Ph.D. - Biomedical Sciences (Genetics - Cell, Molecular and Neuro Sciences)

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    Applications of distribution theory in quantitative genetics
    ( 1976) Yamashita, Toyoko S.
    The present study was designed to develop procedures for estimating genetic parameters in a quantitative trait and detecting association between a quantitative trait and a genetic marker using a random sample of individuals from the population. An exact distribution of phenotypic values of a quantitative trait was used by which parameters such as number of loci involved, gene frequency at each locus, additive and dominant genotypic value, and the variance of environmental effects were estimated. When association between the quantitative trait and a genetic marker occurred, an additional parameter for the degree of linkage disequilibrium was introduced in the exact distribution. For testing the validity and usefulness of the procedures, individual phenotypic values were simulated by the computer with given parameters. The number of loci involved in the quantitative trait was 2, 5, and la, and gene frequency at each locus was chosen at random. Three heritability values were investigated; namely, 0.2, 0.5, and 0.8. The genotypic values were then calculated by setting degree of the dominance either to zero (no dominance) or one (complete dominance). The environmental effects were assumed to be normally distributed; however, the phenotypic distribution of the quantitative trait under investigation was not specified. Five replicate runs were made for most tests to allow variations in gene frequencies, each sample size being set to 5000. Smaller samples of 100 and 500 individuals were also tested. Based on the log likelihood function values, good estimations by the maximum likelihood method were obtained when heritability of the quantitative trait was moderately high (h^2 ≥.5). When the heritability value was low, the phenotypic distribution of the quantitative trait was mainly determined by environmental effects and, therefore, was expected to be normal by assumption. In this case, the method might not give consistent estimates of genetic parameters. As the number of loci increased, the phenotypic distribution would approach normal by the Central Limit Theorem; however, the procedure was still effective for traits with high heritability. Several cases for the association between one of the loci involved in the quantitative trait and a genetic marker were considered, including (1) complete independence (no linkage), (2) partial linkage, and (3) complete linkage or identical locus. The approach employed was mainly based on the distributions of phenotypic values. Data were subgrouped by the genotypes of the marker locus resulting in several phenotypic distributions, one for each genotype of the genetic marker. When association occurred between a quantitative trait locus and a genetic marker locus, the subclass distributions would be expected to be different from one another. The subsequent estimates of parameters including the degree of linkage disequilibrium were then derived from the exact distributions by the maximum likelihood method. The results were generally good. The present study using simulated data has clearly demonstrated that by employing the distribution of phenotypic values of the quantitative trait, it is possible to establish a genetic basis for the character from a random sample of individual data. When information on other genetic markers is available, the association between the quantitative trait and a genetic marker as measured by linkage disequilibrium may be estimated. However, further research would be needed to apply the procedures to real (non-simulated) data and to consider other parameters such as genotype-environmental interactions and epistatic effects in the phenotypic distribution.
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    Demonstration on zymograms and genetic studies of some enzymes from human saliva
    ( 1975) Tan, Soon Guan
    Saliva samples were collected, processed, concentrated and then subjected to polyacrylamide flat slab gel electrophoresis followed by staining to detect enzyme activities. Six different enzyme activities were detected out of the fifty enzyme staining procedures attempted. Repeatable and constant variants were observed in three of these, saliva acid phosphatases, esterases and glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase). Genetic studies were done on these three. Seven phenotypes were found for saliva acid phosphatases. Family and population studies suggested that these phenotypes are the products of two loci, Sap-A with three alleles, A, A' and O, and Sap-B with 1 three alleles, B, B^1 , and O. These loci were found to be polymorphic in Americans of Japanese and Caucasian ancestries living in the State of Hawaii. Saliva esterases show a complex picture on zymograms and had been divided into four major regions. Variants were observed in region 1, the fastest anodal migrating region. Three phenotypes had been observed in region 1. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles, Set-IF and Set-IS. This locus is polymorphic in Americans of Japanese and Caucasian ancestries. Region I esterases are carboxylesterases. Saliva glucose-6-phosphate dehydrogenase (hexose-6-phosphate dehydrogenase) showed three phenotypes on acrylamide gel zymograms. Family and population studies suggested that these phenotypes are the products of an autosomal locus with two alleles, Sgd 1 and Sgd 2. All the above three saliva enzymes in which variants were observed thus allowing genetic studies to be done proved to be different from the analogous enzymes with similar functions in the red blood cell. They constitute previously undescribed polymorphisms in the human species. The three other enzymes whose presence in the saliva had been detected on zymograms were an oxidase, lactate dehydrogenase and superoxide dimutase. No variants were observed for these enzymes which would enable genetic studies to be done. Association and linkage studies were attempted between the three newly described polymorphic saliva enzymes among themselves and between them and the ABH secretor status, Lewis A substance in saliva, ABO blood group, C5, adenosine deaminase, esterase D, PGM I , haptoglobin, Gc protein, MN blood group, P blood group, Duffy blood group, Kell blood group and Kidd blood group loci using data from random Caucasian individuals. Significant associations were only found between Sap-A and haptoglobin locus and between Set-l and MN blood group locus. These significant associations are most probably due to chance. Linkage studies were not very fruitful because of the small numbers of informative families available due to the small family size in most of the sampled families. None of the accumulative lod scores yielded conclusive results.
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    The genetics of pigeon populations on Oahu
    ([Honolulu], 1972) Go, Rodney Chun-Pung
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    A genetic and epidemiological study of cleft lip and cleft palate in Hawaii
    ([Honolulu], 1970) Ching, Gerald Hong Sung
    The purpose of this study was (1) to obtain unbiased estimates of cleft incidences in the major racial groups in Hawaii, (2) to evaluate the extent and nature of racial differences in incidence, (3) to determine how the segregation of cleft defects varies among racial groups, and (4) to estimate the heritability of cleft defects using a multifactorial model of quasicontinuity. Cleft cases were ascertained through birth and death certificates and records of the Crippled Children Branch (Hawaii Health Department) and major Honolulu hospitals. The reference population which provided the basis for the analysis of incidence was composed of live births in Hawaii from 1948 to 1966, as compiled by Drs. Morton, Chung, and Mi. Live birth certificates provided demographic data on all individuals in the reference population. Variables included race and age of each parent, sex of child, birth order, year of birth, and occupation and military status of father. Families of probands were interviewed for additional data on types and numbers of affected and normal relatives and parental consanguinity. The ascertainment of CL(P) was estimated to be 97% complete, and that of CP to be 93% complete. The danger of relying exclusively on birth certificates for the ascertainment of cases was made clear by our findings that only 80.7% of CLP, 66.7% of CL, and 47.7% of CP probands born in 1948-1966 had their defects noted on their birth certificates. A number of factors were found to be related to the ascertainment probabilities of CP and CL(P). In the case of CP, ascertainment appeared more likely when the individual had three or more additional malformations and less likely when his father was actively engaged in military service. The case of CL(P) was more complicated. The probability of ascertainment of CL(P) apparently increased when the defect extended to the palate and when any additional malformation was present; it seemed to decrease with year of birth, age of father, death in infancy, and father in the military. Significantly, however, no racial biases in ascertainment were detected after adjustment for the effects of non-racial factors. After correcting for ascertainment biases, the estimated overall incidence of CP in Hawaii was .78 per thousand live births. In the pure Caucasian group, the estimated incidence was .50; in the pure Japanese group, it was .74. Incidences in excess of one per thousand were found in groups with Hawaiian ancestry. Regression analysis showed a definite association between Hawaiian parentage and high incidence of CP. This strong Hawaiian effect could not be attributed to maternal racial factors or to interracial hybridization. Orientals as a whole did not appear to have a significantly higher incidence of CP than Caucasians. For CL(P), the corrected incidence was estimated at 1.05 per thousand in Caucasians, 1.92 in Japanese, and 1.28 in the general population. Unlike in the case of CP, Hawaiian ancestry was not associated with high incidence of CL(P). Instead, the Oriental groups, particularly the Japanese and Filipinos (1.56), presented the highest risks. No maternal or hybridity racial effects were detected for any of the Oriental groups. The effects of non-racial factors (father's age, mother's age, birth order, year of birth, father's occupational and military status) on incidence were tested by separate regression analyses in the Japanese and in the Caucasian racial . groups. No consistent relationship between incidence and any non-racial variable was detected for either cleft type. However, CP incidence did appear to increase with lower occupational status in the Caucasian group, although not in the Japanese group. Estimates of mean segregation frequencies for CP were 2.2% in families of Hawaiian ancestry and 1.3% in all other families. Although the difference was not statistically significant, the higher risk in Hawaiian families would be expected under the multifactorial hypothesis which equates risk approximately to the square root of the population incidence. Heritability of CP was estimated to be 69%, which possibly indicates that additive genetic factors are very important in the etiology of CP. Mean segregation frequencies for CL(P) were 6.1% in pure Japanese, 6.4% in pure Filipinos, and 4.2% in all other groups. The higher risks in Japanese and Filipinos may reflect their higher incidences of CL(P). Overall heritability of CL(P) was estimated at 86%, with no significant differences among Japanese, Filipinos, and all other groups combined. The weight of evidence now seems to favor a multifactorial hypothesis of CL(P) inheritance, and the present estimates of heritability would suggest a large additive hereditary component.
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    Studies on quasi-continuity
    ([Honolulu], 1969) Campbell, Mary Anne
    This study was an attempt to fit a model of quasi-continuous variation to three sets of data. The model is based on the assumption that the trait under study is completely additive and wholly polygenic. Conditions due to a major gene, in the sense of classical Mendelian inheritance, would give unreasonable parameter estimates and a poor fit on such a model. Information on population prevalence and recurrence in sibs is used to estimate the parameters and predict risk for other degrees of relationship. Records on all surgically corrected cases of pyloric stenosis occurring in the period 1942 to 1966 in Hawaii provided the first set of data. Nearly 200 families were interviewed to obtain a pedigree and family history. Birth certificate numbers were matched with a file of all births for that time period to provide additional information on race and sociological variables. The sex ratio was similar to all earlier reports, approximately 4:1. The segregation analysis was compatible with a major gene hypothesis but the estimates had very large standard errors. The regression analysis of interracial crosses indicated a depression in the frequency in F1, indicative of recessivity. The fit to the model of quasi-continuity was neither good nor consistent. Such a theory cannot be eliminated as the possible genetic mechanism for pyloric stenosis, but the current model is inadequate to explain the data. Males with serum cholesterol levels greater than two standard deviations above the mean of a random sample of 7,000 forty to sixty year old Japanese males were selected for a study of hypercholesterolemia. Sibs were contacted, interviewed and serum cholesterol determinations made. The 219 sibships were analyzed by segregation analysis, providing an estimate of the segregation frequency of 0.52 and the proportion of sporadics as zero, evidence for a dominant gene. Estimates of heritability on the additive model exceeded 1.5. This condition appears to be a dominant gene with nearly complete penetrance. This finding is in agreement with other studies. Information from the Bureau of Identification provided a sample of individuals with a dermal ridge count of zero, an arch pattern on all ten fingers. These individuals were defined as "affected." Ridge counts were made for all available relatives. A control sample was selected from the Bureau files. The frequency of patterns and mean ridge counts for the Caucasian controls agreed well with published studies from England. The Japanese had higher mean counts and a significantly higher frequency of whorls. Correlation coefficients computed in the control sample for sib-sib and parent-child relationships agreed well with complete heritability. Segregation analysis in both the affected families and the controls failed to detect segregation of a major gene. Parameters were estimated for the quasi-continuous model using data from first degree relatives. Fitting these estimates to data from cousins and second degree relatives gave a non-significant deviation. Evidence from earlier studies has shown fairly conclusively that dermal ridge count is additive and multifactorial. The good fit of our data to this model of quasi-continuity was reassuring. Hypercholesterolemia, on the other hand, seems clearly due to a major gene. The failure to fit the data on pyloric stenosis may only reflect inefficient parameter estimates and lack of sufficient sophistication or accuracy in the current model.
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    A genetic study of the amylase isozyme polymorphism in Drosophila melanogaster
    ([Honolulu], 1969) McCune, Thomas Brent
    Many of the recently discovered genetically determined polymorphisms involving isozymes of Drosophila melanogaster have been concerned with identifying the enzyme biochemically and determining the formal genetics of the enzyme. After the survey of several enzyme systems in natural populations of Drosophila pseudoobscura by Lewontin and Hubby (1966), which indicated that isozyme polymorphisms are more frequent than previously expected, there has been greater interest in population studies of these isozymes as is shown by the recent work in Drosophila melanogaster concerning the Esterase 6 polymorphism (Yarbrough and Kojima, 1967) and the alcohol dehydrogenase polymorphism (Kojima and Tobari, 1969b). The purpose of this study was to investigate the frequencies of the amylase isozyme alleles in several natural populations of D. melanogaster, and on the success of this investigation, to attempt to see how the polymorphism might be maintained. The survey consisted of recently collected flies from natural populations of Texas and Wisconsin. In addition, the Odate population (collected in Japan four years ago and since kept in the laboratory) was also examined. For all surveys the frequency of the Amyl allele was clearly the most common (0.80) and was followed by Amy1,3 (about 0.12). Other less frequent alleles observed were Amy2,3, Amyl,2, and hmy1,6. Because of Amyl and Amy1,3 being consistently the most frequent in the natural populations, the selection studies concentrated on these two alleles in the Texas and Wisconsin populations. The results of the viability and fertility experiments (for matings within and between populations) consistently demonstrated higher viability and fertility for the Amyl,3/Amyl,3 genotype followed by Amyl/Amyl,3 and Amyl/Amyl genotypes respectively. The results of the developmental study, which employed the normal sugar containing media and a special media containing starch instead of sugar, were less consistent; however, the Amyl/Amyl genotype had the slowest developmental time with the Amyl/Amyl,3 and Amyl,3/Amyl,3 genotypes being similar to each other. From these results it is difficult to see why the Amyl has such a high gene frequency in natural populations. There was some indication from one of the viability experiments that the recovery frequency of the Amyl allele among progeny is higher than that of the Amyl,3 allele when the parents are very young, thus suggesting a prezygotic mechanism of some sort. This study, however, does not critically test for prezygotic selection and more specific tests are currently being planned.
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    Isolation and characterization of messenger ribonucleic acid for hemoglobin
    ([Honolulu], 1969) Laycock, David Gerald
    An 8s ribonucleic acid (RNA) species, isolated from rabbit reticulocytes, has been purified by salt fractionation and analyzed for characteristics of an information-carrying, messenger RNA. This RNA in reticulocytes is restricted to polyribosome complexes which are active in hemoglobin synthesis and has a base composition similar to that expected of a messenger RNA for rabbit globin. This RNA species has been assayed for the ability to support and direct protein synthesis in bacterial extracts. The 8s RNA from reticulocyte polyribosomes is translated when only N-acetyl-valyl- tRNA is present as an initiation residue. Both rabbit globin polypeptide chains have N-terminal valine residues. The protein produced in bacterial extracts directed by this RNA is identical to rabbit globin by chromatography on diethylaminoethyl-cellulose, carboxymethyl-Sephadex C50, paper electrophoresis and gel-filtration on Sephadex G75. The protein product combines with added hemin and the resultant complex is indistinguishable from native rabbit hemoglobin by the above criteria. The profile of tryptic peptides from chromatography on Dowex 50x2 indicates the product is N-acetyl globin. The implications of these results concerning the universality of the genetic code and of protein biosynthetic mechanisms at a macromolecular level are discussed.
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    Aberrant segregation in human populations
    ([Honolulu], 1969) Grove, John Sinclair
    Three cases of aberrant segregation in humans were considered. The first was a possible case of non-random assortment of chromosomes and meiotic drive. Separating children of fathers of given heterozygous ABO genotypes into sperm classes (classified by the ABO gene received from the father), for children of A1B and A2B fathers a change in association of the sperm type with the sex of the child (which would be determined by the father's sex chromosome received) occurred with advancing father's age. Specifically, the B sperm class was associated with the X chromosome when fathers were old and the Y chromosome when they were young. Mother's age and birth order effects were ruled out by multiple regression. This effect was strikingly non-linear: a fifth order polynomial of father's age was very highly significant (for pooled offspring of A1B and A2B fathers). The sex ratio changed within each sperm class in opposite directions. The segregation frequency of the B sperm class for female offspring increased with father's age (but not mother's age or birth order) and again a fifth order polynomial was very highly significant. The progeny of B0, A2O, and A1O plus A1A2 fathers were not affected. There was a non-linear change in segregation frequency of female offspring of AB mothers as mothers grew older which was not highly significant and may have been due to chance. The second study was on the genetic load of the population in Odate, Japan. Prenatal death was examined with respect to several factors and found to fall into at least three classes: early prenatal death (EPRE), up to four months of gestation; late prenatal death (LPRE), up to nine months; stillbirths, more than nine months. LPRE increased significantly (but non-linearly) with inbreeding but EPRE and stillbirths did not; regressions for LPRE and stillbirths differed significantly. Postnatal death was divided into three classes (which differed in several respects): early postnatal death (less than one month old); middle postnatal death (less than two years old); late postnatal death (two or more years old). Only early postnatal deaths increased significantly with inbreeding (.4 lethal equivalents per gamete; B/A = 14.5) and the regression differed significantly from those of the later stages. The last analysis was on ABO incompatibility in the same population. Although a preliminary analysis had not shown significant effects, it was found that the inclusion of three secretor interactions with ABO incompatibility showed very highly significant effects on the stages of mortality considered above. The secretor interactions were the mother's secretor type, the father's secretor type, and the probability that the zygote was See A and B incompatibility on 0 and non-O mothers were analyzed separately for mortality and were found to behave differently. Interactions with parity and (parity)^2 were often highly significant. The interactions tended to balance each other making simpler models non-significant. Some of the regressions suggested viability differences among genotypes of offspring. Significant environmental interactions were also found.
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    Erythrocyte isozymes, other polymorphisms, and the coefficient of kinship in northeastern Brazil
    ([Honolulu], 1969) Azevêdo, Eliane S.
    Application of zymogram development technique has become a powerful tool in genetics research. It provides easy detection of molecular variation in proteins, leads to the discovery of new polymorphisms, and significantly adds to the knowledgment of man's genetical makeup and population structure. This dissertation comprises a study of isozymes and other polymorphisms, followed by their application to the analysis of population structure. During the year 1962-63 a total of 1068 families was selected from a migrant population of northeastern Brazil. Red blood cells and sera were collected and have been kept in satisfactory condition for study of enzymes and proteins. The red cell enzymes glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, phosphoglucomutase and adenylate kinase were studied by starch gel electrophoresis. Except for G6PD whose gene frequencies depend on the proportion of Negro ancestry, no other association was detected when gene frequency was regressed on other variables such as social level, sex, age, longitude, latitude, and regional pathology (hepatosplenomegaly). Two families carrying an unusual variant in the G6PD system are described, accompanied by biochemical characterization of the new allele v and cross-check with other G6PD mutants. Once proved to be unique, the new all8le is provisionally designated as Gd Minas Gerais (MG), after the Brazilian state of the propositus. Three families carrying rare alleles at the PGMZ locus are also described. A linkage study between isozymes and other genetic markers was done, mainly stimulated by a recent report claiming a probable linkage between the phenylthiocarbamide (PTC) and the phosphoglucomutase (PGM1 ) loci. We studied twenty-eight families where one of the parents was doubly heterozygous, i.e., double and single backcrosses, and four intercross families. We did not detect linkage in our sample either by analyzing the recombination frequencies by sex, nor in the pooled sample. Most lod scores are negative, giving evidence against linkage, and close linkage (θ ≤ .2) may confidently be excluded. By using other segregating families we also tested linkage between hemoglobin and PGM, hemoglobin and Gc, hemoglobin and cholinesterases E1 and E2. None of the observed lod scores were suggestive of linkage. The isozyme, and other polymorphisms which were previously studied in the same sample, are used to bioassay the population structure of northeastern Brazil. The migration model of Malecot was selected because it estimates the most pertinent parameters ruling the structure of a population (migration, systematic pressure, and consanguinity) besides its many-sided applicability to any genetic system (phenotype), isonomy, metric data, and pedigree inbreeding. A computer program DISTAN draws random pairs of individuals, groups them into classes according to the distance between their birth-places, and estimates the coefficient of kinship for every distance class. Estimation of the parameters a (mean coefficient of kinship for individuals born at small distance), b (measure of systematic pressure), and £ (dimensionality of migration) are also given by the program DISTAN. According to Malecot's theory, if the distance between birth-place of individuals I-and J is d, then the coefficient of kinship is given by φ(d) = ae^-bd d^-c , and is defined as the probability that two alleles drawn at random, one from individual I and one from individual J, be identical by descendent from a common ancestor. The monotonic decrease with distance of inbreeding and kinship predicted by Malecot has been observed in northeastern Brazil for polymorphisms, metrics, surname concordance (isonomy), and pedigree inbreeding. Comparison of these indicators with each other and with Malecot's theory indicates that consanguineous marriage at a given distance is preferential, most conspicuously at large distances. The mean coefficient of inbreeding is estimated to be .0080, of which 70 per cent was ascertained through pedigrees. Interpretation of these results is discussed in terms of various concepts of population structure.