Cytogenetics and Genetics

Cytogenetics and Genetics PHILIP L. TOWNES, M.D., Ph.D.

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MORTON S. ADAMS, M.D. n

Human genetics has in recent years become a major discipline in clinical medicine. New developments continue to be reported at a rate which defies description and a comprehensive review of the subject is clearly impossible. Discussion in this review is confined to the current status of several clinically important areas that are presently being intensively studied. HUMAN CYTOGENETICS

A decade of unprecedented progress in human cytogenetics has elapsed since the discovery that mongolism is caused by chromosomal trisomy.83 Further examples of chromosomal aneuploidy rapidly followed the discovery of trisomy 21,46. 52. 84. 106. 124 but the anticipated array of possibly 22 distinct autosomal trisomy syndromes (and an equal number of monosomies) has not materialized. Presumably the undocumented aberrations are not viable. 29 . 34. 128. 129. 139 In recent years, cytogenetic investigations have been devoted to the detailed delineation of the human karyotype,33 to problems of chromosome replication,138. 144. 145 and to more complex variations (translocation, deletion, double aneuploidy, mosaicism) of the various aneuploid syndromes, their incidence, etiology and prognosis. 2. 37. 38. 42. 48. 96. 123. 140. 143 Chromosome Marking Developments in human cytogenetics at first appeared to afford the opportunity to correlate structural and biochemical abnormalities with From the Division of Genetics, Departments of Anatomy and Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, New York "Professor of Anatomy (Genetics), Chairman, Division of Genetics, and Assistant Professor of Pediatrics; Associate Pediatrician, Strong Memorial Hospital (>" Assistant Professor of Anatomy (Genetics) and Senior Instructor in Pediatrics; Assistant Pediatrician, Strong Memorial Hospital Supported by U.S. Public Health Service Grants-in-Aid AM-09247 and GRSGFR-05403. 493 Pediatric Clinics of North America-Vol. 15, No.2, May, 1968

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specific chromosome defects. The limited success in this endeavor may be largely attributed to the considerable similarity of phenotype that occurs in the major trisomy syndromes. The diverse constellation of anomalies observed in the Dl (13-15), E (17-18) and 21 trisomy syndromes 96 , 123 are not sufficiently specific to permit chromosome marking. The defects appear to reflect gross genic imbalance at many loci, and almost any defect observed in one trisomy may occur in others. Chromosome marking has been attempted through studies of deletions,63, 101 by comparing defects in patients with partial trisomy with those observed in complete trisomy. DeGrouchy63 has attempted to map chromosome 18 from deletion data, but the overlap observed makes this exceedingly difficult. Similar difficulties have been encountered in attempts to assign specific loci to trisomic chromosomes by measurement of enzyme levels. The assumption that a trisomic locus may result in increased levels of enzyme and thus permit detection has not yet been confirmed. The critical increment in enzyme activity is difficult to detect because of the considerable variation in normal levels. Brandt22-24 reported increased levels of galactose-I-phosphate uridyl transferase in trisomy 21, and others have reported increased levels of leukocyte alkaline phosphatase,4 erythrocyte phosphohexokinase 5 and other enzymes in this disorder and inferred that the locus is on chromosome 21. 10 , 44, 70, 80, 100, 115 This conclusion has not been substantiated, since glucose-6-phosphate dehydrogenase, which is known to be on the X chromosome, is also elevated. 94 Thus the enzyme elevations observed in trisomy 21 probably reflect a generalized derangement of leukocyte metabolism in this disorder.95 The difficulties encountered in enzyme studies are largely circumvented in the examination of structural proteins. In this regard, Gerald and colleagues 14, 57 have suggested that the structural alpha chain haptoglobin locus is on chromosome 13, based upon the anomalous inheritance of two individuals with a ring D (13) chromosome. The two propositi were found to have inherited only one alpha chain locus. Because the ring represents a deletion on both ends of the chromosome, the authors were not able to determine whether the locus was on the long or the short arm. In a simultaneous publication, BIas and Migeon12 reported no abnormality of haptoglobin inheritance in a family with deletion of the short arm of chromosome 13. The prior suggestion by Hustinx 72 that the haptoglobin locus is on the short arm of a D chromosome translocation carrier has been questioned by BIas and Migeon12 and by BIoom et al. 14 It would appear that the haptoglobin locus is on the long arm of chromosome 13. If these observations are confirmed, they represent the first successful assignment of an autosomal locus in man. Chromosome Breakage Although the major chromosome abnormalities have been well established, the biologic significance of chromosome breakage is less

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evident. Random breaks are regularly observed in routine preparations of human chromosomes and doubtless represent artifacts of cell culture preparation. The breaks may be of several kinds and are present in all preparations to some degree, making it difficult to assess their significance. Differences in cytologic technique between laboratories in the definition and recognition of these subtle abnormalities make comparison of data nearly impossible. Unfortunately, what may be considered a "significantly increased" frequency of breaks in one laboratory may be well within the normal range in another laboratory. Increased frequencies of breaks have been demonstrated in Bloom's syndrome and in Fanconi's constitutional aplastic anemia. 13 , 15, 58, 116, 120, 127 Congenital malformations and a predilection for malignant neoplasm are features of both conditions. The causal relationship between chromosome breakage, neoplasia and teratogenesis in these conditions is presently not understood. Increased frequencies of chromosome breaks have also been associated with viral infection, immunization, x-ray exposure and chemical agents. 28 , 102, 104, 118 Rubella virus, for example, has been clearly associated with chromosome breaks in cell culture and in natural infection by some investigators, although others have been unable to confirm the association. 102 , 104 This lack of agreement has been attributed to possible differences in the age of subjects or the virulence of the virus or the possible coexistence of other unknown viral agents. These speculations underscore the ambiguities and deficiencies in our understanding of chromosome breakage in man. Cohen and co-workers 36 have recently reported an increased frequency of chromosome breaks in leukocyte cultures to which lysergic acid diethylamide (LSD) had been added and in leukocytes from one patient who had been treated with LSD (see also Irwin and Egozcue74 ). However, Loughman and colleagues 87 considered the frequency of breaks in eight LSD users to be no greater than in controls. The limited and contradictory data presently available do not permit any assessment of the possibly deleterious effect of LSD on the chromosomes of man or his progeny. Screening and evaluation of some of our more accepted therapeutic agents would seem a worthy project, since there is little reason to suspect any correlation between the social acceptability of a drug and its potential for chromosome breakage. Chromosomal Monosomy Although monosomy of a complete chromosome has been considered to be lethal, there have been a number of reports of partial monosomy (deletion of a chromosome segment). The first example was reported by Lejeune et al. in the cri-du-chat syndrome. 84 This syndrome has since been confirmed by a number of observers. It is caused by a deletion of a short arm of chromosome 5. Deletions in the short arm of chromosome 4 have also been reported by several workers, but the clinical abnormali-

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ties noted do not yet appear to represent a specific syndrome. 105 Ockey and co-workers 105 have recently reported deletion of the long arm of chromosome 4 in a child with limb and other abnormalities. Apart from the cri-du-chat syndrome, the most common partial monosomy is that involving the short arms of chromosome 18, a condition first reported by de Grouchy et al. 64 The cases have recently been reviewed by Nitowsky and co-workers 103 and collectively do not yet represent a specific syndrome. Although cyclopia was found in two cases, it is not an exclusive feature of this deletion, in that it has also been observed in a child with partial monosomy G (21-22).35 Partial deletion of the long arm of chromosome 18 has been reported in several patients. 73 Partial monosomy G has been reported by Lejeune et al. 82 in a patient wih two other abnormal cell lines, one monosomic (missing a G chromosome) and the other partially monosomic (long arm deletion). The patient evidenced an "antimongoloid" slant of the eyes, which has since been observed in other patients with this deletion. 113 A short arm deletion has been reported in a patient with pyknodysostosis. 47 Until very recently, complete monosomy had not been reported and was considered to be incompatible with life. Even partial monosomy is rare. Two recent observations are therefore of great interest. Thorburn and Johnson13o reported a mentally retarded boy with antimongoloid slant of the eyes and other abnormalities. The child had only 45 chromosomes, the missing member being in the G group. The child died and although cytogenetic analysis was limited to 23 cells the authors considered the possibility of complete monosomy G. Other cytogenetic interpretations were considered because one of the E group members was somewhat larger than normal. AI-Aish3 subsequently reported an extensive study of a 4Yz-yearold mentally retarded girl with monosomy G in cells from peripheral blood, bone marrow and skin cultures. No evidence of mosaicism was found. This patient may represent a translocation in which the missing G chromosome is present but unrecognized, but the detailed studies offer little support for this interpretation. It appears, therefore, that autosomal monosomy has now been convincingly demonstrated and is not necessarily lethal. For a condition long held to be inviable, the patient surprisingly evidenced none of the major abnormalities of the cardiovascular system or genitourinary tract so generally associated with the more viable autosomal trisomy syndromes. Precisely why monosomy G proves to be so rare is a challenging question. Regrettably, the abnormalities exhibited by the patient are in no way unusual and therefore offer no clues for identification of additional cases. Inactivation of Genetic Material Random inactivation of one of the female X chromosomes at an early stage in development, resulting in clones of cells having functionally

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only one X chromosome, was proposed by Lyon. 89 This concept has gained wide acceptance and is increasingly invoked to explain the variable expression of sex-linked genes in heterozygous women. However, the most recent assessment of the available evidence does not support the hypothesis as originally formulated. Gruneberg 65 reviewed the 59 conditions listed by McKusick91 for which X linkage has been reasonably established. Heterozygous manifestations are recognized in 26 of these conditions. For only 17 of these are multicellular effects known; five have recognized cellular effects, and four have only humoral effects and thus provide no information on the Lyon hypothesis. Considering the evidence from genes with multicellular effects which show a mosaic distribution of cells in involved organs (such as anhidrotic ectodermal dysplasia, ocular albinism and color vision), Gruneberg finds that several criteria predicted from the Lyon hypothesis are not fulfilled. The most important is that the mosaics are either nonrandom in distribution or too finely grained to support early (blastula stage) inactivation. This disparity is even more apparent in genes with detectable cellular effects, such as glucose-6-phosphate dehydrogenase (G6PD) deficiency in Negroes. Beutler et al. l l found evidence for two populations of red blood cells in females heterozygous for G6PD deficiency, indicating that inactivation had taken place but providing no information about when the phenomenon occurred. Davidson and co-workers41 examined the electrophoretic behavior of G6PD in clones of cultured fibroblasts from heterozygous females. Within a clone, only one (either) of the two bands normally present in heterozygous Negro females was observed. Similar studies by Linder and Gartler 86 on clones derived from very small (1 cu. rum.) myometrial biopsies demonstrated that clones of both types of cells were present, indicating that mosaicism exists but is of very fine grain. Thus early inactivation is embryologically inconceivable unless the cells are subsequently subjected to a "churning phenomenon"65 as yet unknown. A corollary to these studies is provided by the observation86 that cultured leiomyoma cells from heterozygous females have a single electrophoretic band, suggesting that the tumor arises from a single cell. This observation indicates that a single cell may give rise to a sizable population of surrounding cells and is of considerable basic interest in theories of oncogenesis. Significantly, inactivation may occur in both the autosomal and sexlinked types of Hurler's syndrome,4o suggesting that inactivation of certain genetic material on both autosomal and sex chromosomes may occur. Therefore, although conclusive proof of the Lyon hypothesis is lacking, it is possible that genic inactivation (as contrasted to chromosomal inactivation) may be a general phenomenon.

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Practical Considerations To the physician, human cytogenetic studies have already yielded infonnation of considerable diagnostic and prognostic importance. The major autosomal and sex chromosome abnonnalities have been clearly defined, and the uncertainties that previously existed in the diagnosis of infants with ambiguous genitalia and indeterminate sex no longer exist. The long periods of watchful waiting and uncertainty that previously plagued physicians having patients with questionable Down's, Turner's or Klinefelter's syndromes have been eliminated. The recognition of the D1 and E trisomy syndromes as specific (previously unrecognized) nosological entities within the large heterogeneous group of multiple malfonnation disorders is also of significant diagnostic and prognostic value. Cytogenic studies afford a sound basis for genetic counseling, as the recurrence risks for the various chromosomal aberrations are now well established. The biological and clinical significance of partial and complete monosomy, and chromosome breakage, remain to be detennined. That chromosome aberrations provide a difficult but feasible approach to the problem of chromosome mapping has now been demonstrated. The correlation of chromosome marking through deletion studies with the linkage data obtained from population studies76, 114, 141 should in the future provide an understanding of human genetics that was until recently considered virtually unobtainable. NEW INBORN ERRORS OF METABOLISM

Genetic dis()rders are generally of two types: inborn errors of metabolism, in which the defect is biochemically defined to some degree, and developmental syndromes, in which the specific defect is unknown. This dichotomy is in all likelihood artificial, for both groups of disorders reHect abnormality of genetically detennined enzymatic or structural proteins. Inborn errors of metabolism have been recognized since the tum of the century, but new examples continue to appear. In part, this reHects the widespread current interest in human genetics and the more general availability of laboratory methods which facilitate their recognition and investigation. Waisman137 recently reviewed the newer inborn errors of metabolism, and we will confine our discussion to some of the disorders recognized since then. Hypervalinemia. Hypervalinemia was first reported by Wada,136 in a Japanese patient. The condition is characterized by elevated levels of serum valine, valinuria and failure to thrive. Although it has not yet been reported in other patients, further study of the original patientB9 has demonstrated that the defect is in transamination of valine, a metabolic step immediately preceding the oxidative decarboxylation defect in maple

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syrup urine disease. These studies have clearly defined the metabolic defect in this rare disorder and its relationship to maple syrup urine disease. Isovaleric Acidemia. A new inborn error of leucine metabolism which differs from hypervalinemia and maple syrup urine disease has recently been reported by Budd and colleagues. 27 The disorder is characterized by isovalaric acidemia, periodic acidosis with coma, objectionable body odor and psychomotor retardation. The metabolic defect is one step beyond the defect in maple syrup urine disease and involves the conversion of isovaleryl CoA to ,8-methylcrotonyl CoA by isovaleryl CoA dehydrogenase. Trypsinogen Deficiency Disease. A severe form of malnutrition in early infancy due to specific deficiency of pancreatic trypsinogen has been reported by Townes and co-workers.131. 132 This disorder is characterized by severe failure to thrive, protein deficiency anemia, hypoproteinemia and edema. Hypochromotrichia has been observed, and the clinical picture closely resembles that of kwashiorkor. Although this appears to be a fatal disorder if untreated, dietary therapy (protein hydrolysate and pancreatin) completely corrects all manifestations. Two infants from unrelated families have been studied and evidence completely normal development on continuous therapy. The salutary response to therapy is doubtless due to the fact that the deficient enzyme (trypsin) is one whose normal function is extracellular and therefore replacement is readily accomplished. A third patient with this disorder has recently been reported,98 but response to therapy was less than optimal, presumably because of other abnormalities of a coincidental nature. Limited family studies suggest that trypsinogen deficiency is an autosomal recessive disorder. Hereditary Galactokinase Deficiency. Human galactokinase deficiency was documented by Gitzelmann61 . 62 in a patient originally reported by Fanconi in 1933 to have galactose diabetes and cataracts. A metabolic study of this patient and his sister revealed complete absence of erythrocyte galactokinase. Both patients had cataracts as did another sib who was not studied. Although the propositus had von Recklinghausen's neurofibromatosis, this was considered to be coincidental, and the cataracts appear to be the only major manifestation of galactokinase deficiency. The parents were related, and the disorder appears to be inherited as an autosomal recessive trait. Sulfite Oxidase Deficiency. Laster and co-workers81 have reported deficiency of sulfite oxidase (brain, kidney, liver) in a mentally retarded boy with ectopia lentis who died at 2 years of age. The urine contained increased amounts of S-sulfo-L-cysteine, sulfite and thiosulfate. Three siblings died during the first month of life and may have had the same disease. This disorder joins homocystinuria and cystathioninuria as disorders with defects in metabolism of the sulfur-containing amino acids.

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Triose Phosphate Isomerase Deficiency (TID). A severe form of nonspherocytic hemolytic anemia with deficiency of TID in both erythrocytes and leukocytes has been reported by Schneider et al.119, 134 Other affected individuals were subsequently studied in two other kindreds, believed to be related to the original family. Heterozygotes are biochemically recognizable but phenotypically normal, implying autosomal recessive inheritance. A progressive neurologic disease has been observed in affected individuals which may reRect the importance of the enzyme in metabolism of lipid precursors. Hexokinase Deficiency. Valentine and co-workers133 have reported a congenital anemia in patients with deficiency of erythrocyte hexokinase. Leukocytes and platelets were normal. Detailed studies did not reveal any qualitative abnormality of the hexokinase. There were no other defects and the mode of inheritance is believed to be autosomal recessive. Sphingolipidoses. The sphingolipidoses are a group of disorders characterized by increased tissue glycolipids or phospholipids, and have long been of interest to physicians. 1s Gaucher's disease has recently been shown to be associated with markedly diminished glucocerebrosidase activity;2o,107 Niemann-Pick disease with deficiency of sphingomyelinase,21 and Fabry's disease with ceremidetrihexosidase deficiency.19 These findings have considerably clarified the relationships of these disorders and establish that they are due to specific enzyme deficiencies and represent inborn errors of metabolism. Familial Intrauterine Convulsions. A remarkable example of pyridoxine dependency convulsions affecting three sibs in utero has been reported by Bejsovec and co-workers. 7 The first two infants died in status epilepticus shortly after birth. The mother felt in utero convulsions in the second pregnancy and predicted the outcome. Similar abnormal movements experienced in the third pregnancy responded to pyridoxine therapy. In the neonatal period, the patient convulsed when pyridoxine was withheld, and the seizures responded promptly to pyridoxine therapy. In utero convulsions have been previously reported; however, the study by Bejsovec is the first to demonstrate that intrauterine convulsions can be familial and due to pyridoxine dependency. The relationship of this intrauterine manifestation to familial pyridoxine dependency convulsions remains to be defined. This entity is of interest in that it is one of the few conditions in which a fetal disorder can be specifically diagnosed and treated. Heterogeneity of Phenotype Physicians are accustomed to clinical variability. No two patients are necessarily expected to follow an identical course in a given disorder. In diseases caused by specific enzyme deficiency the variability is frequently assumed to be less extensive and the course considered to be reasonably predictable. This assumption is implicit in our programs for

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screening and treatment of specific inborn errors of metabolism. That this assumption is not always justified has become increasingly apparent. H omocystinuria. A striking example of variability is provided by observations on homo cystinuria, a disorder first reported in two mentally retarded sisters by Carson and Neill32 and in a mentally retarded boy by Gerritsen et a1. 59 . 60 Carson et apo. 31 subsequently discovered 10 cases of homocystinuria in a survey of urines from 2290 mentally deficient patients. From their study, these investigators concluded that "there is a consistent picture of mental retardation, ectopia lentis, fine hair, shuffling gait, thromboembolic episodes and skeletal changes."31 The highest I.Q. in the 10 patients was 53. The only metabolic disorder found in higher frequency in the survey was PKU (69 cases). 31 Other patients have been described,25. 78. 79. 108 but the suggestion that homocystinuria is the second most common metabolic disease responsible for mental retardation31 has been questioned by Spaeth and Barber,125 who found only two cases of homocystinuria in a survey of 10,000 mentally retarded patients from 38 institutions. Schimke et al. 117 described 38 cases ascertained by screening of patients with ectopia lentis and concluded that homocystinuria accounted for 5 per cent of all cases of non traumatic ectopia lentis. Surprisingly, 16 of the 38 patients were found to have normal intelligence, an observation which raises a number of questions concerning the previously established association of mental retardation in this disorder. In part, the discrepancy is due to bias of ascertainment (survey of mentally retarded patients versus survey of persons with a specific ocular disorder). The discrepancy is of more general significance, for it clearly demonstrates the hazards of generalization from observations of highly selected individuals. The suggestion that the mental deficiency in homocystinuria may be prevented by dietary managemenfi'9 is also questionable since almost half of the patients reported by Schimke were of normal intelligence. A valid treatment program will require further understanding of homocystinuria. 25 . 26. 60 Specific deficiency of cystathionine synthetase has been demonstrated in tissues from mentally deficient homocystinuric patients,26. 55.99 but the status of homocystinurics with normal intelligence remains to be clarified. If they should prove to have the same deficiency, the genesis of the mental defect will be a matter of great complexity. Cystathioninuria. Cystathioninuria presents another case in point. Harris and co-workers 67 first reported cystathioninuria in a elderly woman with severe mental retardation, and Frimpter et al. reported a second example of this disorder in a male acromegalic with deafness. 54 The patient was found to have an I.Q. of 100. There have been three subsequent reports of this disorder, in a child with normal intelligence97 and in two retarded persons. 8. 109 The relationship of mental deficiency to the primary disorder is therefore uncertain. Frimpter54 has shown that

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the cystathioninuria is partially corrected by vitamin B6 administration and also that the patients have deficiency of cystathioninase in the liver. The enzyme deficiency is corrected in vitro by addition of vitamin B6, suggesting that the apoenzyme is present but defective in its ability to bind the essential cofactor. 53 Cystathioninuria is therefore considered to be one of the pyridoxine dependency syndromes. 121 Galactosemia. Finally, variability in galactosemia has been recognized for some time. 71 Segal et a1. 6, 122 have demonstrated that there are probably three types of galactosemia: the classic form in which the transferase is deficient and in which the patients have clinical manifestation of the deficiency; the "galactosemic" form in which the transferase is deficient, but the patients are nevertheless able to metabolize galactose reasonably well but still have clinical manifestations; and a third form which metabolically is indistinguishable from the second type, except that the patient has no clinical manifestation whatever. Types 2 and 3 have thus far been found only in Negroes,6, 122 and the ability to utilize galactose has been attributed to alternate metabolic pathways. The prototype of type 3 galactosemia proved to be a sibling of a patient with type 2.6 That two sibs with demonstrated absence of uridyl transferase can differ so remarkably is a sobering realization of our present inability to predict the outcome of a specific enzyme defect. There would have been little reason to doubt the efficacy of treatment had the asymptomatic sib been placed on a restrictive diet. The inconstant association of mental retardation and other defects with seemingly specific errors of metabolism clearly poses many problems for our programs of prevention and therapy. These difficulties have become increasingly apparent on a far greater scale in phenylketonuria. The potential hazards of the PKU screening and treatment program have been extensively· detailed by many investigators and emphasize our limited understanding of the disorder and the critical need for further studies. 9 ,56 Investigation of the structural and functional characteristics of the various enzymes involved in these diseases can only partially fulfill this need. Study of Isolates. The potential genetic heterogeneity underlying a particular clinical expression may be undetected by current diagnostic criteria. This genetic heterogeneity is perhaps best minimized by studies of isolate populations, in which the mutant gene is presumed to be identical by descent in all affected individuals. This permits an assessment of variation in expression of the gene. 142 Two factors common to all isolates 135 make them valuable for the study of recessive disorders. First, chance fluctuations may result in much higher heterozygote frequencies for certain recessive genes than would occur in a large random-breeding population. This increased frequency may occur despite moderate negative selection and lead to a

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relatively greater increase in homozygous individuals. Secondly, a higher level of inbreeding occurs in isolate groups than in the general population. This effect again increases the probability of homozygous expression of a rare gene. The advantages of isolate populations are exemplified in studies of the Amish sect by McKusick and his co-workers.16, 92, 93 A rather large number of new and rare hereditary diseases have been identified in this group. It has often been possible to trace the ancestry of all persons affected with a specific entity to a common ancestor, usually one of the founders of the sect in the United States. Inasmuch as there are often several affected members living at one time it is possible to delineate the extent of variable expression. Occasionally this variation reaches unanticipated levels. This may be the case in some of the inherited anomalies of gamma globulin synthesis which have been studied so extensively in recent years. Ataxia-telangiectasia (AT) was described by Louis-BarSS and has since been characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia and recurrent sinopulmonary infection thought to be related to deficiency of gamma A globulins. While selective depression of gamma A is characteristic of AT there may also be depression of other gamma globulins. There is also usually a variable degree of peripheral lymphopenia. At autopsy AT patients have either an absent or rudimentary thymus. Those affected with this disease generally manifest symptoms at age 1 or 2 years; there is gradual progression, and patients may live to age 20 years or more. They also have an increased tendency to develop malignant neoplasms of lymphoid tissue. Inheritance is autosomal recessive. A more severe form of dysgammaglobulinemia is the Swiss type (SAG) in which there is deficiency of all gamma globulin fractions. Affected children usually succumb within the first year of life. The thymus is either absent or embryonic in appearance and circulating lymphocytes are decreased. Inheritance is autosomal recessive in most families, although an X-linked type exists. McKusick and Cross92 have reported typical cases of both of these diseases in separate but related sibships. Both diseases have not been seen in a single sibship, although a number of children in the AT sibship did die of unknown causes or infection at a young age. All cases are descendants of a single immigrant Amish couple and three are also descendants of another woman thought to be a sister of one of the immigrant couple. McKusick and Cross have suggested that, while distinct genes for both of these disorders may have been present in the founding generation, it is possible that one mutant gene is responsible for both conditions in this family. The genetic implication of these observations in non-Amish cases is problematical.

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Variation in proteins which do not lead to pathologic states are equally important to an understanding of human biology. These are normal variants and their occurrence and significance is only beginning to be appreciated. This type of variation has long been recognized in many blood group factors, in which several alleles coexist at a single locus in a population. Genetic loci showing several discontinuous forms in such proportions that the rarest cannot be maintained merely by recurrent mutation are said to be polymorphic. 51 The recognition of large numbers of polymorphisms in human populations for which no selective value is recognized represents a focal point of current investigation in human genetics. If the polymorphisms are adaptively neutral-that is, have no selective advantage or disadvantage-extinction will result unless opposed by recurrent mutation, migration or genetic drift. The effective breeding size of human populations (except in the case of isolates) is so large that little differentiation can result from genetic drift. Likewise recurrent mutation seems to be an unlikely explanation unless these loci mutate at inconceivably high rates. If the polymorphisms are not adaptively neutral, selection may favor heterozygotes, as in the well-documented case of sickle cell anemia, and thus maintain the balance. However, if many loci were maintained by balanced heterotic forces, the total amount of selection in a population would be clearly incompatible with survival of the population. 85 Systematic study of various enzymes by electrophoretic methods has shown that many exist in two or more forms at frequencies greater than 1 per cent (the level that recurrent mutation alone can maintain). The following polymorphisms illustrate the type of genetic variability present in human populations. As yet no sufficient mechanisms have been advanced to explain the maintenance of these genes at the levels they are observed. Erythrocyte Phosphoglucomutase (PGM) This enzyme is found in many body tissues and is an important catalyst in the transfer of phosphate groups between the 1 and 6 positions of glucose. In the red blood cell seven proteins with PGM activity have been described. 126 Three phenotypes, 1-1,2-1 and 2-2 result from segregation of two codominant autosomal alleles and occur in the English population in frequencies of 0.550, 0.376 and 0.074, respectively. This gives a gene frequency of PGMI = 0.74 and PGM2 = 0.26. It seems that each of these phenotypes is equally efficient enzymatically and no factors of selective importance have been identified.

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Erythrocyte Acid Phosphatase Although acid phosphatase is ubiquitously distributed in human tissues there are almost certainly structural differences in the enzyme derived from diverse sources. The acid phosphatases cleave phosphoric monoesters optimally at acid pH. The phenotypes described in red blood cells 68 are designated A, BA, B, CA and CB. Family studies show that three codominant alleles, pA, pB and pc, at a single autosomal locus, are responsible for the observed phenotypes. These alleles have gene frequencies of 0.36, 0.60 and 0.04, respectively, in the English population. The hypothetical C phenotype has not been observed, presumably because of the rarity of the gene PC. The distribution of red cell acid phosphatase activity in a random population sample shows a continuous unimodal curve (that is, it appears to be a quantitative trait). However, the enzymatic activity of the various phenotypes varies as follows: A > BA > B = CA > CB,69 and this distribution has been shown to be discontinuous. Analogous examples presumably exist for other seemingly continuously variable traits. Again no selective importance has been attributed to this polymorphism and all phenotypes seem to function adequately. Erythrocyte Adenylate Kinase The enzyme is a phosphotransferase which catalyzes the reaction AMP ATP ~ 2ADP. Three phenotypes are recognized49 and are designated AK 1, AK 2-1 and AK 2. These represent the autosomal codominant alleles AK1 and AK2, which have a gene frequency in the English population of approximately 0.95 and 0.05, respectively. No selective forces are recognized. Similar polymorphisms have been demonstrated in several other enzymes, including serum alkaline phosphatase,17 serum cholinesterase,66, 77 6-phosphogluconate dehydrogenase43 and acetyl transferase,112 The proportion of all enzymes which are polymorphic is unknown but an appreciable number seem quite invariant. Many other polymorphisms have been demonstrated,111 including the blood group antigens, phenylthiocarbamide (PTC) tasting ability, f3 aminoisobutyric aciduria (BAIB), color blindness, the hemoglobinopathies, and various plasma proteins such as group specific component (Gc), haptoglobin, transferrin, lipoprotein and gammaglobulins (Gm and Inv). Although the precise mechanism maintaining these polymorphisms is unknown several authors have attempted to correlate them with specific diseases. 9o The direct association of the Rhesus factor and erythroblastosis fetalis is well known. The mechanism by which this particular genotype is related to a specific clinical phenotype is understood. There are, however, other well-established associations between the human blood groups and disease in which the mechanisms are less evident.

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The extensively studied association between duodenal ulcer and blood groups 0 and A, first suggested by Aird et al.,l has since been confirmed by many investigators in many populations. However, other factors, both genetic and environmental, affect the clinical expression of this disorder. Edwards 45 has provided a model for the analysis of such associations. This model is based on the assumption that susceptibility to duodenal ulcer is different in persons of different blood groups. It is assumed that persons of one blood group have a distribution of liability to manifest duodenal ulceration which differs from persons of another blood group. The mathematical model permits an estimate of the relative contribution of a person's blood type to the expression of a specific disease. In duodenal ulcer the contribution of the ABO blood group system is estimated to be 1 per cent. This infers that 99 per cent of the causation is attributable to other factors. The magnitude of the ABO effect is so slight as to imply an inconsequential genetic effect. Of course other genetic factors may prove to be important, but blood group and disease associations per se have not yielded insight either into the causation of disease or into the selective forces which maintain the blood group polymorphisms.

SUMMARY

Selected aspects of several areas of investigation in human genetics are reviewed. The principal aneuploid syndromes are well defined and current cytogenetic studies are concerned with problems of chromosome marking, chromosome breakage and partial and complete monosomy. Certain limitations of the X-inactivation hypothesis are considered and evidence for genic inactivation on both autosomal and X chromosomes is discussed. The variable expression of seemingly specific metabolic disorders is discussed with regard to the problems posed for large scale screening and treatment programs. Of particular importance is the somewhat inconstant association of mental retardation in several well-defined inborn errors of metabolism. Although genetic variability is usually associated with pathologic states, recent studies have revealed a considerable degree of genetically determined biochemical variability (polymorphism) in normal populations.

REFERENCES 1. Aird, I., Bentall, H. H., Mehigan, J. A., and Roberts, J. A. F.: The blood groups in relation to peptic ulceration and carcinoma of colon, rectum, breast and bronchus. Brit. Med. J., 2:315, 1954.

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