Michael J. Chao

Expression of the Multiple Sclerosis-Associated MHC Class II Allele HLA-DRB1*1501 Is Regulated by Vitamin D

Multiple sclerosis (MS) is a complex trait in which allelic variation in the MHC class II region exerts the single strongest effect on genetic risk. Epidemiological data in MS provide strong evidence that environmental factors act at a population level to influence the unusual geographical distribution of this disease. Growing evidence implicates sunlight or vitamin D as a key environmental factor in aetiology. We hypothesised that this environmental candidate might interact with inherited factors and sought responsive regulatory elements in the MHC class II region. Sequence analysis localised a single MHC vitamin D response element (VDRE) to the promoter region of HLA-DRB1. Sequencing of this promoter in greater than 1,000 chromosomes from HLA-DRB1 homozygotes showed absolute conservation of this putative VDRE on HLA-DRB1*15 haplotypes. In contrast, there was striking variation among non–MS-associated haplotypes. Electrophoretic mobility shift assays showed specific recruitment of vitamin D receptor to the VDRE in the HLA-DRB1*15 promoter, confirmed by chromatin immunoprecipitation experiments using lymphoblastoid cells homozygous for HLA-DRB1*15. Transient transfection using a luciferase reporter assay showed a functional role for this VDRE. B cells transiently transfected with the HLA-DRB1*15 gene promoter showed increased expression on stimulation with 1,25-dihydroxyvitamin D3 (P = 0.002) that was lost both on deletion of the VDRE or with the homologous “VDRE” sequence found in non–MS-associated HLA-DRB1 haplotypes. Flow cytometric analysis showed a specific increase in the cell surface expression of HLA-DRB1 upon addition of vitamin D only in HLA-DRB1*15 bearing lymphoblastoid cells. This study further implicates vitamin D as a strong environmental candidate in MS by demonstrating direct functional interaction with the major locus determining genetic susceptibility. These findings support a connection between the main epidemiological and genetic features of this disease with major practical implications for studies of disease mechanism and prevention.

An extremes of outcome strategy provides evidence that multiple sclerosis severity is determined by alleles at the HLA-DRB1 locus

Multiple sclerosis (MS) is a common inflammatory disease of the central nervous system unsurpassed for variability in disease outcome. A cohort of sporadic MS cases (n = 163), taken from opposite extremes of the distribution of long-term outcome, was used to determine the role of the HLA-DRB1 locus on MS disease severity. Genotyping sets of benign and malignant MS patients showed that HLA-DRB1*01 was significantly underrepresented in malignant compared with benign cases. This allele appears to attenuate the progressive disability that characterizes MS in the long term. The observation was doubly replicated in (i) Sardinian benign and malignant patients and (ii) a cohort of affected sibling pairs discordant for HLA-DRB1*01. Among the latter, mean disability progression indices were significantly lower in those carrying the HLA-DRB1*01 allele compared with their disease-concordant siblings who did not. The findings were additionally supported by similar transmission distortion of HLA-DRB1*04 subtypes closely related to HLA-DRB1*01. The protective effect of HLA-DRB1*01 in sibling pairs may result from a specific epistatic interaction with the susceptibility allele HLA-DRB1*1501. A high-density (>700) SNP examination of the MHC region in the benign and malignant patients could not identify variants differing significantly between the two groups, suggesting that HLA-DRB1 may itself be the disease-modifying locus. We conclude that HLA-DRB1*01, previously implicated in disease resistance, acts as an independent modifier of disease progression. These results closely link susceptibility to long-term outcome in MS, suggesting that shared quantitative MHC-based mechanisms are common to both, emphasizing the central role of this region in pathogenesis.

Epistasis among HLA-DRB1, HLA-DQA1, and HLA-DQB1 loci determines multiple sclerosis susceptibility

Multiple sclerosis (MS), a common central nervous system inflammatory disease, has a major heritable component. Susceptibility is associated with the MHC class II region, especially HLA-DRB5*0101–HLA-DRB1*1501–HLA-DQA1*0102–HLA-DQB1*0602 haplotypes(hereafter DR2), which dominate genetic contribution to MS risk. Marked linkage disequilibrium (LD) among these loci makes identification of a specific locus difficult. The once-leading candidate, HLA-DRB1*15, localizes to risk, neutral, and protective haplotypes. HLA-DRB1*15 and HLA-DQB1*0602, nearly always located together on a small ancestral chromosome segment, are strongly MS-associated. One intervening allele on this haplotype, viz. HLA-DQA1*0102, shows no primary MS association. Two Canadian cohorts (n = 830 and n = 438 trios) genotyped for HLA-DRB1, HLA-DQA1 and HLA-DQB1 alleles were tested for association using TDT. To evaluate epistasis involving HLA-DRB1*15, transmissions from HLA-DRB1*15-negative parents were stratified by the presence/absence of HLA-DRB1*15 in affected offspring. All 3 alleles contribute to MS susceptibility through novel epistatic interactions. HLA-DQA1*0102 increased disease risk when combined with HLA-DRB1*1501 in trans, thereby unambiguously implicating HLA-DQ in MS susceptibility. Three-locus haplotypes demonstrated that HLA-DRB1*1501 and HLA-DQB1*0602 each influence risk. Transmissions of rare morcellated DR2 haplotypes showed no interaction with HLA-DQA1*0102. Incomplete haplotypes bearing only HLA-DRB1*1501 or HLA-DQB1*0602 did not predispose to MS. Balanced reciprocal transmission distortion can mask epistatic allelic association. These findings implicate epistasis among HLA class II alleles in human immune responses generally, provide partial explanation for intense linkage disequilibrium in the MHC, have relevance to animal models, and demonstrate key roles for DR2-specific interactions in MS susceptibility. MHC disease associations may be more generally haplotypic or diplotypic.

Epigenetics in multiple sclerosis susceptibility: difference in transgenerational risk localizes to the major histocompatibility complex

Multiple sclerosis (MS) susceptibility demonstrates a complex pattern of inheritance. Haplotypes containing HLA-DRB1*1501 carry most of the genetic risk. Epidemiological evidence implicating epigenetic factors includes complex distortion of disease transmission seen in aunt/uncle-niece/nephew (AUNN) pairs. Unexpectedly, in AUNN families we found that allele frequencies for HLA-DRB1*1501 were different between the first and second generations affected. Affected aunts had significantly lower HLA-DRB1*15 frequency compared with their affected nieces (chi(2) = 9.90, P = 0.0016), whereas HLA-DRB1*15 frequency in affected males remains unaltered across the two generations (chi(2) = 0.23, P = 0.63). We compared transmissions for the HLA-DRB1*15 allele using a family-based transmission disequilibrium test approach in 1690 individuals from 350 affected sibling pair (ASP) families and 960 individuals from 187 AUNN families. Transmissions differed between the ASP and the AUNN families (chi(2) = 6.92; P = 0.0085). The risk carried by HLA-DRB1*15 was increased in families with affected second-degree relatives (AUNN: OR = 4.07) when compared with those consisting only first-degree relatives (ASP: OR = 2.17), establishing heterogeneity of risk among HLA-DRB1*15 haplotypes based on whether collateral parental relatives are affected. These observations strongly implicate gene-environment interactions in susceptibility and more specifically, that epigenetic modifications differentiate among human leukocyte antigen class II risk haplotypes and are involved in the determination of the gender bias in MS. These data strongly suggest that the female-specific increasing risk of MS is mediated through these alleles or adjacent variation. The comparison of transmission of the same allele in vertically affected pedigrees (AUNN) to collinear sibling pairs (ASP) may provide a useful screen for putative epigenetic marks.

HLA class I alleles tag HLA-DRB1*1501 haplotypes for differential risk in multiple sclerosis susceptibility.

The major locus for multiple sclerosis (MS) susceptibility is located within the class II region of the Major Histocompatibility Complex (MHC). HLA-DRB1 alleles, constituting the strongest MS susceptibility factors, have been widely exploited in research including construction of transgenic animal models of MS. Many studies have concluded that HLA-DRB1*15 allele itself determines MS-associated susceptibility. If this were true, haplotypes bearing this allele would confer equal risk. If HLA-DRB1*15 bearing haplotypes differed for risk, roles for other loci in this region would be implied and further study of the fine structure of this locus would be compelling. We have tested the hypothesis comparing haplotypes stratified by HLA class I tagging. We show here that HLA-DRB1*15-bearing-haplotypes in 1970 individuals from 494 MS families are indeed heterogeneous. Some HLA-DRB1*15 haplotypes determine susceptibility while others do not. Three groups of class I tagged HLA-DRB1*15 haplotypes were not over-transmitted: (i) HLA-DRB1*15-HLA-B*08 (TR = 25, NT = 23, Odds Ratio = 1.09), (ii) -HLA-B*27 (TR = 18, NT = 17, Odds Ratio = 1.06), and (iii) rare HLA-DRB1*15 haplotypes (frequency <0.02). Rare haplotypes were significantly different from common haplotypes, and transmissions were remarkably similar to those for class-I-matched non-HLA-DRB1*15 haplotypes. These results unambiguously indicate that HLA-DRB1*15 is part of a susceptibility haplotype but cannot be the susceptibility allele itself, requiring either epistatic interactions, epigenetic modifications on some haplotypes, or nearby structural variation. These findings strongly imply that differences among HLA-DRB1*15 haplotypes will furnish the basis for MHC-associated susceptibility in MS and raise the possibility that the MHC haplotype is the fundamental unit of genetic control of immune response.

Parent-of-origin effects in MS Observations from avuncular pairs

Background: Multiple sclerosis (MS) is a complex neurologic disease of unknown etiology and inheritance pattern, but with increasing incidence among females. The study of aunt/uncle–niece/nephew (AUNN) pairs has potential to shed light on the on complex trait inheritance as this group can be divided into eight different pair types by gender, MS status, and parent of origin. Methods: Using a cohort of 807 avuncular MS families with 938 affected AUNN pairs ascertained from a longitudinal, population-based Canadian database, we examined differential MS transmission by separating affected pairs into likely maternal and paternal trait origin. Results: We observed an increased number of avuncular pairs connected through unaffected mothers compared to unaffected fathers (p = 0.008). To restrict confounders introduced by families with multiple pairs the overall number of maternal and paternal families were compared, to reveal a significantly higher number of maternal families (p = 0.038). Female-to-male sex ratios were higher among affected nieces/nephews when compared to the sex ratio for aunts/uncles (0.00042). Conclusions: This observation independently confirms previous findings of a “maternal parent-of-origin” effect in multiple sclerosis (MS) susceptibility. These findings highlight the special contribution that can be derived from avuncular pairs. These underutilized pairings can compare transmission by the gender of affected aunt–uncle, the unaffected transmitting parent, and by that of the affected offspring. This strategy may be especially profitable in diseases where parent-of-origin effects are being sought. These findings also independently confirm the increasing rate of MS in females, demonstrating that familial cases are influenced by the same environmental factors as the general MS population.

MHC transmission: insights into gender bias in MS susceptibility.

Objective: Major histocompatibility complex (MHC) genes dominate genetic susceptibility factors in multiple sclerosis (MS). Given the general consensus that incidence and prevalence of MS has been rising and specifically in women, we evaluated MHC–gender interactions. Methods: In a large family-based cohort consisting of 7,093 individuals (2,127 affected individuals) from 1,055 MS families, we examined MHC transmission by family structure and gender stratified by genetic distance of affected relatives from the MS proband. Results: We found that affected individuals with HLA-DRB1*15-positive genotypes have higher female-to-male ratios as compared with affected individuals with HLA-DRB1*15-negative genotypes (χ2 = 9.97, p = 0.0015) with the exception of multiplex families with 3 or more affected across 2 generations. Transmission disequilibrium test results show that HLA-DRB1*15 transmission was more distorted in collateral families vs nuclear families (χ2 = 8.030, p = 0.0046), exclusively in affected female-female pairs (χ2 = 7.81, p = 0.0051), but not in mixed gender pairs (χ2 = 1.58, p = 0.21) or matched male pairs (Fisher p = 0.21). Conclusions: These observations implicate the MHC as the site of interactions and modifications mediating the female-to-male gender ratio in MS and its progressive increase. They further suggest this occurs via gene–environment interactions and epigenetic modifications in this region. The difference between collateral and nuclear families provides some insight into the inheritance, decay, and gender specificity of putative epigenetic marks.

Parental transmission of MS in a population-based Canadian cohort

Objective: Genetic and environmental factors have important roles in multiple sclerosis (MS) susceptibility. The precise nature of these factors and mode of inheritance remains unknown. A female predominance is universally found. Recently, offspring of affected fathers were reported to be more likely to have MS than those of affected mothers. This was attributed to the Carter effect, which is seen in polygenic disorders. The Carter effect predicts that affected parents of the sex lesser affected by a disease/trait are more genetically loaded for risk alleles and thus transmit these more often to their offspring. This hypothesis was tested in a population-based Canadian MS cohort. Methods: Using the longitudinal Canadian database, we identified 3,088 nuclear families with one affected parent and a total of 8,401 offspring, of which 798 had MS. Transmission to daughters and sons from affected mothers and fathers was compared. Results: There was equal transmission of MS from affected fathers vs affected mothers (9.41% vs 9.76%). Stratifying by gender of affected parent there were no differences in the female:male sex ratio of affected (2.46% vs 2.41%, p = 0.88) or unaffected offspring (0.91% vs 0.95%, p = 0.46). Conclusions: We observed1 equal disease transmission to offspring from affected mothers and affected fathers,2 no difference in the female:male sex ratio of affected offspring, and previously3 no difference in sibling recurrence risk by gender of parent affected. These findings show no evidence for the Carter effect and do not support the hypothesis of polygenic inheritance of multiple sclerosis susceptibility by parent.

Parent-of-origin effects at the major histocompatibility complex in multiple sclerosis

Multiple sclerosis (MS) susceptibility is characterized by maternal parent-of-origin effects and increased female penetrance. In 7796 individuals from 1797 MS families (affected individuals n = 2954), we further implicate epigenetic modifications within major histocompatibility complex (MHC) class II haplotypes as mediating these phenomena. Affected individuals with the main MS-associated allele HLA-DRB1*15 had a higher female-to-male ratio versus those lacking it (P = 0.00023). Distorted transmission of MHC haplotypes by both parent-of-origin and gender-of-affected-offspring was most evident in the maternal HLA-DRB1*15 transmission to affected female offspring (OR = 3.31, 95% CI = 2.59-4.24) contrasting with similarity among maternal transmission to affected male offspring (OR = 2.13, 95% CI = 1.44-3.14), paternal transmissions to affected female (OR = 2.14, 95% CI = 1.64-2.78) and male (OR = 2.16, 95% CI = 1.37-3.39) offspring. Significant parent-of-origin effects were observed in affected females (maternal: P = 9.33 x 10(-42); paternal: P = 1.12 x 10(-15); comparison: P = 0.0014), but not in affected males (maternal: P = 6.70 x 10(-8); paternal: P = 2.54 x 10(-6); comparison: P = 0.95). Conditional logistic regression analysis revealed further differential risk of HLA diplotypes. Risks for HLA-DRB1*15 and likely for other HLA-DRB1 haplotypes were restricted by (i) parent-of-origin, (ii) gender-of-offspring and (iii) trans epistasis in offspring. These findings may illuminate the gender bias characterizing autoimmunity overall. They raise questions about the concept of restricted antigen presentation in autoimmunity and suggest that gender-specific epigenetic interactions may be the driving forces behind the MHC haplotypic associations. Haplotype-specific epigenetic modifications at MHC class II and their decay appear to be at the heart of MS pathogenesis and inheritance of risk, providing the focus for gene-environment interactions that determine susceptibility and resistance.