Exome sequencing identifies novel dysferlin mutation in a family with pauci-symptomatic heterozygous carriers.

BACKGROUND: We investigated a South African family of admixed ancestry in which the first generation (G1) developed insidious progressive distal to proximal weakness in their twenties, while their offspring (G2) experienced severe unexpected symptoms of myalgia and cramps since adolescence. Our aim was to identify deleterious mutations that segregate with the affected individuals in this family. METHODS: Exome sequencing was performed on five cases, which included three affected G1 siblings and two pauci-symptomatic G2 offspring. As controls we included an unaffected G1 sibling and a spouse of one of the G1 affected individuals. Homozygous or potentially compound heterozygous variants that were predicted to be functional and segregated with the affected G1 siblings, were further evaluated. Additionally, we considered variants in all genes segregating exclusively with the affected (G1) and pauci-symptomatic (G2) individuals to address the possibility of a pseudo-autosomal dominant inheritance pattern in this family. RESULTS: All affected G1 individuals were homozygous for a novel truncating p.Tyr1433Ter DYSF (dysferlin) mutation, with their asymptomatic sibling and both pauci-symptomatic G2 offspring carrying only a single mutant allele. Sanger sequencing confirmed segregation of the variant. No additional potentially contributing variant was found in the DYSF or any other relevant gene in the pauci-symptomatic carriers. CONCLUSION: Our finding of a truncating dysferlin mutation confirmed dysferlinopathy in this family and we propose that the single mutant allele is the primary contributor to the neuromuscular symptoms seen in the second-generation pauci-symptomatic carriers.

Exome sequencing identifies targets in the treatment-resistant ophthalmoplegic subphenotype of myasthenia gravis.

Treatment-resistant ophthalmoplegia (OP-MG) is not uncommon in individuals with African genetic ancestry and myasthenia gravis (MG). To identify OP-MG susceptibility genes, extended whole exome sequencing was performed using extreme phenotype sampling (11 OP-MG vs 4 control-MG) all with acetylcholine receptor-antibody positive MG. This approach identified 356 variants that were twice as frequent in OP-MG compared to control-MG individuals. After performing probability test estimates and filtering variants according to those 'suggestive' of association with OP-MG (p < 0.05), only three variants remained which were expressed in extraocular muscles. Validation in 25 OP-MG and 50 control-MG cases supported the association of DDX17delG (p = 0.014) and SPTLC3insACAC (p = 0.055) with OP-MG, but ST8SIA1delCCC could not be verified by Sanger sequencing. A parallel approach, using a semantic model informed by current knowledge of MG-pathways, identified an African-specific interleukin-6 receptor (IL6R) variant, IL6R c.*3043 T>C, that was more frequent in OP-MG compared to control-MG cases (p = 0.069) and population controls (p = 0.043). A weighted genetic risk score, derived from the odds ratios of association of these variants with OP-MG, correlated with the OP-MG phenotype as opposed to control MG. This unbiased approach implicates several potentially functional gene variants in the gangliosphingolipid and myogenesis pathways in the development of the OP-MG subphenotype.

The role of microRNAs in the therapeutic action of D-cycloserine in a post-traumatic stress disorder animal model: an exploratory study.

OBJECTIVES: Post-traumatic stress disorder is characterized by impaired fear extinction and excessive anxiety. D-Cycloserine (DCS) has previously been shown to facilitate fear extinction and decrease anxiety in animal and human studies. This study utilized a contextual fear-conditioning animal model to investigate the involvement of microRNAs (miRNAs) in fear extinction and the reduction of anxiety, as mediated by the co-administration of DCS and behavioural fear extinction. METHODS: Fear conditioning consisted of an electric foot shock; fear extinction consisted of behavioural fear extinction co-administered with either DCS or saline. The light/dark avoidance test was used to evaluate anxiety-related behaviour subsequent to fear conditioning and was used to evaluate anxiety-related behaviour following fear conditioning and to subsequently group animals into well-adapted and maladapted subgroups. These subgroups also showed significant differences in terms of fear extinction. Small RNAs extracted from the left dorsal hippocampus were sequenced using next-generation sequencing to identify differentially expressed miRNAs associated with DCS-induced fear extinction and reduction of anxiety. In-silico prediction analyses identified mRNA targets (from data of the same animals) of the differentially expressed miRNAs. Two of the predicted mRNA-miRNA interactions were functionally investigated. RESULTS: Overall, 32 miRNAs were differentially expressed between rats that were fear conditioned, received DCS and were well adapted and rats that were fear conditioned, received saline and were maladapted. Nineteen of these miRNAs were predicted to target and regulate the expression of 63 genes differentially expressed between fear-conditioned, DCS-administered, well-adapted and fear-conditioned, saline-administered, and maladapted groups (several of which are associated with neuronal inflammation, learning and memory). Functional luciferase assays indicated that rno-mir-31a-5p may have regulated the expression of interleukin 1 receptor antagonist (Il1rn) and metallothionein 1a (Mt1a). CONCLUSION: These differentially expressed miRNAs may be mediators of gene expression changes that facilitated decreased neuronal inflammation, optimum learning and memory and contributed towards effective fear extinction and reduction of anxiety following the co-administration of DCS and behavioural fear extinction.

A practical guide to filtering and prioritizing genetic variants.

Next-generation sequencing (NGS) of whole genomes and exomes is a powerful tool in biomedical research and clinical diagnostics. However, the vast amount of data produced by NGS introduces new challenges and opportunities, many of which require novel computational and theoretical approaches when it comes to identifying the causal variant(s) for a disease of interest. While workflows and associated software to process raw data and produce high-confidence variant calls have significantly improved, filtering tens of thousands of candidates to identify a subset relevant to a specific study is still a complex exercise best left to bioinformaticists. However, as this prioritization procedure requires biological/biomedical reasoning, biologists and clinicians are increasingly motivated to handle the task themselves. Here, we describe a set of guidelines, tools, and online resources that can be used to identify functional variants from whole-genome and whole-exome variant calls and then prioritize these variants with potential associations to phenotypes of interest. Insights gained from a recently published analysis of protein-coding gene variation in >60,000 humans by the Exome Aggregation Consortium (ExAC) are also taken into account.

Semantic interrogation of a multi knowledge domain ontological model of tendinopathy identifies four strong candidate risk genes.

Tendinopathy is a multifactorial syndrome characterised by tendon pain and thickening, and impaired performance during activity. Candidate gene association studies have identified genetic factors that contribute to intrinsic risk of developing tendinopathy upon exposure to extrinsic factors. Bioinformatics approaches that data-mine existing knowledge for biological relationships may assist with the identification of candidate genes. The aim of this study was to data-mine functional annotation of human genes and identify candidate genes by ontology-seeded queries capturing the features of tendinopathy. Our BioOntological Relationship Graph database (BORG) integrates multiple sources of genomic and biomedical knowledge into an on-disk semantic network where human genes and their orthologs in mouse and rat are central concepts mapped to ontology terms. The BORG was used to screen all human genes for potential links to tendinopathy. Following further prioritisation, four strong candidate genes (COL11A2, ELN, ITGB3, LOX) were identified. These genes are differentially expressed in tendinopathy, functionally linked to features of tendinopathy and previously implicated in other connective tissue diseases. In conclusion, cross-domain semantic integration of multiple sources of biomedical knowledge, and interrogation of phenotypes and gene functions associated with disease, may significantly increase the probability of identifying strong and unobvious candidate genes in genetic association studies.

Population-specific common SNPs reflect demographic histories and highlight regions of genomic plasticity with functional relevance.

BACKGROUND: Population differentiation is the result of demographic and evolutionary forces. Whole genome datasets from the 1000 Genomes Project (October 2012) provide an unbiased view of genetic variation across populations from Europe, Asia, Africa and the Americas. Common population-specific SNPs (MAF > 0.05) reflect a deep history and may have important consequences for health and wellbeing. Their interpretation is contextualised by currently available genome data. RESULTS: The identification of common population-specific (CPS) variants (SNPs and SSV) is influenced by admixture and the sample size under investigation. Nine of the populations in the 1000 Genomes Project (2 African, 2 Asian (including a merged Chinese group) and 5 European) revealed that the African populations (LWK and YRI), followed by the Japanese (JPT) have the highest number of CPS SNPs, in concordance with their histories and given the populations studied. Using two methods, sliding 50-SNP and 5-kb windows, the CPS SNPs showed distinct clustering across large genome segments and little overlap of clusters between populations. iHS enrichment score and the population branch statistic (PBS) analyses suggest that selective sweeps are unlikely to account for the clustering and population specificity. Of interest is the association of clusters close to recombination hotspots. Functional analysis of genes associated with the CPS SNPs revealed over-representation of genes in pathways associated with neuronal development, including axonal guidance signalling and CREB signalling in neurones. CONCLUSIONS: Common population-specific SNPs are non-randomly distributed throughout the genome and are significantly associated with recombination hotspots. Since the variant alleles of most CPS SNPs are the derived allele, they likely arose in the specific population after a split from a common ancestor. Their proximity to genes involved in specific pathways, including neuronal development, suggests evolutionary plasticity of selected genomic regions. Contrary to expectation, selective sweeps did not play a large role in the persistence of population-specific variation. This suggests a stochastic process towards population-specific variation which reflects demographic histories and may have some interesting implications for health and susceptibility to disease.