Investigation of NOTCH4 coding region polymorphisms in sporadic inclusion body myositis

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Abstract

The NOTCH4 gene, located within the MHC region, is involved in cellular differentiation and has varying effects dependent on tissue type. Coding region polymorphisms haplotypic of the sIBM-associated 8.1 ancestral haplotype were identified in NOTCH4 and genotyped in two different Caucasian sIBM cohorts. In both cohorts the frequency of the minor allele of rs422951 and the 12-repeat variation for rs72555375 was increased and was higher than the frequency of the sIBM-associated allele HLA-DRB1*0301. These NOTCH4 polymorphisms can be considered to be markers for sIBM susceptibility, but require further investigation to determine whether they are directly involved in the disease pathogenesis.

Introduction

Sporadic inclusion body myositis (sIBM) is a chronic inflammatory myopathy which occurs in individuals over the age of 40 years. Pathologically, the disease is characterised by a combination of a mixed inflammatory infiltrate with myofibre invasion by CD8 + T-cells and macrophages (Arahata and Engel, 1984), and degenerative changes in muscle fibres including vacuolation, congophilic filamentous inclusions (Dalakas, 2006) and greater than expected numbers of cytochrome c oxidase (COX) deficient fibres suggesting an acceleration of the normal aging process (Needham et al., 2007). The pathogenesis of sIBM remains elusive, but genetic factors are considered to play an important role in determining susceptibility to the disease (Needham et al., 2007).

The most consistent genetic association in Caucasians with sIBM is with the Class II MHC allele HLA-DRB1*0301 (Garlepp et al., 1994), which in Caucasian populations is carried on the 8.1 ancestral haplotype (AH) (Price et al., 2004) as defined by the alleles HLA-A*0101, B*0801, DRB1*0301, and DQB1*0201 (Cattley et al., 2000). The sIBM susceptibility region in the MHC has been refined to the border of the Class II and III regions, specifically the region between PBX2 and HLA-DRB1 (Price et al., 2004). In a recent recombination mapping study we further refined the 8.1AH-derived sIBM susceptibility region to a 172 kb segment containing three genes — BTNL2, HLA-DRA and HLA-DRB3 (Scott et al., 2011). However, as the number of patients was relatively small, this requires further verification and other genes in the immediate vicinity, such as NOTCH4, cannot be disregarded for a possible role in sIBM susceptibility.

The NOTCH gene family acts as a driver of general cell development and differentiation in many cell types, including skeletal muscle, from embryonic to adult life and with effects dependent on the conditions and cell type (Li et al., 1998, Artavanis-Tsakonas et al., 1999, Kadesch, 2004). These molecular targets and binding partners of NOTCH4 in skeletal muscle may therefore be different from those in other tissues, presenting the possibility of a muscle-specific effect of NOTCH4 variants. Murine Notch4 is known to enhance stem cell activity and to reduce cellular differentiation and alter lymphoid cell development (Vercauteren and Sutherland, 2004). In sIBM, myogenic differentiation of mesoangioblasts into myotubes in vitro is known to be severely impaired, suggesting that myofibre regeneration in vivo may also be defective (Morosetti et al., 2006). Genes involved in regulating cellular differentiation, such as NOTCH4, are thus candidates for playing a role in this aspect of sIBM pathology. Given the expression of NOTCH4 in skeletal muscle (Li et al., 1998) and its role in regulating cellular differentiation, NOTCH4 variants could play a part in the pathogenesis of sIBM by inhibiting myofibre regeneration, or by generating antigenic peptides that are specific to muscle. Genetic variations within NOTCH4 could also influence susceptibility to sIBM through other mechanisms.

We have therefore investigated the association of NOTCH4 functional variants with sIBM in two different patient cohorts. The focus of this analysis has been NOTCH4 variants most likely to affect function, specifically non-synonymous polymorphisms within the coding region. Specific polymorphisms were also selected where the minor allele was characteristic of the 8.1AH. Polymorphisms likely to alter gene expression, such as those in the promoter region, were not considered in view of the finding in a previous study that NOTCH4 mRNA expression is not altered in sIBM muscle tissue (Greenberg et al., 2002).

Section snippets

Patients

An Australian cohort of sIBM patients was expanded from 42 patients studied previously (Price et al., 2004). DNA was obtained from a total of 77 Caucasian sIBM patients from the Inflammatory Myopathies Clinic at the Australian Neuromuscular Research Institute (ANRI), the Department of Neurology at Royal Melbourne Hospital, the Neuromuscular Clinic at the Monash Medical Centre and the Neuromuscular Clinic at Concord Hospital. All had biopsy-proven disease and fulfilled the criteria for definite

Selection of polymorphisms and genotyping

NOTCH4 polymorphisms were identified from a sequence alignment of four homozygous cell lines — COX (8.1AH), PGF (7.1AH), QBL (18.2AH) and SSTO (44.XAH). Comparison of the NOTCH4 coding region identified five polymorphisms where the minor allele was found in the COX cell line (8.1AH), but not PGF (7.1AH), QBL (18.2AH) or SSTO (44.1AH). These were rs422951 (A  G, Thr320Ala), rs915894 (A  C, Lys117Gln), rs3134942 (C  A, Val1384Val), rs443198 (T  C, Gly111Gly), and rs72555375 (CTG6–14, Leun6Leun)

The CTG

Discussion

Given the presence of NOTCH4 in the immediate vicinity of the previously defined sIBM susceptibility region in the MHC and its role in cellular differentiation, polymorphisms within the NOTCH4 coding region were identified and assessed in two separate Caucasian sIBM patient cohorts, one Australian and the other American. Four coding region polymorphisms for which the minor allele was characteristic of the sIBM-associated 8.lAH were investigated. The rs422951, rs915894, and rs443198

Acknowledgements

We thank Professor A. Corbett from Concord Hospital in Sydney and Associate Professors T. Day and L. Kiers from the Royal Melbourne Hospital in Melbourne who referred cases of sIBM for the study. We also thank the DNA sequencing service provided through the Department of Clinical Immunology and Biochemical Genetics at Royal Perth Hospital. This study was supported by the Australian National Health and Medical Research Council (NH&MRC) Fellowship Grants 403941 and 392500.

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