Ian James

Background. Inclusions of Sequestosome1 (SQSTM1/p62) within muscle fibers are a pathological hallmark of sporadic inclusion body myositis (sIBM) with p62 overexpression reported in patients. Mounting evidence suggests a role for p62 expression and/or variation in sIBM pathology, due to the presence of rare and potentially pathogenic missense variants (A117V, G194R, K238E, P392L). Consequently, we hypothesized that genetic modifiers of SQSTM1 may present a critical missing link for sIBM pathology and contribute to disease expres¬sion. Short structural variants (SSVs) are a class of genetic variation that can be difficult to characterized due to their highly repetitive and complex nature. Evidence that SSVs play an important role in complex diseases such as Alzhei¬mer’s Disease, Amyotrophic lateral sclerosis, Spinocerebellar Ataxia type 2, and Huntington’s disease is now confirmed and further investigations of this type of genetic variation is necessary to uncover missing heritability in complex dis¬eases. We and others have previously reported an SSV within SQSTM1 that is associated with altered expression of p62. The SSV rs60327661 is a CAAA in¬sertion/deletion within intron 5 of SQSTM1, which also confers risk for familial Amyotrophic lateral sclerosis. Due to the role of the SSV in Amyotrophic lateral sclerosis and altered p62 expression, we hypothesized the SSV rs60327661 may have disease-modifying effects in a longitudinal cohort of sIBM patients.

Methods. DNA samples from 218 sIBM patients and 242 healthy controls were received from The Institute for Immunology and Infectious Diseases, Murdoch, Western Australia, and the NINDS Repository, Coriell Institute for Medical Research, New Jersey. Genomic DNA samples were systematically assessed through polymerase chain reaction, capillary separation, and Sanger sequencing for rs60327661 allele genotyping.

Results: In the present study, when controlling for self-declared ancestry, car¬riage of the D/D genotype is associated with sIBM disease expression (p<0.05). Both the case and control groups did not violate Hardy-Weinberg equilibrium (p=0.99, p=0.98; respectively. Intriguingly, patients who were CN1A seroposi¬tive were more likely carry the D allele (n=18) when compared to patients with-out a D allele (n=3; p<0.047). Patients classified as fast progressors (n=2) carried only the D/D genotype.

Conclusion. In this study, we present the first report of an association between the SQSTM1 insertion/deletion and sIBM disease expression. We provide evi¬dence that the investigation of genetic variants outside of the HLA region is war¬ranted, and that such investigations are likely to uncover critical information for sIBM. We present the SSV rs60327661 as a novel disease modifying variant for sIBM which is functionally linked to p62 by altering protein expression. Our data adds to growing evidence that examination of SSVs may uncover novel genetic risk markers, and consequently further understanding of the pathogenic mechanisms at play.

Abacavir (ABC) binds non-covalently to the floor of the peptide-binding groove of HLA-B*57:01, altering the chemistry and shape of the antigen binding cleft. This allows previously untolerized self-peptides to be presented by HLA-B*57:01 which upon T-cell receptor binding initiates a CD8+ T-cell response and a clinical hypersensitivity reaction(HSR). Endoplasmic reticulum aminopeptidases (ERAPs) trim peptides for MHC Class I presentation, influencing the degree and specificity of the CD8+ T-cell response. Genetic variation within ERAP adds to the positive predictive value (PPV) of the HLA class I risk allele in autoimmune diseases such as HLA-B27 positive ankylosing spondylitis. Considering the altered peptide repertoire mechanism of ABC HSR we hypothesize that variation in ERAP may help explain why 45% carrying HLA-B*57:01 can tolerate ABC. SNPs within ERAP1 (rs27044, rs17482078, rs30187, rs27434, rs2287987) were examined in HLA-B*57:01+ ABC HSR patch test positive (PT+)[n=53] and HLA-B*57:01+ ABC tolerant[n=22] with sequence-based typing. Rs2248374, a tag SNP for functional ERAP2 haplotypes was also examined. Haplotype A is tagged by the (A) allele, while haplotype B is tagged by rs2248374(G). Fisher exact tests and multiple logistic regressions were used to compare genotypes between the ABC HSR PT+ and tolerant groups. HLA-B*57:01+ ABC tolerance was associated with rs27434(GG) (18/22(82%) vs 24/53(45%) in ABC HSR PT+, p=0.005). This SNP maps to the active site within ERAP1 (AA356). For an HLA-B*57:01 positive population the estimated PPV for rs27434 genotypes for ABC HSR PT+ is AA(100%), AG(77%) and GG(40%). A missense mutation within the domain junction (rs30187(C)) important in conformation change of ERAP1 (AA528), was overrepresented in HLA-B*57:01+ ABC tolerant individuals (p=0.04). Analysis indicated linkage between rs27044 and rs30187, rs17482078 and rs2287987, and between rs30187 and rs27434 (all p < 0.0001). In a multivariable model with rs27434(GG), the ERAP2 SNP (rs2248372(G)) that tags haplotype B which is characterized by a truncated protein, was decreased in tolerant individuals (p = 0.005). ERAP and particularly ERAP1 variants are important in the development of ABC HSR. ERAP activity may influence the repertoire of peptides presented by HLA-B*57:01 or influence early changes in immunodominant epitope selection. This provides a potential pathogenic mechanism for the development of ABC HSR or ABC tolerance in HLA-B*57:01 carriers.

Hepatitis C is a global health issue with approximately 3% of the worlds’ population estimated to be infected with the hepatitis C virus (HCV) Inefficiencies in treatment has led to development of direct-acting antivirals (DAAs) that specifically target HCV proteins involved in the virus’s lifecycle1. One of the major concerns arising from the use of the DAAs is the emergence of resistance-associated variants (RAVs) that affect the efficacy of the drugs. RAVs are generally associated with a fitness cost and the use of ultra-deep pyrosequencing technology has shown that in most treatment naïve subjects low frequency circulating strains carry RAVs2. The aim of the study was to investigate i) the clinical relevance of low frequency RAVs; ii) the persistence of RAVs and iii) compensatory mutations in a subset of subjects who had failed boceprevir (SCH503034; protease inhibitor).