The affected sib method. IV. Sib trios

The classical sib pair method uses the expected and observed HLA (human leukocyte antigen) haplotype sharing distribution in sib pairs, who are affected with an HLA associated disease, to make inferences about the inheritance of the disease. In this paper we present the expected HLA haplotype sharing distributions in affected sib trios, and sib pairs, from families with three or more affected sibs. The underlying model for both distributions, as for the classical sib pair method, is that disease predisposition is determined by a single allele at an HLA-linked locus. The sib trio tests of hypotheses (additive and recessive), and disease parameter estimates (additive, recessive and intermediate), can be compared with those obtained from the classical sib pair analysis. In addition, the sib trio data allow parameter estimation for a general disease model to be made, if the data fall within the bounds of the expectation. This study forms the basis of later investigations which show that haplotype sharing of affected sib trios for two susceptibility alleles (negative complementation) model, which appears appropriate for insulin dependent diabetes mellitus (IDDM), moves outside the bound of the single susceptibility expectations outlined here, whereas haplotype sharing values for sib pairs are bound by the single susceptibility allele expectations. Available Caucasian IDDM data have been analysed. The results support genetic heterogeneity of IDDM.     

Gene dosage and susceptibility to insulin-dependent diabetes

1. All members of 33 families in which two or more sibs have insulin-dependent diabetes mellitus (IDDM) were HLA-typed. The results strongly support the hypothesis that, closely linked to the HLA region, there is a locus ( S ) for susceptibility to IDDM. We use S ^d for alleles at this locus which confer susceptibility to disease, and S ^a for all other alleles.
2. Published prevalence (recurrence) rates for relatives of an IDDM proband, which are essential for the analysis, are reviewed critically, and a new estimate of prevalence at age 30 in the general population is calculated from published data.
3. The analysis allows for greater risk (penetrance) of the genotype S ^a S ^d (two doses of S^d) than of S ^a S ^d (one dose of S^d) and for recombination between the HLA region and S. With either our own data on 26 affected sib pairs or a much larger sample assembled by pooling with published data, the maximum-likelihood estimate of the two-dose penetrance is 71%, of the one dose penetrance 6·5%. The hypothesis of differential penetrance according to gene dosage provides a significantly better explanation than either simple dominant or simple recessive inheritance.
4. Estimates of the recombination fraction between HLA and S are very sensitive to the proportion of affected sib pairs sharing neither HLA haplotype, which varies considerably among the samples pooled. With the pooled data the recombination fraction is estimated as 3%, but in view of the strong associations with particular HLA alleles seen in population data, this should probably be considered an upper limit.     

The Affected Sib Pair IBD Distribution for HLA-Linked Disease Susceptibility Genes

The distribution of identity by descent (IBD) scores for sib pairs affected with a disease determined by a disease susceptibility (DS) locus tightly linked to the HLA complex is derived. It is shown that the sib pair IBD distribution differs from its a priori distribution and, moreover, is completely specified by three observable population parameters-the additive and dominance variances and the prevalence of the disease in the population. An application of the model is illustrated using data on juvenile diabetes mellitus.     

The affected sib method. V. Testing the assumptions

The affected sib methods, which are used to make inferences about the genetic components of HLA associated diseases, have many underlying assumptions which may not always be realistic. These include no selective disadvantage of affected individuals, little or no recombination between the marker loci and the 'disease' locus, a single panmictic population, Mendelian segregation of the disease locus alleles and random distribution of individuals over environments. The effects of breaking these assumptions have been investigated. We have explicitly derived the haplotype sharing identity by descent (IBD) expectations for the cases of selection against affected individuals and recombination between the HLA marker loci and the 'disease' predisposing locus for affected sib trios (as was previously done for affected sib pairs). We have also derived, for both affected sib pairs and trios, the haplotype sharing expectations for non-random mating (positive assortative), admixture, meiotic drive (of disease allele carrying haplotypes), and a random versus shared environmental component for sibs. In order to assess the sensitivity of the affected sib methods to perturbations in the assumptions, the expectation spaces of haplotype sharing in affected sib pairs and sib trios under the single diallelic locus model with varying penetrances and allele frequencies are fully described. The effects on haplotype sharing and subsequent disease parameter estimation are different for each of the factors we have considered. The affected sib methods are found to be robust in many situations.     

The generalized sib pair IBD distribution: its use in the detection of linkage

General expressions for the distribution of identity by descent (IBD) scores at a marker locus have been derived given neither, one or both sibs affected with a disorder determined by a linked trait locus with arbitrary gene frequency and penetrance vector. It is shown that the distribution of IBD scores depends only on the additive and dominance variances and the population prevalence of the disorder. A one-sided test is suggested as an appropriate means of statistically testing the hypothesis that the recombination fraction is significantly less than. This sib pair approach is designed primarily to detect the presence of a critical disease susceptibility locus but when the assumptions of the incompletely penetrant single locus model are correct the methodology proposed here results in consistent estimates of the recombination fraction. The affected sib pair methodology seems especially suited to traits determined by single loci with non-Mendelian transmission. We wish to express our gratitude to Mr P. Van Eerdewegh and to Drs P. Fishman and S. Hodge for their many helpful comments and criticisms. This work has been supported by HEW grants MH 25430, MH 22521, MH 14677, MH 13002 and AA 00209.     

A three-allele model for heterogeneity of juvenile onset insulin-dependent diabetes

A three-allele model is presented for the inheritance of ‘juvenile’ insulin-dependent diabetes mellitus (IDDJI). The model postulates a susceptibility locus S tightly linked to the HLA complex. The model incorporates relative-risk and HLA-association data from the literature which suggest genetic heterogeneity within IDDM, together with population prevalence, twin and sib concordance rates, and distortions in HLA haplotype concordance values for affected sib pairs. The model appears to provide a reasonable fit to existing data. It predicts low penetrances for some, but not all, genotypes and a strong association between HLA B8 and one of the S alleles. The model makes additional specific predictions about how different forms of IDDX are distributed in sporadic and familial cases. These predictions can be tested in future genetic-epidemiologic studies.     

HLA and susceptibility to multiple sclerosis

The study of the joint segregation of multiple sclerosis and HLA, using affected sib pairs as well as whole pedigrees, shows that these two traits are not independently transmitted. The hypothesis of a single susceptibility locus inside HLA region could explain all the observed data, only if a high gene frequency, a very low penetrance, and some environmental correlation between relatives are assumed. Linkage analysis performed on the basis of this hypothesis for 58 multiple sclerosis families concludes to a strict linkage. We obtained a maximum score of 3.11 at theta = 0.00 for a dominant gene of frequency 0.18 and penetrance of 0.02. This result contrasts with the large recombination fraction obtained by other authors and the discrepancy is explained by the very low gene frequency used in their analysis. Some environmental correlation, in addition to the genetic determinant in HLA region, may explain the overall familial aggregation, but an alternative is the existence of additional genetic determinants.     

Interactive effect of HLA and Gm tested in a study of 135 juvenile insulin-dependent diabetic families

Interaction between HLA and Gm for susceptibility to insulin-dependent diabetes (IDD) has been studied in 135 IDD families comparing HLA and Gm phenotype distributions in patients and sibling controls. The association analysis comparing the 135 index cases to 124 sibling controls did not show any significant interaction, but only a tendency for Gm 4,5 phenotypes to be more frequent in IDD patients, particularly in those who carried DR3/X. The sib pair analysis of 16 pairs of affected sibs did not show any distortion of segregation of Gm. On the other hand, among 98 sib pairs of one affected and one nonaffected sib, there was a significant distortion in favour of sib pairs with non-identical Gm phenotypes. However, no interaction between HLA and Gm was found. These results provide suggestive evidence that Gm or a locus very close to Gm could be involved in susceptibility to IDD.     

Predictions of a 2-locus model for disease heterogeneity: application to adrenoleukodystrophy.

Adrenoleukodystrophy (ALD) is an X-linked disorder that exhibits a wide range of phenotypic variability within individuals in a single family carrying the mutant allele. A 2-locus epistatic model has been proposed to explain the inheritance of the severe childhood form of ALD and the milder adult-onset adrenomyloneuropathy (AMN). Under a dominant epistatic model, a single M allele at an autosomal modifier locus ameliorates the most severe effects of the disease allele leading to the milder AMN phenotype; only males with genotype mm would have ALD. Under a recessive epistatic model, 2 copies of the M allele would be necessary to have the milder AMN phenotype. Here, we show that recurrence risks for a second affected male depend on the frequency of the protective allele at this modifier locus. Whereas it is most likely that 2 affected brothers will be concordant for their disease phenotypes, discordant pairs of affected brothers are possible at all frequencies of M. Within a narrow range of modifier allele frequencies, the predicted distribution of affected sib pairs (over all families) is consistent with empiric data from a large clinic population. Here we suggest sampling discordant affected sib pairs as a strategy for detecting linkage between a polymorphic DNA marker and a possible modifier gene. Since both epistatic models predict that discordant affected pairs should not share 2 alleles at the modifier locus, we expect that departures from the null distribution could be detected with relatively small numbers of sib pairs.     

Predictions of a 2-locus model for disease heterogeneity: Application to adrenoleukodystrophy

Adrenoleukodystrophy (ALD) is an X-linked disorder that exhibits a wide range of phenotypic variability within individuals in a single family carrying the mutant allele. A 2-locus epistatic model has been proposed to explain the inheritance of the severe childhood form of ALD and the milder adult-onset adrenomyloneuropathy (AMN). Under a dominant epistatic model, a single M allele at an autosomal modifier locus ameliorates the most severe effects of the disease allele leading to the milder AMN phenotype; only males with genotype mm would have ALD. Under a recessive epistatic model, 2 copies of the M allele would be necessary to have the milder AMN phenotype. Here, we show that recurrence risks for a second affected male depend on the frequency of the protective allele at this modifier locus. Whereas it is most likely that 2 affected brothers will be concordant for their disease phenotypes, discordant pairs of affected brothers are possible at all frequencies of M. Within a narrow range of modifier allele frequencies, the predicted distribution of affected sib pairs (over all families) is consistent with empiric data from a large clinic population. Here we suggest sampling discordant affected sib pairs as a strategy for detecting linkage between a polymorphic DNA marker and a possible modifier gene. Since both epistatic models predict that discordant affected pairs should not share 2 alleles at the modifier locus, we expect that departures from the null distribution could be detected with relatively small numbers of sib pairs. © 1992 Wiley-Liss, Inc.     

The effects of reduced fertility, method of ascertainment, and a second unlinked locus on affected sib-pair marker allele sharing

The method of affected sib-pair marker allele sharing has been used not only for the detection of linkage, but also for discerning inheritance. The application of this method has included the tacit assumption of no selective disadvantage for affected individuals. For some of the disorders analyzed by this method, this assumption is not tenable. Also, the method of ascertainment of affected sib pairs is not taken into account. It is shown here that the ascertainment procedure and reduced reproductivity of affected individuals can alter the expected distribution of affected sib-pair marker allele sharing. The effect is greatest for a recessive disease susceptibility locus. While the effect of reduced reproductivity is to inflate the gene frequency necessary to account for the observed distribution, a second unlinked disease susceptibility locus can diminish this effect. Application to type 1 diabetes shows that according to a simple recessive model with 50% penetrance, the observed distribution of affected sib-pair HLA haplotype sharing requires a gene frequency in the range of 0.55-0.60, much greater than estimates previously proposed. According to such a model, the frequency of disease would be around 15%, clearly out of range of the observed frequency of 0.4%.     

Age of diagnosis-based linkage analysis in type 1 diabetes

Genetic linkage studies of type 1 diabetes have produced a number of conflicting results, suggesting a high degree of locus heterogeneity in this disease. Approaches which model such heterogeneity will increase the power to fine map susceptibility loci. Here, using data from a genome scan of 356 affected sib pairs with type 1 diabetes, we performed heterogeneity analysis based on similarity of age at diagnosis of the sib pairs. We observed linkage to the region on chromosome 4p16.3 in sib pairs both diagnosed over the age of 10 years, whilst there was no evidence for linkage in sib pairs diagnosed before age 10 years. In contrast the sib pairs diagnosed before the age of 10 years demonstrated linkage to IDDM10, on chromosome 10p. Age of diagnosis-based heterogeneity analyses in complex diseases may be particularly helpful in mapping some susceptibility loci.     

Investigation of the genetics of allergic children, the second report--family study to determine the major gene in allergic disease using affected sib pair method

Twenty-six families with allergic siblings were investigated to define the mode of inheritance of asthma and DF specific IgE antibody production in children. The affected sib pair method was used to establish the linkage between disease susceptibility genes and HLA antigens. The affected sib pair method revealed that in asthmatic families chi 2 = 4.9 (no significance) and in DF-specific IgE positive families chi 2 = 6.2 (p less than 0.05). This study suggests that the gene of DF-specific IgE antibody production is linked to HLA haplotypes, but the mode of inheritance of DF-specific IgE antibody production couldn't be defined.     

Contribution of the MHC region to the familial risk of coeliac disease.

Susceptibility to coeliac disease is genetically determined by possession of specific HLA-DQ alleles, acting in concert with one or more non-HLA linked genes. The pattern of risk seen in sibs and twins in coeliac disease is most parsimonious with a multiplicative model for the interaction between the two classes of genes. Based on a sib recurrence risk for coeliac disease of 10% and a population prevalence of 0.0033, the sib relative risk is 30. To evaluate the contribution of the MHC region to the familial risk of coeliac disease, we have examined haplotype sharing probabilities across this region in 55 coeliac disease families. Based on these probabilities the sib relative risk of coeliac disease associated with the MHC region is 3.7. Combining these results with published data on allele sharing at HLA, the estimated sib relative risk associated with the MHC region is 3.3. Therefore, the MHC genes contribute no more than 40% of the sib familial risk of coeliac disease and the non-HLA linked gene (or genes) are likely to be the stronger determinant of coeliac disease susceptibility.     

Application of the lod score method to detection of linkage between HLA and juvenile insulin-dependent diabetes

The lod score method has been applied to 28 informative families with at least one child suffering from juvenile insulin-dependent diabetes (JIDD), assuming autosomal recessive inheritance, for detection of linkage between HLA and a susceptibility locus for this disease. These 28 families were pooled with 21 other families from the literature. The maximum lod scores were obtained for recombination fractions from 4 to 16 %, according to the level of penetrance (10 to 90 %). These high estimates of the recombination fraction are not in agreement with the hypothesis that the association between JIDD and specific HLA haplotypes is due to a simple linkage disequilibrium between the HLA region and a susceptibility locus for JIDD.     

A simple estimate of the general population frequency of the MHC susceptibility gene for autoimmune polygenic disease

We wished to determine the frequencies of the MHC and non-MHC susceptibility genes for polygenic autoimmune diseases like type 1 diabetes (IDDM). We used Mendelian inheritance and the Hardy-Weinberg equilibrium to calculate the frequencies of mating pairs and susceptible offspring under classical recessive and dominant inheritance of the MHC susceptibility gene. We then analyzed the distribution of haplotype sharing by affected sib pairs of the 4 MHC haplotypes in each of the kinds of mating pairs in terms of the frequency of the disease susceptibility gene. For IDDM, the analysis was consistent with a recessive, but not a dominant, MHC susceptibility gene of frequency 0.525 at a distribution of 55, 38 and 7% of affected sib pairs who share 2, 1 and 0 MHC haplotypes, respectively. A simple relationship was obtained: if inheritance is recessive, the MHC susceptibility gene frequency is the square root of the fraction of affected sib pairs who share no MHC haplotypes multiplied by 4. For recessive inheritance, affected sib pairs who share no haplotypes are solely in families where both parents are homozygous MHC-susceptible. Although homozygous MHC susceptibles represent over 25% of the population, only 2-3% of them are IDDM-susceptible at non-MHC susceptibility loci, also required for disease expression. Predictions from our analysis fit all published observations of the familial occurrence of disease. The analysis is general, simple and provides a single estimate (not a range) of the MHC susceptibility gene frequency. This approach should be applicable to other MHC-determined polygenic diseases.     

A simple method to detect linkage for rare recessive diseases: An application to juvenile diabetes

A simple procedure designed specifically to detect linkage for rare recessive diseases is described. The method uses information on identity by descent scores for a pair of sibs at a marker locus conditioned on the number of affected sibs in the pair. A procedure for estimating the recombination fraction is described, and a table facilitating the likelihood ratio test of linkage is provided. The method, when applied to a collection of multiplex families segregating for juvenile diabetes mellitus, suggests the possibility that this disease is linked to the HLA complex. The method is found to compare favorably to the maximum likelihood approach, for which the computer program LIPED gives a maximum lod score of 2.48 at a male and female recombination fraction of theta = 0.20.     

HLA haplotype segregation in families with allergic asthma

In order to evaluate genetic linkage between Allergic Asthma (AA) and the HLA system, we studied 20 families having AA affected sib pairs and 8 families with Intrinsic Asthma (IA) affected sib pairs. All AA patients had a strong IgE immune response to the mite D. farinae. Serological HLA typing (A, B, C, DR, and DQ antigens) was performed by the standard microcytotoxicity assay. Genetic analysis was made by means of the "Affected sib pairs" method. Out of the 20 affected sib pairs, 14 shared two HLA haplotypes, five shared one HLA haplotype and one was HLA-non-identical. These results differed significantly from the random ratio 1:2:1 for sharing, 2, 1 or 0 haplotypes (p less than 0.0001), and was very close to that expected for a recessive mode of inheritance. In contrast, among the IA sib pairs there was not an important distortion in the pattern of haplotypes segregation. However, a significant association between any of the HLA alleles and the two types of Asthma studied was not found. The results suggest the existence of an HLA-linked recessive gene controlling the IgE immune responsiveness to mite allergens and conferring susceptibility to AA.     

Power of association test for detecting minor histocompatibility gene causing graft-versus-host disease following bone barrow transplantation

Incompatibility of minor histocompatibility antigen (mHa) is a major cause of acute graft-versus-host disease (GVHD) following bone marrow transplantation in human leukocyte antigen (HLA)-matched donor-recipient pairs. To avoid acute GVHD, as many mHa genes as possible need to be identified . In this study, we introduce a comparison of two proportions as an association test for detecting mHa genes in HLA-matched pairs with and without GVHD. Assuming multiple mHa loci, each with two alleles, we evaluated the effects of (1) minor allele frequency of the mHa locus of interest (denoted by p), and (2) probability of GVHD developing in a donor-recipient pair being incompatible at an mHa locus (denoted by r) on the powers of association tests for unrelated pairs and for sib pairs. Our results showed that based on a candidate gene approach, an mHa gene with high p and r values can be detected by the association test with a small sample size. Application of the present method to the Japanese population revealed that the association test for unrelated pairs is more suitable for detecting an mHa gene with a high r value than that for sib pairs. The present method will be helpful to researchers who evaluate the power of association study in advance.     

HLA Haplotypes in Familial Graves'Disease

In order to further elucidate the genetics of Graves'disease, we studied two families with several affected members, as well as tested the degree to which HLA haplotypes were shared in affected sibpairs. Further, we sought to identify the disease related haplotypes by determining the haplotypes shared among affected parent-child combinations. In one family, two affected sibs differed at four possible parental HLA haplotypes; no evidence of recombination was observed which could account for the result. In the other family, five siblings were affected. Four out of the five affected sibs shared the maternal haplotype HLA-A11, Bw51, Cw5, Cw-, DRw5, Bfs, GLO1, whereas three shared the paternal haplotype HLA-Al, B8, Cw-, DRw3, Bf and GLO1. Looking at haplotype sharing, two pairs of sibs were found to be HLA identical, whereas the fifth sib shared one haplotype with one of these pairs but not with the other. Out of 14 (eight of our own and six from the literature) affected sibpairs examined, nine were found to be HLA identical and four shared one haplotype, suggesting that the contribution of both paternal haplotypes may be necessary for the susceptibility to the disease. Fourteen parent-child combinations were studied; in only three out of 13 in which the shared haplotype could be ascertained was the haplotype B8 positive; this distribution is similar to controls. However, of the remaining 10 combinations which did not share a B8 positive haplotype, five were B8 positive at one or the other of the nonshared haplotypes.