Genetic analysis of IDDM: summary of GAW5 IDDM results

This paper summarizes the analyses by participants in the insulin-dependent diabetes mellitus (IDDM) component of Genetic Analysis Workshop 5 (GAW5). The data were obtained from 94 families with two or more IDDM sibs. Topics treated in the Workshop analysis included the following: methods for detecting associations and linkage, the contribution by HLA-linked and -unlinked loci to IDDM susceptibility, the role of subtypes of the serologically defined HLA specificities, the implications of associated diseases other than IDDM in the families, the significance of antibodies to Coxsackie viruses, and of autoantibodies to pancreatic islet cells and insulin, and the use of genetic models to analyze the inheritance of IDDM. There was agreement that an explanation for the data on multiplex IDDM families must include the following features: 1) There is a susceptibility locus (or loci) in the HLA region. 2) The HLA-linked factor(s) are more complex than a single locus with one disease and one nondisease allele. 3) There is additional familial correlation beyond that explained by HLA-linked susceptibility, which may be genetic and/or environmental. With regard to the third feature, IDDM-GAW5 included data on variation in Gm haplotypes and at the insulin gene, two regions unlinked to HLA. However, there was no direct evidence (i.e., from marker segregation) that the additional factor, if genetic, is linked to either Gm or the insulin gene. Nevertheless, a significant difference was found between "diabetic" and "control" insulin genes with respect to frequency of class 1 alleles for the 5' flanking polymorphism, strongly suggesting linkage.     

HLA antigens and acute rheumatic fever: evidence for a recessive susceptibility gene linked to HLA

From 60 probands with acute rheumatic fever (ARF), 19 multiplex families segregating for ARF were ascertained. The parents and rheumatic and normal sibs of the probands in these 19 families were also studied. HLA typing using the microlymphocytotoxic assay was then performed on the 60 unrelated probands, the multiplex families, and 234 unrelated controls using 23 antigens from the HLA-A and -B loci. The controls lacked a past history of ARF and were from the same geographic locality. Calculations of relative risk demonstrate an increase of HLA-B5 antigen in the 60 patients, but the result might not be significant from the point of view of multiple comparisons. Nevertheless, affected sib pairs from the multiplex families show 93% concordance for both or one HLA haplotype. A formal linkage analysis demonstrates that a recessive etiology is most likely (lod score of 3.3) with approximately 68% of cases being due to a gene closely linked to HLA and in linkage disequilibrium with HLA-B5. The remaining 32% of cases are due to other familial factors such as polygenic inheritance or common environmental factors. The results confirm a strong genetic predisposition to ARF and its heterogeneous nature in families.     

Investigating the HLA component in rheumatoid arthritis: an additive (dominant) mode of inheritance is rejected, a recessive mode is preferred.

We examined the mode of inheritance of rheumatoid arthritis (RA) and estimated the genetic contribution of the HLA-linked locus to the development of RA using data from 111 multiplex families (54 London, 57 Cleveland), and 43 randomly ascertained patients (Seattle). HLA-DR4 was present in 78 multiplex probands (70%); a further 16 probands who were negative for DR4 were positive for DR1. Both DR4 and DR1 were significantly in excess when compared to control population frequencies (P less than 0.001); an additional finding was an excess of DR7, although the numbers of probands with DR7 were small. Despite the well-established HLA association with RA, neither recessive nor additive (dominant) modes of inheritance, nor any intermediate models have been ruled out using affected sib-pair and antigen genotype frequency among patients (AGFAP) methods. However, in our study the AGFAP data for HLA-DR4 and DR1 were close to recessive expectations (P = ns) while an additive (dominant) mode of inheritance was rejected (P less than 0.001). The same results were obtained by an independent method which considered HLA-DR transmission from affected parents to their affected children. The affected sib-pair haplotype sharing method showed deviation from random expectations but did not allow discrimination between recessive and additive (dominant) modes. The effect of the HLA-linked locus on familiarity accounted for only a 1.61-fold increased risk to sibs over the population prevalence, compared to an observed value of 3.90. This indicated that there could be at least one other non-HLA locus predisposing to RA with a weight that is slightly greater than that of HLA.     

Evidence for a dominant gene mechanism underlying coeliac disease in the west of Ireland

Although coeliac disease (CD) is strongly associated with the HLA alleles B8 and DR3, the genetic basis of this illness remains obscure. Recent studies show that at least two unlinked loci are involved. Most studies agree on recessivity at the HLA-unlinked locus but differ with respect to dominance or recessivity at the HLA-linked disease susceptibility locus. To address this controversy, we examined the association of CD with HLA in 39 families from the West of Ireland. Previous studies have shown that the prevalence of CD and the frequencies of the HLA antigens associated with it are higher in this population than in most others. Analysis of the data revealed a significant excess of concordant sib-pairs with two HLA haplotypes in common and an excess of discordant pairs with no haplotype in common. Chi-square tests confirmed a highly significant association between HLA-B8 and CD. Both heterozygotes and homozygotes for B8 had a significantly increased risk of CD. The risk for homozygotes was slightly higher than for heterozygotes, although not significantly so. The segregation ratio for disease occurrence among sibs of probands was estimated to be 0.185 when neither parent is affected. We estimated a gene frequency of 0.003 for the disease allele (C) at the HLA-linked locus and of 0.648 for the disease allele (d) at the HLA-unlinked locus. Assuming that CCdd homozygotes are always affected and that only carriers of C who are homozygous dd can be affected, the disease was found to be completely penetrant in Ccdd heterozygotes. These results support dominance at the HLA-linked locus conferring susceptibility to CD. Possible reasons for the discrepancy between the West of Ireland and other populations are discussed.     

HLA may be involved in resistance and susceptibility to affective disorders

The role of HLA in susceptibility to affective disorders was assessed by sib pair linkage analysis and by association studies. Unipolar Disorder (UD) and Bipolar Disorder (BD) were studied separately. Both the sib pair data and the antigen frequency distribution suggested an HLA-linked susceptibility to UD. For BD however, the HLA haplotype sharing distribution in sib pairs was random, but the antigen frequencies suggested at least one positive association and one negative association with HLA. The lack of evidence for linkage to HLA from sib pairs may have been due to the genetic heterogeneity of BD.     

Linkage analysis and genetic models for IDDM

The multiplex insulin-dependent diabetes mellitus (IDDM) families have been examined for linkage with the human leukocyte antigen (HLA), Gm, Km, and insulin loci. For the last three, no evidence of linkage (as measured by haplotype sharing in affected siblings) was found. For HLA, strong evidence of linkage was demonstrated, as expected from previous studies of both haplotype sharing and HLA association. The haplotype sharing in affected siblings from this and previous studies would explain a relative risk of about 3.7 in the sibling of an affected individual (95% confidence limits 2.8 to 5.4), considerably less than the values of 10-15 given by epidemiological studies. This discrepancy may be explained by other predisposing genes unlinked to HLA, or be due to common environmental factors. Analysis conditional on the affection status of all individuals, and on the parental HLA haplotypes clearly rejects both a dominant and recessive mode of inheritance in favor of a two-allele model with a reduced risk for heterozygotes, and provides some support for a three-allele model. Some evidence of age effects was found; in particular there was some suggestion of greater haplotype sharing in siblings both affected at a young age, and there was some suggestion that individuals with the predisposing HLA-DR3/DR4, DR3/DRx, and DR4/DRx genotypes were affected at a younger age. These results may be diagnostic artifacts, however, and need investigation in future studies.     

HLA DQ beta 3.2 identifies subtypes of DR4+ haplotypes permissive for IDDM

The HLA class II-related susceptibility to type I insulin-dependent diabetes mellitus (IDDM) is examined in 94 multiplex families sorted by the presence or absence of a DR4+ haplotype in at least one diabetic family member. The families with DR4+ haplotypes are then sorted by the presence or absence of a DR4-linked DQ beta 3.2 allele. Further analysis assumes each multiplex family to represent a single diabetic genetic event and identifies the HLA class II haplotype(s) present in all affected members. The DQ beta 3.2 allele is present in over 95% of the multiplex families where DR4+ haplotypes segregate with IDDM, implying a major permissive role in determining susceptibility to IDDM.     

Tests for covariate-associated heterogeneity in IBD allele sharing of affected relatives

Linkage studies that aim to map susceptibility genes for complex diseases commonly test for excess allele sharing among affected relatives. Conventional methods based on identical-by-descent IBD allele sharing do not allow for possible differences among families, such as arise in the case of locus heterogeneity, and thus have reduced ability to detect linkage in the presence of such heterogeneity. We investigated two approaches to test for heterogeneity in allele sharing, using a family-level covariate that may be associated with different disease mechanisms leading to differences in allele sharing. Likelihood ratio tests for heterogeneity were formulated based on an extension of the linear and exponential likelihood models developed by Kong and Cox. Alternatively, we examined the asymptotic and permutation distributions of T-tests for differences between mean allele-sharing linkage scores from two covariate-defined family subgroups, assuming exchangeability. The size and power of heterogeneity tests were evaluated for S(all) and S(pairs) allele-sharing scoring functions using data sets of families with affected sibling and cousin pairs, generated under a model of locus heterogeneity. In certain simulation scenarios, the likelihood ratio test statistics did not follow the expected asymptotic distributions. The type I error estimates for the T-statistics conformed to nominal 5 and 1% levels in all scenarios considered, and corresponding power was comparable to that of the likelihood ratio tests. Application of these tests for heterogeneity detected significant differences in allele sharing between subgroups of families with inflammatory bowel disease.     

Characteristics of a multiplex IDDM sample: unexplained differences with other samples

Characteristics of a multiplex sample of families with insulin-dependent diabetes mellitus (IDDM) are studied and contrasted with similar characteristics in other, more conventionally sampled data sets. Some characteristics remain consistent with earlier observations including the high frequency of human leukocyte antigen (HLA) DR3,4 in affected individuals and the greater than expected percentage of HLA haplotype sharing among affected sib pairs. In other respects, however, differences are seen between this sample and others. "Control" haplotypes, i.e., those not transmitted to the first affected offspring, had a higher frequency of DR3 and DR4 than expected, and a rather high frequency of affected parents was observed. Differences between the first affected and later affected offspring reported in other samples were absent from these families. No effect of the sampling scheme and the resulting distribution of parental phenotypes could be shown to explain this difference.     

Inferring disease risk genes from sequencing data in multiplex pedigrees through sharing of rare variants

We previously demonstrated how sharing of rare variants (RVs) in distant affected relatives can be used to identify variants causing a complex and heterogeneous disease. This approach tested whether single RVs were shared by all sequenced affected family members. However, as with other study designs, joint analysis of several RVs (e.g., within genes) is sometimes required to obtain sufficient statistical power. Further, phenocopies can lead to false negatives for some causal RVs if complete sharing among affected is required. Here, we extend our methodology (Rare Variant Sharing, RVS) to address these issues. Specifically, we introduce gene-based analyses, a partial sharing test based on RV sharing probabilities for subsets of affected relatives and a haplotype-based RV definition. RVS also has the desirable feature of not requiring external estimates of variant frequency or control samples, provides functionality to assess and address violations of key assumptions, and is available as open source software for genome-wide analysis. Simulations including phenocopies, based on the families of an oral cleft study, revealed the partial and complete sharing versions of RVS achieved similar statistical power compared with alternative methods (RareIBD and the Gene-Based Segregation Test), and had superior power compared with the pedigree Variant Annotation, Analysis, and Search Tool (pVAAST) linkage statistic. In studies of multiplex cleft families, analysis of rare single nucleotide variants in the exome of 151 affected relatives from 54 families revealed no significant excess sharing in any one gene, but highlighted different patterns of sharing revealed by the complete and partial sharing tests.     

Linkage studies of HLA and insulin gene restriction fragment length polymorphisms in families with IDDM

Linkage analysis of HLA DR antigen as well as DR and DQ restriction fragment length polymorphism (RFLP) data using the LIPED computer program and various three-allele disease locus models showed very close linkage to an insulin-dependent diabetes mellitus (IDDM)-susceptibility locus. RFLP data alone were equal or superior to conventional HLA antigen typing in the linkage analysis. Insulin gene restriction fragment data were analyzed for evidence of either a susceptibility locus linked to the insulin gene or an effect of alleles at the insulin locus on the HLA-linked susceptibility gene. No evidence was found of any effect of the insulin gene, and it is suggested that alternative explanations of the reported population associations between the insulin gene and IDDM should be considered.     

Genotypic and phenotypic similarities in pulmonary function among family members of adult monozygotic and dizygotic twins

Population studies have demonstrated that obstructive airways disease aggregates within families. The authors used a twin family model of analysis to estimate the genetic and environmental influences on pulmonary function. A total of 1,635 members of 414 families of adult twins (252 monozygotic, 162 dizygotic) enrolled in the Greater Boston Twin Registry were studied between 1981 and 1982. Correlations in levels of forced expiratory volume in one second (FEV1) and forced vital capacity (FVC), adjusted for age, sex, height, and current smoking status, were compared among 16 groups of relatives sharing various degrees of genetic relatedness. A direct relation between shared genotype and the magnitude of the familial correlations for pulmonary function was observed. For FEV1, the correlations were 0.71 for monozygotic twins (100% shared genotype), 0.16 to 0.29 for relatives with 50% shared genotype, 0.09 to 0.27 for relatives with 25% shared genotype, 0.06 for cousins with 12.5% shared genotype, and -0.14 to 0.14 for unrelated family members. Correlations for FVC were similar. Stratification of the analysis by concordance or discordance for passive tobacco smoke exposure or for frequency with which families visited one another did not systematically alter these relations. These data suggest that phenotypic similarities in pulmonary function relate directly to genetic similarities, and are consistent with a multifactorial mode of inheritance.     

The insulin gene and susceptibility to IDDM

The association between insulin-dependent diabetes mellitus (IDDM) and an allele of a restriction fragment length polymorphism (RFLP) 5' to the coding region of the insulin gene has raised the possibility that variation in the vicinity of the insulin gene confers susceptibility to IDDM. To test this hypothesis, the distribution of insulin gene sharing in affected sib pairs (ASPs) from the Genetic Analysis Workshop 5 (GAW5) families has been compared with that expected on the basis of random assortment. There is no deviation from random expectation in insulin gene sharing among 95 ASPs from families fully informative for the insulin gene. This is also true when insulin gene sharing is conditioned on HLA sharing, on the particular HLA DR types in ASPs, or on the parents' insulin allele classes. These results thus provide no evidence that variation at or near the insulin gene confers susceptibility to IDDM. However, we also used computer simulation to investigate how the insulin gene region could contribute susceptibility to IDDM without yielding evidence for distortion in insulin gene sharing in a sample comparable to that of GAW5. We found that various levels of insulin gene involvement in IDDM could generate a population association between the insulin gene RFLP and IDDM comparable to that reported in the literature, without producing significant distortion in insulin gene sharing of ASPs.     

THE DETECTION OF EARLY HEMOCHROMATOSIS

The detection of early latent disease in the relatives of individuals with idiopathic hemochromatosis is a matter of importance. A study of iron metabolism suggests that no one test is diagnostic and several are needed. Another study evaluated the concordance of HLA — haplotypes in affected and nonaffected relatives and gives evidence of a promising new approach.     

Increased risk for hepatitis B-related liver cirrhosis in relatives of patients with hepatocellular carcinoma in northern Taiwan

BACKGROUND: There is a tendency for familial aggregation of hepatocellular carcinoma (HCC). The aims of this study were to assess the degree to which familial aggregation of hepatitis B surface antigen (HBsAg) carriers accounts for familiality of HCC in families of hepatitis B-related HCC patients, and whether HCC shares a familial predisposition with liver cirrhosis among HBsAg carriers. METHODS: A total of 671 first-degree relatives of HBsAg-positive HCC cases were recruited using abdominal ultrasonography and tests for HBsAg and serum aminotransferases. They were from 165 simplex families defined as having only one HCC case and 72 multiplex families with more than one case. In analyses of family history of HCC and cirrhosis, the data set consisted of 4,471 unrelated asymptomatic HBsAg carriers recruited in a prospective study. RESULTS: There was no significant difference in the HBsAg-positive rate among relatives between multiplex (55.7%) and simplex (48.1%) families. Sonographic evidence of liver cirrhosis was present in 14.4% of HBsAg-positive relatives from multiplex families but in only 7.8% of HBsAg-positive relatives from simplex families (multiplex versus simplex families: adjusted odds ratio [OR] = 2.29; 95% CI: 1.10-4.77). Among unrelated asymptomatic HBsAg carriers, the adjusted OR of liver cirrhosis associated with a first-degree family history of HCC was 2.80 (95% CI: 1.68-4.66). This association was stronger in HBsAg carriers <50 years. No association was seen between family history of HCC and hepatitis activity based on elevated levels of aminotransferases. CONCLUSIONS: Familial aggregation of HCC in HBsAg carriers is associated with familial clustering of liver cirrhosis.     

A family study of panic disorder

Panic disorder, as defined by the DSM III diagnostic criteria, was diagnosed in 117 probands for whom age of onset ranged from 10 to 59 years, with a mean of 26.6 years. Diagnosis of parents and siblings was based on interviews with the probands, and only those with “definite” panic disorder by the FISC criteria were considered to be affected. The pattern of concordances for panic across different groups of relatives was estimated concurrently by a log-linear model for binary pedigree data, assuming different values for the cumulative risk. When an adjustment for age was made, based on the age of onset of probands, there was no significant difference between parent-offspring concordance and sibling concordance. There was a negative, but not significant, concordance between spouse pairs. Assuming the lifetime cumulative risk was 1.9% for males and 4.7% for females, values considered appropriate for this population, our model predicted that the presence of an affected parent or sibling incurs an approximately five times increase in the risk of developing panic disorder. Our model assumes in effect that this risk is multiplied for each further affected relative. Although the common concordance across relationship groups is consistent with a genetic hypothesis, it can also be explained by common family environmental factors. There is a need for further pedigree studies, using twins and relatives, for example, and reliable information on the cumulative risk.     

Analysis of the Toronto-Rochester Depression Study follow-up data confirms an HLA-region gene contribution to susceptibility to affective disorder

Analysis of HLA haplotype distributions in relation to major affective disorder in affected sibling pairs and affected aunt or uncle and niece or nephew pairs confirmed that HLA-region genes do contribute to susceptibility to affective disorder. The data indicated that this effect may be greater in unipolar than in bipolar disorder, and more apparent in families with few affected members than in heavily loaded families. Nonrandom assortment of HLA haplotypes to affected and unaffected offspring in "low load" families occurred principally for the haplotype transmitted from the side of the family without affective disorder. We conclude that HLA-region genes contribute to but are not the only factor in susceptibility to major depression.     

Testing genetic models for IDDM by the MASC method

The MASC method has been applied to the GAW5 data. The method uses the simultaneous information on association and segregation of the HLA marker with the disease and the segregation of the HLA marker in affected families. It also takes into account the differential risk for parents of a patient, as well as the different HLA haplotype sharing, according to the HLA genotype of the patient. The goodness of fit of several genetic models has been tested. The observed data are not compatible with a two-allele, one-locus model, but they fit a three-allele, one-locus model and a complementation two-locus model if additional familial correlation is allowed.     

Regression models for allele sharing: analysis of accumulating data in affected sib pair studies

Advances in human genome mapping have led to the identification of large numbers of genetic markers that allow systematic searches for multiple disease susceptibility genes for complex traits. A common design involves the recruitment of families with at least two children affected with the disease of interest. The objective is to find chromosomal regions that harbour susceptibility genes for the disease. The affected children, their parents if available, and sometimes other, unaffected, siblings are genotyped using sets of microsatellite DNA markers representing chromosomal sites distributed across the genome. Each marker can occur in several different variants known as alleles, and a pair of alleles constitutes the marker genotype. Each child randomly inherits one of their mother's two alleles and one of their father's two alleles. If a marker is close to a disease susceptibility gene, then affected siblings are expected to have more sharing of the same maternal and/or paternal marker alleles. Statistical methods are used to estimate the distribution of allele sharing in each affected sib pair (ASP) using the set of markers typed across each chromosome, and to test for the presence of excess sharing in the families as a group at each point across the genome. Regression models that allow the allele sharing proportions to depend on characteristics of the family such as diagnostic subtype or ethnic background have been developed to address the heterogeneity that is characteristic of complex disease, but these have not yet been widely applied. In this paper, we apply regression modelling to investigate variation associated with family-level covariates and with the order in which families are recruited and genotyped. We also discuss how some of the concepts of group sequential analysis apply to accumulating data from genome scans of complex disease.     

Does Simultaneous Consideration of Multiple Regions Improve Disease Gene Localization?

Improvement in localization of disease susceptibility genes by simultaneous consideration of multiple interacting loci was assessed using the Genetic Analysis Workshop 12 simulated data. Evidence of linkage at primary loci was used to weight families for analyses at secondary loci. To identify regions linked to disease susceptibility genes, parametric and allele‐sharing genome scans were performed in the extended pedigrees and nuclear families, respectively. The position of the peak allele‐sharing lod was used as the estimate of a disease gene location. In weighted analyses, the positions where the greatest lod increases occurred were taken as alternative estimates of the gene locations. Variability of the location estimates of disease genes given by the unweighted and weighted analyses was compared. Similar analyses were carried out using true disease loci to determine weights. Weighted analyses did not in general improve the localization of disease genes in this data set, even with a large sample of 1,928 nuclear families, due to the features of the underlying additive liability threshold model. © 2001 Wiley‐Liss, Inc.