Richard Allcock

Mutations in KLHL40 Are a Frequent Cause of Severe Autosomal-Recessive Nemaline Myopathy

Nemaline myopathy (NEM) is a common congenital myopathy. At the very severe end of the NEM clinical spectrum are genetically unresolved cases of autosomal-recessive fetal akinesia sequence. We studied a multinational cohort of 143 severe-NEM-affected families lacking genetic diagnosis. We performed whole-exome sequencing of six families and targeted gene sequencing of additional families. We identified 19 mutations in KLHL40 (kelch-like family member 40) in 28 apparently unrelated NEM kindreds of various ethnicities. Accounting for up to 28% of the tested individuals in the Japanese cohort, KLHL40 mutations were found to be the most common cause of this severe form of NEM. Clinical features of affected individuals were severe and distinctive and included fetal akinesia or hypokinesia and contractures, fractures, respiratory failure, and swallowing difficulties at birth. Molecular modeling suggested that the missense substitutions would destabilize the protein. Protein studies showed that KLHL40 is a striated-muscle-specific protein that is absent in KLHL40-associated NEM skeletal muscle. In zebrafish, klhl40a and klhl40b expression is largely confined to the myotome and skeletal muscle, and knockdown of these isoforms results in disruption of muscle structure and loss of movement. We identified KLHL40 mutations as a frequent cause of severe autosomal-recessive NEM and showed that it plays a key role in muscle development and function. Screening of KLHL40 should be a priority in individuals who are affected by autosomal-recessive NEM and who present with prenatal symptoms and/or contractures and in all Japanese individuals with severe NEM.

The human TREM gene cluster at 6p21.1 encodes both activating and inhibitory single IgV domain receptors and includes NKp44

We have characterized a cluster of single immunoglobulin variable (IgV) domain receptors centromeric of the major histocompatibility complex (MHC) on human chromosome 6. In addition to triggering receptor expressed on myeloid cells (TREM)‐1 and TREM2, the cluster contains NKp44, a triggering receptor whose expression is limited to NK cells. We identified three new related genes and two gene fragments within a cluster of ∼200 kb. Two of the three new genes lack charged residues in their transmembrane domain tails. Further, one of the genes contains two potential immunotyrosine inhibitory motifs in its cytoplasmic tail, suggesting that it delivers inhibitory signals. The human and mouse TREM clusters appear to have diverged such that there are unique sequences in each species. Finally, each gene in the TREM cluster was expressed in a different range of cell types.

Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy

Nemaline myopathy (NM) is a rare congenital muscle disorder primarily affecting skeletal muscles that results in neonatal death in severe cases as a result of associated respiratory insufficiency. NM is thought to be a disease of sarcomeric thin filaments as six of eight known genes whose mutation can cause NM encode components of that structure, however, recent discoveries of mutations in non-thin filament genes has called this model in question. We performed whole-exome sequencing and have identified recessive small deletions and missense changes in the Kelch-like family member 41 gene (KLHL41) in four individuals from unrelated NM families. Sanger sequencing of 116 unrelated individuals with NM identified compound heterozygous changes in KLHL41 in a fifth family. Mutations in KLHL41 showed a clear phenotype-genotype correlation: Frameshift mutations resulted in severe phenotypes with neonatal death, whereas missense changes resulted in impaired motor function with survival into late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of Klhl41 results in highly diminished motor function and myofibrillar disorganization, with nemaline body formation, the pathological hallmark of NM. These studies expand the genetic heterogeneity of NM and implicate a critical role of BTB-Kelch family members in maintenance of sarcomeric integrity in NM.

Multiple immune factors are involved in controlling acute and chronic chikungunya virus infection

The recent epidemic of the arthritogenic alphavirus, chikungunya virus (CHIKV) has prompted a quest to understand the correlates of protection against virus and disease in order to inform development of new interventions. Herein we highlight the propensity of CHIKV infections to persist long term, both as persistent, steady-state, viraemias in multiple B cell deficient mouse strains, and as persistent RNA (including negative-strand RNA) in wild-type mice. The knockout mouse studies provided evidence for a role for T cells (but not NK cells) in viraemia suppression, and confirmed the role of T cells in arthritis promotion, with vaccine-induced T cells also shown to be arthritogenic in the absence of antibody responses. However, MHC class II-restricted T cells were not required for production of anti-viral IgG2c responses post CHIKV infection. The anti-viral cytokines, TNF and IFNγ, were persistently elevated in persistently infected B and T cell deficient mice, with adoptive transfer of anti-CHIKV antibodies unable to clear permanently the viraemia from these, or B cell deficient, mice. The NOD background increased viraemia and promoted arthritis, with B, T and NK deficient NOD mice showing high-levels of persistent viraemia and ultimately succumbing to encephalitic disease. In wild-type mice persistent CHIKV RNA and negative strand RNA (detected for up to 100 days post infection) was associated with persistence of cellular infiltrates, CHIKV antigen and stimulation of IFNα/β and T cell responses. These studies highlight that, secondary to antibodies, several factors are involved in virus control, and suggest that chronic arthritic disease is a consequence of persistent, replicating and transcriptionally active CHIKV RNA.

SPEG interacts with myotubularin, and its deficiency causes centronuclear myopathy with dilated cardiomyopathy.

Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy.

Susceptibility to Multiple Sclerosis Mediated by HLA-DRB1 is Influenced by a Second Gene Telomeric of the TNF Cluster

Susceptibility to multiple sclerosis (MS) is clearly associated with human leukocyte antigen (HLA)-DRB1*1501, but some studies show associations with HLA-B7 and -B18. These are often co-expressed with DRB1*1501 in the ancestral haplotypes (AH) denoted 7.1 (HLA-A3, B7, tumor necrosis factor [TNF]a11b4, DRB1*1501) and 18.1 (HLA-A25, B18, TNFa10b4, DRB 1*1501). Here we present a systematic study of 218 patients and 274 controls typed at all standard class II and TNF microsatellite loci, and a novel non-synonymous polymorphism in the central major histocompatibility complex gene, inhibitor of kappa B-like protein (IKBL). The C allele at IKBL+738 is only found on the 7.1 haplotype. HLA-DRB1*1501 was associated with disease, as expected. When subjects expressing DRB 1*501 were analyzed separately, TNFa11b4 and IKBL+738C were less common in the patients and, hence, mark an allele that mediates resistance which lies telomeric of IKBL. TNFa10b4 and TNFa1b5 were more common in DRB1*1501 patients than in controls. These alleles have been associated with the 18.1 and 18.2 AH, respectively. Since no component of these haplotypes was an independent risk factor in this study, it appears likely that a gene linked to TNFa10b4 and TNFa1b5 modifies the effect of the susceptibility locus marked by HLA-DRB1*1501. Potential candidate genes telomeric of the TNF cluster are discussed.

Polymorphisms of the CRP gene inhibit inflammatory response and increase susceptibility to depression: The Health in Men Study

BACKGROUND Depression has been associated with chronic changes in the serum concentration of C-reactive protein (CRP) in observational studies, but it is unclear if this association is causal or is due to confounding and bias. Genetic studies are less subject to this type of error and offer an opportunity to investigate if CRP is causally linked to depression, particularly because known polymorphisms of the CRP gene have been associated with high- and low-basal serum concentrations of CRP [single nucleotide polymorphisms (SNPs) rs1130864 and rs1205, respectively]. The aim of this study is to determine if polymorphisms of SNPs rs1130864 and rs1205 are associated with prevalent depression. METHODS We completed a cross-sectional study of a community sample of 3700 men aged > or = 70 years, and used the 15-item Geriatric Depression Scale (GDS-15) to assess depressive symptoms. A GDS-15 score 7 or more indicates the presence of clinically significant depressive symptoms. Physical morbidity was assessed with the physical component summary score (PCS) of the SF-36 Health Survey. We collected fasting blood samples to measure high sensitivity CRP and to extract DNA for the genotyping of SNPs rs1130864 and rs1205 of the CRP gene. RESULTS One hundred and eighty-two men were depressed (4.9%). The odds of depression increased by 2% (95% CI = 1-4%) for every unit (mg/l) increase of CRP and nearly doubled for men with CRP > or = 3 mg/l vs <1 mg/l [odds ratio (OR) = 1.95, 95% confidence interval (CI) = 1.27-2.98]. However, the association between high CRP (> or = 3 mg/l) and depression was no longer significant after the analyses were adjusted for smoking, age, body mass index (BMI) and PCS. Men with the CT and TT genotypes of rs1130864 had 1.36 (95% CI = 1.13-1.63) and 2.31 (95% CI = 1.65-3.24) greater odds of CRP > or = 3 mg/l than CC carriers, but there was no association between this polymorphism and the presence of prevalent depression. The G > A polymorphism of SNP rs1205 was associated with 24% (95% CI = 16-32%) lower concentration of CRP compared with other genotypes. Men with the rs1205 AA genotype had 1.66 (95% CI = 1.07-2.57) and 1.67 (95% CI = 1.08-2.58) greater odds of having clinically significant depression than participants with the GA and GG genotypes, respectively. CONCLUSION Our study shows that clinically significant depressive symptoms in later life are unlikely to be caused by an increase in the serum concentration of CRP. Instead, we found that the risk of depression was greater amongst people who carry the rs1205 G > A genetic polymorphism of the CRP gene, which was associated with approximately 20% lower serum concentration of CRP compared with other genotypes. This suggests that CRP may be a compensatory response to external insults that predispose to depression, and that an increase in the concentration of CRP might be adaptive.

The central MHC gene, BAT1, may encode a protein that down-regulates cytokine production.

BAT1 belongs to the DEAD‐box family of RNA‐binding proteins and is encoded in the central MHC. To determine whether it affects immune responses and hence diseases influenced by MHC haplotypes, U937, THP1 and Jurkat cells were stably transfected with anti‐sense DNA corresponding to exons 2–5 of BAT1 using a retroviral vector.

16(th) IHIW : review of HLA typing by NGS.

Human leucocyte antigen (HLA) genes play an important role in the success of organ transplantation and are associated with autoimmune and infectious diseases. Current DNA‐based genotyping methods, including Sanger sequence‐based typing (SSBT), have identified a high degree of polymorphism. This level of polymorphism makes high‐resolution HLA genotyping challenging, resulting in ambiguous typing results due to an inability to resolve phase and/or defining polymorphisms lying outside the region amplified. Next‐generation sequencing (NGS) may resolve the issue through the combination of clonal amplification, which provides phase information, and the ability to sequence larger regions of genes, including introns, without the additional effort or cost associated with current methods. The NGS HLA sequencing project of the 16IHIW aimed to discuss the different approaches to (i) template preparation including short‐ and long‐range PCR amplicons, exome capture and whole genome; (ii) sequencing platforms, including GS 454 FLX, Ion Torrent PGM, Illumina MiSeq/HiSeq and Pacific Biosciences SMRT; (iii) data analysis, specifically allele‐calling software. The pilot studies presented at the workshop demonstrated that although individual sequencers have very different performance characteristics, all produced sequence data suitable for the resolution of HLA genotyping ambiguities. The developments presented at this workshop clearly highlight the potential benefits of NGS in the HLA laboratory.

Sporadic inclusion body myositis: HLA-DRB1 allele interactions influence disease risk and clinical phenotype

Susceptibility to sIBM is strongly associated with the HLA-DRB1*03 allele and the 8.1 MHC ancestral haplotype (HLA-A1, B8, DRB1*03) but little is known about the effects of allelic interactions at the DRB1 locus or disease-modifying effects of HLA alleles. HLA-A, B and DRB1 genotyping was performed in 80 Australian sIBM cases and the frequencies of different alleles and allele combinations were compared with those in a group of 190 healthy controls. Genotype-phenotype correlations were also investigated. Amongst carriers of the HLA-DRB1*03 allele, DRB1*03/*01 heterozygotes were over-represented in the sIBM group (p<0.003) while. DRB1*03/*04 heterozygotes were under-represented (p<0.008). The mean age-at-onset (AAO) was 6.5 years earlier in DRB1*03/*01 heterozygotes who also had more severe quadriceps muscle weakness than the rest of the cohort. The findings indicate that interactions between the HLA-DRB1*03 allele and other alleles at the DRB1 locus can influence disease susceptibility and the clinical phenotype in sIBM.