Combined Linkage and Association Studies Show that HLA Class II Variants Control Levels of Antibodies against Epstein-Barr Virus Antigens

Over 95% of the adult population worldwide is infected with Epstein-Barr virus (EBV). EBV infection is associated with the development of several cancers, including Hodgkin lymphoma (HL). Elevated levels of anti-EBV antibodies have been associated with increased risk of HL. There is growing evidence that genetic factors control the levels of antibodies against EBV antigens. Here, we conducted linkage and association studies to search for genetic factors influencing either anti-viral capsid antigen (VCA) or anti-Epstein Barr nuclear antigen-1 (EBNA-1) IgG levels in a unique cohort of 424 individuals of European origin from 119 French families recruited through a Hodgkin lymphoma (HL) patient. No major locus controlling anti-VCA antibody levels was identified. However, we found that the HLA region influenced anti-EBNA-1 IgG titers. Refined association studies in this region identified a cluster of HLA class II variants associated with anti-EBNA-1 IgG titers (e.g. p = 5×10–5 for rs9268403). The major allele of rs9268403 conferring a predisposition to high anti-EBNA-1 antibody levels was also associated with an increased risk of HL (p = 0.02). In summary, this study shows that HLA class II variants influenced anti-EBNA-1 IgG titers in a European population. It further shows the role of the same variants in the risk of HL.     

LILRB2 Interaction with HLA Class I Correlates with Control of HIV-1 Infection

Natural progression of HIV-1 infection depends on genetic variation in the human major histocompatibility complex (MHC) class I locus, and the CD8⁺ T cell response is thought to be a primary mechanism of this effect. However, polymorphism within the MHC may also alter innate immune activity against human immunodeficiency virus type 1 (HIV-1) by changing interactions of human leukocyte antigen (HLA) class I molecules with leukocyte immunoglobulin-like receptors (LILR), a group of immunoregulatory receptors mainly expressed on myelomonocytic cells including dendritic cells (DCs). We used previously characterized HLA allotype-specific binding capacities of LILRB1 and LILRB2 as well as data from a large cohort of HIV-1-infected individuals (N = 5126) to test whether LILR-HLA class I interactions influence viral load in HIV-1 infection. Our analyses in persons of European descent, the largest ethnic group examined, show that the effect of HLA-B alleles on HIV-1 control correlates with the binding strength between corresponding HLA-B allotypes and LILRB2 (p = 10⁻²). Moreover, overall binding strength of LILRB2 to classical HLA class I allotypes, defined by the HLA-A/B/C genotypes in each patient, positively associates with viral replication in the absence of therapy in patients of both European (p = 10⁻¹¹–10⁻⁹) and African (p = 10⁻⁵–10⁻³) descent. This effect appears to be driven by variations in LILRB2 binding affinities to HLA-B and is independent of individual class I allelic effects that are not related to the LILRB2 function. Correspondingly, in vitro experiments suggest that strong LILRB2-HLA binding negatively affects antigen-presenting properties of DCs. Thus, we propose an impact of LILRB2 on HIV-1 disease outcomes through altered regulation of DCs by LILRB2-HLA engagement.     

Influence of HLA Class I Haplotypes on HIV-1 Seroconversion and Disease Progression in Pumwani Sex Worker Cohort

We examined the effect of HLA class I haplotypes on HIV-1 seroconversion and disease progression in the Pumwani sex worker cohort. This study included 595 HIV-1 positive patients and 176 HIV negative individuals. HLA-A, -B, and -C were typed to 4-digit resolution using sequence-based typing method. HLA class I haplotype frequencies were estimated using PyPop 32-0.6.0. The influence of haplotypes on time to seroconversion and CD4+ T cell decline to <200 cells/mm³ were analyzed by Kaplan-Meier analysis using SPSS 13.0. Before corrections for multiple comparisons, three 2-loci haplotypes were significantly associated with faster seroconversion, including A*23∶01-C*02∶02 (p = 0.014, log rank(LR) = 6.06, false-discovery rate (FDR) = 0.056), B*42∶01-C*17∶01 (p = 0.01, LR = 6.60, FDR = 0.08) and B*07∶02-C*07∶02 (p = 0.013, LR = 6.14, FDR = 0.069). Two A*74∶01 containing haplotypes, A*74∶01-B*15∶03 (p = 0.047, LR = 3.942, FDR = 0.068) and A*74∶01-B*15∶03-C*02∶02 (p = 0.045, LR = 4.01, FDR = 0.072) and B*14∶02-C*08∶02 (p = 0.021, LR = 5.36, FDR = 0.056) were associated with slower disease progression. Five haplotypes, including A*30∶02-B*45∶01 (p = 0.0008, LR = 11.183, FDR = 0.013), A*30∶02-C*16∶01 (p = 0.015, LR = 5.97, FDR = 0.048), B*53∶01-C*04∶01 (p = 0.010, LR = 6.61, FDR = 0.08), B*15∶10-C*03∶04 (p = 0.031, LR = 4.65, FDR = 0.062), and B*58∶01-C*03∶02 (p = 0.037, LR = 4.35, FDR = 0.066) were associated with faster progression to AIDS. After FDR corrections, only the associations of A*30∶02-B*45∶01 and A*30∶02-C*16∶01 with faster disease progression remained significant. Cox regression and deconstructed Kaplan-Meier survival analysis showed that the associations of haplotypes of A*23∶01-C*02∶02, B*07∶02-C*07∶02, A*74∶01-B*15∶03, A*74∶01-B*15∶03-C*02∶02, B*14∶02-C*08∶02 and B*58∶01-C*03∶02 with differential seroconversion or disease progression are due to the dominant effect of a single allele within the haplotypes. The true haplotype effect was observed with A*30∶02-B*45∶01, A*30∶02-C*16∶02, B*53∶01-C*04∶01 B*15∶10-C*03∶04, and B*42∶01-C*17∶01. In these cases, the presence of both alleles accelerated the disease progression or seroconversion than any of the single allele within the haplotypes. Our study showed that the true effects of HLA class I haplotypes on HIV seroconversion and disease progression exist and the associations of HLA class I haplotype can also be due to the dominant effect of a single allele within the haplotype.     

Presentation of Phagocytosed Antigens by MHC Class I and II

Phagocytosis provides innate immune cells with a mechanism to take up and destroy pathogenic bacteria, apoptotic cells and other large particles. In some cases, however, peptide antigens from these particles are preserved for presentation in association with major histocompatibility complex (MHC) class I or class II molecules in order to stimulate antigen‐specific T cells. Processing and presentation of antigens from phagosomes presents a number of distinct challenges relative to antigens internalized by other means; while bacterial antigens were among the first discovered to be presented to T cells, analyses of the cellular mechanisms by which peptides from phagocytosed antigens assemble with MHC molecules and by which these complexes are then expressed at the plasma membrane have lagged behind those of conventional model soluble antigens. In this review, we cover recent advances in our understanding of these processes, including the unique cross‐presentation of phagocytosed antigens by MHC class I molecules, and in their control by signaling modalities in phagocytic cells.     

Additive Contribution of HLA Class I Alleles in the Immune Control of HIV-1 Infection

Previous studies have identified a central role for HLA-B alleles in influencing control of HIV infection. An alternative possibility is that a small number of HLA-B alleles may have a very strong impact on HIV disease outcome, dominating the contribution of other HLA alleles. Here, we find that even following the exclusion of subjects expressing any of the HLA-B class I alleles (B*57, B*58, and B*18) identified to have the strongest influence on control, the dominant impact of HLA-B alleles on virus set point and absolute CD4 count variation remains significant. However, we also find that the influence of HLA on HIV control in this C-clade-infected cohort from South Africa extends beyond HLA-B as HLA-Cw type remains a significant predictor of virus and CD4 count following exclusion of the strongest HLA-B associations. Furthermore, there is evidence of interdependent protective effects of the HLA-Cw*0401-B*8101, HLA-Cw*1203-B*3910, and HLA-A*7401-B*5703 haplotypes that cannot be explained solely by linkage to a protective HLA-B allele. Analysis of individuals expressing both protective and detrimental alleles shows that even the strongest HLA alleles appear to have an additive rather than dominant effect on HIV control at the individual level. Finally, weak but significant frequency-dependent effects in this cohort can be detected only by looking at an individual''s combined HLA allele frequencies. Taken together, these data suggest that although individual HLA alleles, particularly HLA-B, can have a strong impact, HIV control overall is likely to be influenced by the additive effect of some or all of the other HLA alleles present.HIV-specific CD8⁺ T cells play a central role in resolution of primary viremia and the long-term suppression of viral replication (13). Supporting this notion is the observed correlation between possession of particular human leukocyte antigen (HLA) class I alleles and control of HIV, measured both directly by time-to-AIDS (5, 6) and indirectly via clinical markers of disease progression (viral load [VL] and CD4 count) (15, 26, 28). Specific HLA class I alleles have been associated with relatively successful control of viral replication and slow disease progression, most notably, alleles HLA-B*57 and HLA-B*27 (1, 7, 12, 15, 21, 23), and also with relatively ineffective control of viral replication and rapid disease progression [B*35(Px), B*5802, and B*18] (5, 15, 17, 23). In addition, general trends suggesting an HLA class I heterozygote advantage (5) and rare allele advantage (28) and, most recently, a correlation between levels of surface expression linked to certain HLA-Cw alleles (11, 27) and HIV control has also been described.Among the different HLA class I loci, the HIV-specific CD8⁺ T-cell responses restricted by HLA-B alleles are thought to play the central role in determining disease outcome: the majority of detectable HIV-specific CD8⁺ T-cell responses are restricted by HLA-B alleles (3, 15, 16), HLA-B-restricted responses typically express a more effective “polyfunctional” phenotype (14), the strongest HLA-associations with either slow or rapid progression are with HLA-B alleles (5, 10, 11, 15), and HLA-B-restricted CD8⁺ T cells exert the strongest selection pressure on the virus (15, 19, 24). However, whether this apparent association between HIV immune control and HLA-B is a general and causal trend or, rather, is biased by the coincidence that the strongest HLA associations with either extreme of disease control happen, by chance, to involve HLA-B alleles remains uncertain.In order to further investigate the correlation between HLA type and HIV infection control, we here examine a cohort now comprising >1,200 chronically HIV C-clade-infected, treatment-naïve subjects from Durban, South Africa, in an extended analysis following from our previous studies of a smaller cohort (15). We first address the question of whether the dominant role of HLA-B in this population compared to the roles of HLA-A or HLA-C results from the influence of HLA-B alleles in general or is dependent on a few known strong associations, such as that between HLA-B*57 alleles and low viremia. Second, in light of recent data (11, 27), we assess the impact of HLA-C alleles on HIV disease outcome and examine the effect of HLA haplotypes on observed HLA associations with disease control. Third, we investigate the question of whether the impact of certain HLA-B alleles on HIV outcome dominates that of other HLA-B alleles to negate the contribution of the latter or whether the impact of individual HLA alleles can be additive. Finally, we compare the impact of individual HLA alleles on HIV on immune control to the impact of heterozygote and rare allele advantage in this cohort.     

HLA Class I and Class II Associations with ESRD in Saudi Arabian Population

Background

Chronic renal failure (CRF) leads in the majority of instances to end stage renal disease (ESRD) requiring renal replacement therapy. Our interest was to evaluate the possible associations of HLA class I and class II antigens with ESRD independent of other factors, in Saudi Arabia population.

Methodology

A retrospective study to determine the HLA class I and class II polymorphisms and their association with ESRD, was performed on 350 patients with ESRD, and 105 healthy unrelated control. Patients and control groups were typed by SSOP lumenix techniques. The alleles positively associated to the ESRD were: HLA-B*15, B*18, B*49 - DRB1*03, negatively associated alleles were A*26, HLA-B*39, B*50. The haplotypes positively associated with ESRD were: HLA-A*01-DRB1*13 and HLA-A*30-DRBI*03. The negatively associated haplotypes were: HLA-A*02-B*39, A*02-B*50, A*24-B*35, A*24-B*58, A*24-DRB1*16, A*68-DRB1*04, A*02-DQB1*03, A*29-DQB1*02, A*29-DOB1*05 and B*27-DRB1*07 and the last one is the most significant protective haplotypes.

Conclusion

The high Relative Risk (RR) observed and its statistical correlation reflect the strength of the described association between HLA antigens and ESRD.     

HIV-1 Gag Cytotoxic T Lymphocyte Epitopes Vary in Presentation Kinetics Relative to HLA Class I Downregulation

Although CD8⁺ cytotoxic T lymphocytes (CTLs) are protective in HIV-1 infection, the factors determining their antiviral efficiency are poorly defined. It is proposed that Gag targeting is superior because of very early Gag epitope presentation, allowing early killing of infected cells before Nef-mediated downregulation of human leukocyte antigen class I (HLA-I). To study Gag epitope presentation kinetics, three epitopes (SL9_77-85, KF11_162-172, and TW10_240-249) were genetically translocated from their endogenous location in the Rev-dependent (late) gag gene into the Rev-independent (early) nef gene with concomitant mutation of the corresponding endogenous epitopes to nonrecognized sequences. These viruses were compared to the index virus for CTL-mediated suppression of replication and the susceptibility of this antiviral activity to Nef-mediated HLA-I downregulation. SL9-specific CTLs gained activity after SL9 translocation to Nef, going from Nef sensitive to Nef insensitive, indicating that translocation accelerated infected cell recognition from after to before HLA-I downregulation. KF11-specific CTL antiviral activity was unchanged and insensitive to HLA-I downregulation before and after KF11 translocation, suggesting that already rapid recognition of infected cells was not accelerated. However, TW10-specific CTLs that were insensitive to Nef at the baseline became sensitive with reduced antiviral activity after translocation, indicating that translocation retarded epitope expression. Cytosolic peptide processing assays suggested that TW10 was inefficiently generated after translocation to Nef, compared to SL9 and KF11. As a whole, these data demonstrate that epitope presentation kinetics play an important role in CTL antiviral efficiency, that Gag epitopes are not uniformly presented early, and that the epitope context can play a major role in presentation kinetics.     

Estimating the Impact of Plasma HIV-1 RNA Reductions on Heterosexual HIV-1 Transmission Risk

Background

The risk of sexual transmission of HIV-1 is strongly associated with the level of HIV-1 RNA in plasma making reduction in HIV-1 plasma levels an important target for HIV-1 prevention interventions. A quantitative understanding of the relationship of plasma HIV-1 RNA and HIV-1 transmission risk could help predict the impact of candidate HIV-1 prevention interventions that operate by reducing plasma HIV-1 levels, such as antiretroviral therapy (ART), therapeutic vaccines, and other non-ART interventions.

Methodology/Principal Findings

We use prospective data collected from 2004 to 2008 in East and Southern African HIV-1 serodiscordant couples to model the relationship of plasma HIV-1 RNA levels and heterosexual transmission risk with confirmation of HIV-1 transmission events by HIV-1 sequencing. The model is based on follow-up of 3381 HIV-1 serodiscordant couples over 5017 person-years encompassing 108 genetically-linked HIV-1 transmission events. HIV-1 transmission risk was 2.27 per 100 person-years with a log-linear relationship to log₁₀ plasma HIV-1 RNA. The model predicts that a decrease in average plasma HIV-1 RNA of 0.74 log₁₀ copies/mL (95% CI 0.60 to 0.97) reduces heterosexual transmission risk by 50%, regardless of the average starting plasma HIV-1 level in the population and independent of other HIV-1-related population characteristics. In a simulated population with a similar plasma HIV-1 RNA distribution the model estimates that 90% of overall HIV-1 infections averted by a 0.74 copies/mL reduction in plasma HIV-1 RNA could be achieved by targeting this reduction to the 58% of the cohort with plasma HIV-1 levels ≥4 log₁₀ copies/mL.

Conclusions/Significance

This log-linear model of plasma HIV-1 levels and risk of sexual HIV-1 transmission may help estimate the impact on HIV-1 transmission and infections averted from candidate interventions that reduce plasma HIV-1 RNA levels.     

Immunogenicity of HLA Class I and II Double Restricted Influenza A-Derived Peptides

The aim of the present study was to identify influenza A-derived peptides which bind to both HLA class I and -II molecules and by immunization lead to both HLA class I and class II restricted immune responses. Eight influenza A-derived 9-11mer peptides with simultaneous binding to both HLA-A*02:01 and HLA-DRB1*01:01 molecules were identified by bioinformatics and biochemical technology. Immunization of transgenic HLA-A*02:01/HLA-DRB1*01:01 mice with four of these double binding peptides gave rise to both HLA class I and class II restricted responses by CD8 and CD4 T cells, respectively, whereas four of the double binding peptides did result in HLA-A*02:01 restricted responses only. According to their cytokine profile, the CD4 T cell responses were of the Th2 type. In influenza infected mice, we were unable to detect natural processing in vivo of the double restricted peptides and in line with this, peptide vaccination did not decrease virus titres in the lungs of intranasally influenza challenged mice. Our data show that HLA class I and class II double binding peptides can be identified by bioinformatics and biochemical technology. By immunization, double binding peptides can give rise to both HLA class I and class I restricted responses, a quality which might be of potential interest for peptide-based vaccine development.     

The Effect of HLA Polymorphisms on the Recognition of Gag Epitopes in HIV-1 CRF01_AE Infection

Introduction

The design of a globally effective vaccine rests on the identification of epitopes capable of eliciting effective cytotoxic T lymphocyte (CTL) responses across multiple HIV clades in different populations. This study aims to discern the effect of HLA polymorphisms and the cross-clade reactivity or clade-specificity of epitopes in Thailand where HIV-1 CRF01_AE is circulating.

Materials and Methods

14 peptides based on consensus HIV-1 CRF01_AE amino acid sequences were designed for use in IFN-γ ELISpot assays and ⁵¹Cr release assays among 66 HIV-1 CRF01_AE-infected Thai patients. For ELISpot responders carrying HLA alleles currently unknown to restrict CRF01_AE epitopes, in silico epitope-HLA prediction was performed.

Results

29/66 (43.9%) patients recognized at least one peptide. In total 79 responses were seen against all 14 peptides. 28/79 (35.4%) of the responses were in patients with HLA alleles previously reported to restrict CRF01_AE epitopes, 24/79 (30.4%) responses were in individuals with HLA alleles previously reported to restrict epitopes of HIV clades other than CRF01_AE, and the remaining 27/79 (34.2%) responses were not associated with HLA alleles previously known to restrict HIV epitopes. In silico epitope prediction detected 19 novel, epitope-HLA combinations, and 11/19 (57.9%) were associated with HLA-C alleles. We further confirmed a novel HLA restriction of a previously identified HIV-1 Gag epitope [p24_122–130: PPIPVGDIY (PY9)] by HLA-B*40:01 with a standard ⁵¹Cr release assay.

Discussion

CTL recognition sites in HIV-1 Gag were similar among different clades but the HLA restriction differed in Thai patients. This disparity in HLA restriction along different populations illustrated the importance of clade- and population-specific HLA analysis prior to CTL vaccine design.     

Widespread impact of HLA restriction on immune control and escape pathways of HIV-1

The promiscuous presentation of epitopes by similar HLA class I alleles holds promise for a universal T-cell-based HIV-1 vaccine. However, in some instances, cytotoxic T lymphocytes (CTL) restricted by HLA alleles with similar or identical binding motifs are known to target epitopes at different frequencies, with different functional avidities and with different apparent clinical outcomes. Such differences may be illuminated by the association of similar HLA alleles with distinctive escape pathways. Using a novel computational method featuring phylogenetically corrected odds ratios, we systematically analyzed differential patterns of immune escape across all optimally defined epitopes in Gag, Pol, and Nef in 2,126 HIV-1 clade C-infected adults. Overall, we identified 301 polymorphisms in 90 epitopes associated with HLA alleles belonging to shared supertypes. We detected differential escape in 37 of 38 epitopes restricted by more than one allele, which included 278 instances of differential escape at the polymorphism level. The majority (66 to 97%) of these resulted from the selection of unique HLA-specific polymorphisms rather than differential epitope targeting rates, as confirmed by gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISPOT) data. Discordant associations between HLA alleles and viral load were frequently observed between allele pairs that selected for differential escape. Furthermore, the total number of associated polymorphisms strongly correlated with average viral load. These studies confirm that differential escape is a widespread phenomenon and may be the norm when two alleles present the same epitope. Given the clinical correlates of immune escape, such heterogeneity suggests that certain epitopes will lead to discordant outcomes if applied universally in a vaccine.     

Identification of Disease-Promoting HLA Class I and Protective Class II Modifiers in Japanese Patients with Familial Mediterranean Fever

ObjectivesThe genotype-phenotype correlation of MEFV remains unclear for the familial Mediterranean fever (FMF) patients, especially without canonical MEFV mutations in exon 10. The risk of FMF appeared to be under the influence of other factors in this case. The contribution of HLA polymorphisms to the risk of FMF was examined as strong candidates of modifier genes.MethodsGenotypes of HLA-B and -DRB1 loci were determined for 258 mutually unrelated Japanese FMF patients, who satisfied modified Tel-Hashomer criteria, and 299 healthy controls. The effects of carrier status were evaluated for the risk of FMF by odds ratio (OR). The HLA effects were also assessed for clinical forms of FMF, subsets of FMF with certain MEFV genotypes and responsiveness to colchicine treatment.ResultsThe carriers of B*39:01 were increased in the patients (OR = 3.25, p = 0.0012), whereas those of DRB1*15:02 were decreased (OR = 0.45, p = 0.00050), satisfying Bonferroni’s correction for multiple statistical tests (n = 28, p<0.00179). The protective effect of DRB1*15:02 was completely disappeared in the co-existence of B*40:01. The HLA effects were generally augmented in the patients without a canonical MEFV variant allele M694I, in accordance with the notion that the lower penetrance of the mutations is owing to the larger contribution of modifier genes in the pathogenesis, with a few exceptions. Further, 42.9% of 14 colchicine-resistant patients and 13.5% of 156 colchicine-responders possessed B*35:01 allele, giving OR of 4.82 (p = 0.0041).ConclusionsThe differential effects of HLA class I and class II polymorphisms were identified for Japanese FMF even in those with high-penetrance MEFV mutations.     

HLA Class I and Class II Alleles and Haplotypes Confirm the Berber Origin of the Present Day Tunisian Population

In view of its distinct geographical location and relatively small area, Tunisia witnessed the presence of many civilizations and ethnic groups throughout history, thereby questioning the origin of present-day Tunisian population. We investigated HLA class I and class II gene profiles in Tunisians, and compared this profile with those of Mediterranean and Sub-Sahara African populations. A total of 376 unrelated Tunisian individuals of both genders were genotyped for HLA class I (A, B) and class II (DRB1, DQB1), using reverse dot-blot hybridization (PCR-SSO) method. Statistical analysis was performed using Arlequin software. Phylogenetic trees were constructed by DISPAN software, and correspondence analysis was carried out by VISTA software. One hundred fifty-three HLA alleles were identified in the studied sample, which comprised 41, 50, 40 and 22 alleles at HLA-A,-B,-DRB1 and -DQB1 loci, respectively. The most frequent alleles were HLA-A*02:01 (16.76%), HLA-B*44:02/03 (17.82%), HLA-DRB1*07:01 (19.02%), and HLA-DQB1*03:01 (17.95%). Four-locus haplotype analysis identified HLA-A*02:01-B*50:01-DRB1*07:01-DQB1*02:02 (2.2%) as the common haplotype in Tunisians. Compared to other nearby populations, Tunisians appear to be genetically related to Western Mediterranean population, in particular North Africans and Berbers. In conclusion, HLA genotype results indicate that Tunisians are related to present-day North Africans, Berbers and to Iberians, but not to Eastern Arabs (Palestinians, Jordanians and Lebanese). This suggests that the genetic contribution of Arab invasion of 7^th-11^th century A.D. had little impact of the North African gene pool.     

AIDS restriction HLA allotypes target distinct intervals of HIV-1 pathogenesis

An effective acquired immune response to infectious agents mediated by HLA-restricted T-cell recognition can target different stages of disease pathogenesis. We show here that three distinct HLA alleles known to alter the overall rate of AIDS progression act during distinct intervals after HIV-1 infection. The discrete timing of HLA allele influence suggests alternative functional mechanisms in immune defense against this dynamic and chronic immunosuppressive disease.     

Class I and II HLA genes are associated with susceptibility and age at onset in Finnish families with type 1 diabetes

OBJECTIVES: We explored the properties of the long-term survivor model (LTS) in the genetic association studies and studied allelic and haplotypic associations between the age at onset and partially latent susceptibility of type 1 diabetes (T1DM) and Human Leucocyte Antigen (HLA) A, B and DR loci. METHODS: The authors applied the long-term survivor model (LTS) for sibships collected in a population-based registry during a calendar time period. The method uses sibs that could not become probands and includes the proband's age at onset during the recruitment period. Association between the candidate gene and the partially latent susceptibility is modeled with logistic regression and the age at onset with a two-parameter gamma distribution, where a scale parameter depends on the candidate genotypes. We also performed a simulation study of nuclear families to compare the power of the likelihood ratio tests of the genetic association based on the LTS model with those obtained using family-based association method (FBAT) and bias of the case-pseudo control design. In addition, we analysed allele and haplotype associations between HLA A, B and DR loci (IDDM1) with T1DM, using population-based ascertainment of 705 sibships with complete HLA information. RESULTS: A simulation study showed that the estimates of the genetic association using an ascertainment-corrected LTS model are virtually unbiased and that the relative risk estimates obtained from case-pseudo control design (TDT) are negatively biased. In the analysis of the Finnish T1DM families we found that only B62 (p < 0.05) is positively significantly associated with susceptibility after adjusting for the haplotype effects. Five alleles were significantly associated with age at onset (B8 and DR3, p < 0.01; A2, B60 and DR6, p < 0.05). No significant three-locus haplotype associations with the susceptibility were found, but A3B18DR4 (p < 0.001) haplotype was associated with older age at onset than average. CONCLUSIONS: Estimates of genetic relative risk obtained from the case-pseudo control design are negatively biased and the prospective LTS model is an appropriate choice, when there are non-susceptible subjects in the population with variable age at onset. Based on the analysis of T1DM, we conclude that there are gene(s) in the HLA region that are associated with susceptibility and/or age at onset of T1DM, and this should be taken into account in future studies.