Patricia Martinez

Genomics of the major histocompatibility complex: haplotypes, duplication, retroviruses and disease

Summary: The genomic region encompassing the Major Histocompatibility Complex (MHC) contains polymorphic frozen blocks which have developed by local imperfect sequential duplication associated with insertion and deletion (indels), In the alpha block surrounding HLA‐A, there are ten duplication units or beads on the 62,1 ancestral haplotype. Each bead contains or contained sequences representing Class 1, PERB11 (MHC Class I chain related (MIC)) and human endogenous retrovirus (HERV) 16, Here we consider explanations for co‐occurrence of genomic polymorphism, duplication and HERVs and we ask how these features encode susceptibility to numerous and very diverse diseases. Ancestral haplotypes differ in their copy number and indels in addition to their coding regions. Disease susceptibility could be a function of all of these differences. We propose a model of the evolution of the human MHC. Population‐specific integration of retroviral sequences could explain rapid diversification through duplication and differential disease susceptibility. If HERV sequences can be protective, there are exciting prospects for manipulation. In the mean‐while, it will be necessary to understand the function of MHC genes such as PEKB11 (MIC) and many others discovered by genomic sequencing.

High-resolution HLA-DRB1 genotyping in an Australian inclusion body myositis (s-IBM) cohort: An analysis of disease-associated alleles and diplotypes

We performed high-resolution (4-digit) HLA-DRB1 genotyping in an Australian cohort of 105s-IBM patients and 189 controls. Our findings showed that whilst the strongest association was with the HLA-DRB1*03:01 allele and the HLA-DRB1*03:01/*01:01 diplotype, HLA-DRB1*01:01 and HLA-DRB1*13:01 are also risk alleles. A number of other alleles, HLA-DRB1*04:01, *04:04, *07:01, *09:01, *11:01 and *15:01, as well as the HLA-DRB1*03:01/*04:01 and HLA-DRB1*03:01/*07:01 diplotypes were reduced in s-IBM cases and may be protective. The HLA-DRB1*03:01 and HLA-DRB1*13:01 alleles also appear to have an influence on the age at onset of the disease and severity of muscle weakness. Our findings indicate that the influence of HLA-DRB1 in s-IBM is complex and that epistatic interactions at the HLA-DRB1 locus contribute both to disease susceptibility and to the clinical phenotype.

Alu polymorphism within the MICB gene and association with HLA-B alleles

We describe the finding of an Alu repeat dimorphism within the first intron of the MICB gene. The frequencies of the two AluyMICB alleles, AluyMICB*0 (absence of insertion) and AluyMICB*1 (presence of insertion), and their associations with the highly polymorphic HLA-B locus were determined for 51 human cell lines and for 109 and 200 Caucasians and northeastern Thais, respectively. Analysis of the AluyMICB and HLA-B allelic relationships revealed that AluyMICB*1 occurred at relatively low gene frequency (0.113–0.118) but was strongly associated with HLA-B17 (HLA-B57,HLA-B58) and HLA-B13. The AluyMICB locus provides a useful dimorphic marker for investigations on the level of linkage disequilibrium between MICB, MICA, and HLA-B loci.

The Association Between HLA-A Alleles and an Alu Dimorphism Near HLA-G

The AluYb8 sequences are a subfamily of short interspersed Alu retroelements that have been amplified within the human genome during recent evolutionary time and are useful polymorphic markers for studies on the origin of human populations. We have identified a new member of the Yb8 subfamily, AluyHG, located between the HLA-H and -G genes and 88-kb telomeric of the highly polymorphic HLA-A gene within the alpha block of the major histocompatibility complex (MHC). The AluyHG element was characterised with a view to examining the association between AluyHG and HLA-A polymorphism and reconstructing the history of the MHC alpha block. A specific primer pair was designed for a simple PCR assay to detect the absence or presence (dimorphism) of the AluyHG element within the DNA samples prepared from a panel of 46 homozygous cell-lines containing complete or recombinant ancestral haplotypes (AH) of diverse ethnic origin and 92 Caucasoid and Asian subjects on which HLA-A typing was available. The AluyHG insertion was most strongly associated with HLA-A2 and, to a lesser degree with HLA-A1, -A3, -A11, and A-19. The gene frequency of the AluyHG insertion for 146 Caucasians and 94 Chinese-Han was 0.30 and 0.32 and there was no significant difference between the observed and expected frequencies. The results of the association studies and the phylogenetic analysis of HLA-A alleles suggest that the AluyHG sequence was integrated within the progenitor of HLA-A2, but has been transferred by recombination to other human ancestral populations. In this regard, the dimorphic AluyHG element is an important diagnostic marker for HLA association studies and could help in elucidating the evolution and functions of the MHC alpha block and polymorphism within and between ancestral haplotypes.

Frequency of autoantibodies and correlation with HLA-DRB1 genotype in sporadic inclusion body myositis (s-IBM): A population control study

The frequency of myositis-associated and myositis-specific autoantibodies (AAb) was compared in 51 s-IBM patients and a population control group. Non-organ specific AAb (ANA, anti-Ro52, anti-Ro60, anti-La, anti-RNP) but not anti-thyroid peroxidase, anti-tissue transglutaminase or myositis-specific antibodies, were more frequent in s-IBM patients, and 14/51 (27%) had another autoimmune disease (Sjögrens syndrome, thyroiditis, psoriasis, vitiligo). The presence of AAb did not correlate with carriage of HLA-DRB1*0301, but there was a negative correlation between ANA/anti-Ro52 and carriage of HLA-DRB1*1301. The findings in this cohort confirm that patients with sIBM do not show evidence of a muscle-specific humoral immune process but have an increased frequency of non-organ specific AAb and other autoimmune disorders.

Analysis of HLA-DRB3 alleles and supertypical genotypes in the MHC Class II region in sporadic inclusion body myositis

We compared the carriage frequencies of HLA-DRB3 and its major alleles and of HLA-DRB4 and HLA-DRB5 in an Australian sIBM cohort and a population control group who had previously been genotyped for the HLA-DRB1*03:01 risk allele. There was a strong disease association with the carriage of the DRB3*01:01 allele which was accounted for by its linkage disequilibrium with DRB1*03:01. The carriage of HLA-DRB4 was found to be strongly protective and abrogated the risk effect of HLA-DRB1*03:01. The findings indicate that haplotypic combinations of alleles at the HLA-DRB1 and secondary HLA-DRB loci have important risk modifying effects in sIBM.

Establishment of gene copy number-specific normal ranges for serum C4 and its utility for interpretation in patients with chronically low serum C4 concentrations.

To establish gene copy number (GCN)–specific normal ranges for serum C4 genes and to determine their utility with respect to the interpretation of chronically low serum C4 concentrations in patients with clinically quiescent systemic lupus erythematosus (SLE).

Insights on the relationship between complement component C4 serum concentrations and C4 gene copy numbers in a Western Australian systemic lupus erythematosus cohort

The relationship between serum concentration of complement C4 ([C4]) and C4 gene copy number (GCN) was investigated in 56 systemic lupus erythematosus (SLE) patients and 33 age and sex-matched controls in a Western Australian population. C4A and C4B gene copy numbers (C4A & B GCN) together with the presence or absence of the ≈6.4-kb human endogenous retroviral element type K (hereafter HERV-K) in intron 9 were estimated by two TaqMan™ real-time PCR (RT-PCR) assays that measured total C4 and HERV-K GCNs, respectively. There was good correlation between the two methods; however, the HERV-K GCN method showed a positive bias (≈6%) relative to the C4A & B total GCN. Despite individual variation, excellent correlation between total C4 GCN and mean [C4] per GCN was observed for both the SLE and control cohorts (R2 = 88% and R2 = 99%, respectively). It was noted that serum [C4] was significantly lower in the SLE patients than the controls (p = 0.006) despite there being no difference between C4A and C4B GCN in both cohorts. The data therefore confirm previous reports that the C4A genes are preferentially associated with the presence of the HERV-K insertion relative to C4B genes and does not support the hypothesis that low [C4] in SLE is explained by low C4A GCNs. There was no evidence also that the presence of the HERV-K insertion in C4 genes influenced [C4]. This study supports the view that low [C4] in SLE patients is due to consumption rather than deficient synthesis related to lower C4A & B GCN.

G.P.63 High-resolution analysis of HLA-DRB1 alleles and diplotypes in an Australian inclusion body myositis cohort

In Caucasians, susceptibility to sporadic inclusion body myositis (s-IBM) is associated with the HLA-DR3 serotype and the HLA-DRB1∗0301 allele which encodes it, and with the extended 8.1 MHC ancestral haplotype. Most previous HLA studies in s-IBM have used serological typing or low-resolution (2-digit) genotyping to identify disease-associated alleles, and the contribution of alleles other than HLA-DRB1∗0301 and of the complementary in trans allele at the HLA-DRB1 locus has received little attention. In the present study we performed high-resolution (4-digit) HLA-DRB1 genotyping in an Australian cohort of 105 s-IBM patients and 189 controls from the Busselton Population Study and analysed risk-associated alleles and effects on age-at-onset (AAO) of the disease. Case–control logistic regression analysis showed that, apart from the strongly associated HLA-DRB1∗0301 allele (OR 13.6), HLA-DRB1∗0101 (OR 3.65) and HLA-DRB1∗1301 (OR 3.31) are independent risk alleles. A number of other alleles, HLA-DRB1∗0401, ∗0404, ∗0701, ∗0901, ∗1101 and ∗1501 were reduced in frequency in s-IBM cases and may be protective. We evaluated the influence of allele combinations on disease risk and found that the strongest estimated risk is with the HLA-DRB1∗0301/∗0101 diplotype, while other allele combinations have diminishing risk effects. HLA-DRB1∗0301 was associated with an AAO 4.3 years earlier than other alleles and DRB1∗1301 with an AAO 4.5 years later, despite it being a risk factor. The findings indicate that the influence of HLA-DRB1 in s-IBM is complex, influencing both disease risk and AAO, and that allelic interactions at the HLA-DRB1 locus contribute to disease susceptibility.

Potential for paralogous mapping to simplify the genetics of diseases and functions associated with MHC haplotypes

The major histocompatibility complex (MHC) region contains several hundred genes in addition to the well-known HLA Class I and II loci. Some of these will explain MHC-disease associations. We have used paralogous and syntenic mapping to short list candidates and conclude that non-HLA genes present on human 6,1 and 19 and murine 17, 4 and 7 regulate immune responses and autoimmune disease. These genes are likely to be components of the early pre-HLA MHC and include tenascin and possibly notch inter alia.