locus tested. (We cannot ies did not arise. reproduce this calculation.) In the present series of control children, 236 rare variants were encountered in 108,963 de- FINDINGS terminations (215,609 locus tests), a frequency of 0.00109 + Table 3 lists the types of children included in the study. All 0.00007 per locus tested (ref. 4 and unpublished data). In the children with karyotypic abnormalities exhibited major phe- series of children who were premature or were found to have notypic findings, and they could be considered as a subset of major congenital defects, rare electrophoretic variants were children with congenital defects. On the other hand, the pre- encountered in 15 of 5341 determinations (10,603 locus mature infants did not exhibit gross defects and, in fact, for tests), a frequency of0.00141 per locus tested, with 95% con- the most part were ultimately discharged from the hospital as fidence intervals of 0.00079 and 0.00233. We are thus unable normal healthy infants. Most of these children have under- to confirm the finding of an excess of rare variants in chil- gone extensive medical studies. Of the total of 178 children, dren with severe congenital defects and/or prematurity. 31 entered the series because of prematurity, 27 because of We have also failed to detect any mutations resulting in an chromosomal abnormality associated with physical defects, electrophoretic variant in the aforementioned material, nor and 120 because of a variety of congenital defects not known have we encountered any mutations resulting in a loss of en- to be associated with a chromosomal abnormality. zyme activity in this series. By contrast, the Russian investi- The electrophoretic findings in both the Study Series and gators reported that in all five instances in which they were the Control Series are summarized in Table 1. Differing able to do family studies on the parents of a defective child numbers of determinations per protein result from the intro- exhibiting a variant (among 14 such children), in no case did Downloaded by guest on September 27, 2021 5502 Genetics: Neel and Mohrenweiser Proc. NatL Acad ScL USA 81 (1984) Table 3. Defects that formed the basis for this study Category Total System n Diagnosis Prematurity 31 Congenital defect without known 120 Cardiovascular 12 Transposition of great vessels with/without other defects chromosomal defect 7 Tetralogy of Fallot with/without other defects 9 Ventricular septal defect 3 Ventricular septal defect, pulmonary valve stenosis (atresia) 3 Aortic valvular stenosis 3 Total anomalous pulmonary venous return 2 Double-chambered right ventricle with ventricular septal defect 5 Atrial septal defect 11 Miscellaneous cardiovascular defects 55 Gastrointestinal 4 Imperforate anus 4 Atresia/stenosis of intestine (ileal, jejunal, rectal, or anal canal) 3 Cleft lip and/or palate 2 Hirschsprung disease 1 Biliary atresia 14 Central nervous system 11 Myelomeningocoele (myelodysplasia) with/without hydrocephalus 4 Hydrocephalus 2 Encephalocoele 1 Optic atrophy with other central nervous system defects 18 Genitourinary 1 Cystic kidney disease 1 Hypoplastic kidneys 1 Severe hypospadias and epispadias 3 Multiple complex defects 6 Musculoskeletal 3 Gastroschisis 1 Omphalocele 1 Sternal fusion defect 1 Polydactyly and syndactyly, severe (fingers and toes) 1 Anomalies of upper and lower limbs, shoulder, and pelvic girdles 1 Jarcho-Levin syndrome and spondylothoracic dysplasia 8 Multisystem defects* 19

Congenital defect with known 27 Sex chromosome aneuploids chromosomal defect 3 Turner syndrome 1 Klinefelter syndrome Autosomal trisomy (partial or complete) 13 Down syndrome 3 Other 3 Autosomal deletion syndromes 3 Translocation syndromes 1 Monosomy 21, 15p+ *These defects were as follows: (1) two myelomeningocoele with renal defect; (2) clinodactyly, syndactyly, ptosis, brittle hair, mild mental retardation, and unusual facies; (3) congenital glomerulonephritis, multiple epiphyseal dysplasia, and congenital deformed ear; (4) tetralogy of Fallot and atresia and stenosis of small intestine; (5) atrial and ventricuiar septal defect, pulmonic valvular stenosis, cleft palate, and Wilms tumor; (6) imperforate anus and absent right auditory canal; (7) spina bifida, aortic stenosis, and severe scoliosis; (8) absent left kidney and Hirschsprung disease; (9) rectovaginal fistula, sacral and vertebral anomalies, and bilateral pes equinovarus; (10) congenital mitral regurgita- tion and malrotation of bowel with volvulus; (11) holoprosencephaly, hypoteleorism, hypoplastic nose, multiple facial skin tags, low-set ears, choanal atresia, ventricular septal defect, anomalous venous return to heart, and left renal agenesis; (12) interventricular septal defect, micrognathia, pes equinovarus, and undescended testes; (13) first and second branchial arch syndrome, brachycephaly, plagiocephaly, and micrognathia; (14) gastroschisis and jejunal atresia; (15) double aortic arch, multiple abnormalities of vertebral and thoracic skeleton, and bilateral inguinal hernia; (16) congenital aortic regurgitation and Wilms tumor; (17) right porencephalic cyst, bilateral choreoretinal and optic nerve colobomas, hypoplastic thoracic vertebrae, and hemivertebra; (18) interventricular septal defect, abnormal sacral vertebrae, clinodacty- ly, dysplastic nails, congenital dislocation of hip, and failure to thrive. either the father or the mother exhibit a similar variant. Lim- lation. If we accept the authors' estimate of =2 x 10- per ited "paternity exclusion" studies failed to reveal any evi- locus per generation, then the expectation in our series dence of a discrepancy between legal and biological parent- would be for more than 20 variants arising from mutation to age, so that the variants were presumed to result from a mu- have been detected. Clearly there is an enormous discrepan- tational event in one or the other parent. It is difficult to cy. extract a mutation rate from these data for this general popu- It should be pointed out that even had the report proven Downloaded by guest on September 27, 2021 Genetics: Neel and Mohrenweiser Proc. Nadl. Acad Sci USA 81 (1984) 5503 correct, this demonstration would not have simplified genet- K., Kageoka, T., Fujita, M., Neriishi, S. & Asakawa, J. (1980) ic monitoring to the extent implied by the authors. Numera- Proc. Nati. Acad. Sci. USA 77, 4221-4225. tors without denominators are of little value in genetic epide- 4. Neel, J. V., Mohrenweiser, H. W. & Meisler, M. M. (1980) miology. It would still be necessary to define the absolute Proc. Nati. Acad. Sci. USA 77, 6037-6041. frequency of defective children in a carefully defined study 5. Suskov, I. I. (1982) Mutat. Res. 97, 225-226. 6. Mohrenweiser, H. W. (1981) Proc. Natl. Acad. Sci. USA 78, population, and, indeed, given the cause-and-effect relation- 5046-5050. ship of biochemical mutation and congenital defect implied 7. Mohrenweiser, H. W. (1983) Isozymes: Curr. Top. Biol. Med. by the Russian studies, if the finding were correct then in Res. 10, 51-68. most settings it would be more efficient simply to monitor 8. Mohrenweiser, H. W. (1983) in Utilization ofMammalian Spe- the frequency of defective children. cific Locus Studies in Hazard Evaluation and Estimation of Genetic Risk, eds. de Serres, F. J. & Sheridan, W. (Plenum, The technical assistance of P. T. Wade and K. H. Wurzinger is New York), pp. 55-69. gratefully acknowledged, as is the role played by Dr. W. J. Oliver in 9. Neel, J. V., Mohrenweiser, H. W., Hanash, S., Rosenblum, facilitating the collection of the samples. This study was supported B., Sternberg, S., Wurzinger, K., Rothman, E., Satoh, C., by the National Institute of Environmental Health Sciences. Goriki, K., Krasteff, T., Long, M., Skolnick, M. & Krzesicki, R. (1983) in Utilization ofMammalian Specific Locus Studies 1. Dubinin, N. P. & Altukhov, Y. P. (1979) Proc. Natl. Acad. in Hazard Evaluation and Estimation ofGenetic Risk, eds. de Sci. USA 76, 5226-5229. Serres, F. J. & Sheridan, W. (Plenum, New York), pp. 71-93. 2. Neel, J. V., Mohrenweiser, H., Satoh, C. & Hamilton, H. B. 10. Fielek, S. & Mohrenweiser, H. W. (1979) Clin. Chem. (Win- (1979) in Genetic Damage in Man Caused by Environmental ston-Salem, NC) 25, 384-388. Agents, ed. Berg, K. (Academic, New York), pp. 29-47. 11. Mohrenweiser, H. W. & Fielek, S. (1982) Pediatr. Res. 16, 3. Neel, J. V., Satoh, C., Hamilton, H. B., Otake, M., Goriki, 960-963. Downloaded by guest on September 27, 2021