Phylogenetic analysis of ferlin genes reveals ancient eukaryotic origins

<p>Abstract</p> <p>Background</p> <p>The ferlin gene family possesses a rare and identifying feature consisting of multiple tandem C2 domains and a C-terminal transmembrane domain. Much currently remains unknown about the fundamental function of this gene family, however, mutations in its two most well-characterised members, dysferlin and otoferlin, have been implicated in human disease. The availability of genome sequences from a wide range of species makes it possible to explore the evolution of the ferlin family, providing contextual insight into characteristic features that define the ferlin gene family in its present form in humans.</p> <p>Results</p> <p>Ferlin genes were detected from all species of representative phyla, with two ferlin subgroups partitioned within the ferlin phylogenetic tree based on the presence or absence of a DysF domain. Invertebrates generally possessed two ferlin genes (one with DysF and one without), with six ferlin genes in most vertebrates (three DysF, three non-DysF). Expansion of the ferlin gene family is evident between the divergence of lamprey (jawless vertebrates) and shark (cartilaginous fish). Common to almost all ferlins is an N-terminal C2-FerI-C2 sandwich, a FerB motif, and two C-terminal C2 domains (C2E and C2F) adjacent to the transmembrane domain. Preservation of these structural elements throughout eukaryotic evolution suggests a fundamental role of these motifs for ferlin function. In contrast, DysF, C2DE, and FerA are optional, giving rise to subtle differences in domain topologies of ferlin genes. Despite conservation of multiple C2 domains in all ferlins, the C-terminal C2 domains (C2E and C2F) displayed higher sequence conservation and greater conservation of putative calcium binding residues across paralogs and orthologs. Interestingly, the two most studied non-mammalian ferlins (Fer-1 and Misfire) in model organisms <it>C. elegans </it>and <it>D. melanogaster</it>, present as outgroups in the phylogenetic analysis, with results suggesting reproduction-related divergence and specialization of species-specific functions within their genus.</p> <p>Conclusions</p> <p>Our phylogenetic studies provide evolutionary insight into the ferlin gene family. We highlight the existence of ferlin-like proteins throughout eukaryotic evolution, from unicellular phytoplankton and apicomplexan parasites, through to humans. We characterise the preservation of ferlin structural motifs, not only of C2 domains, but also the more poorly characterised ferlin-specific motifs representing the DysF, FerA and FerB domains. Our data suggest an ancient role of ferlin proteins, with lessons from vertebrate biology and human disease suggesting a role relating to vesicle fusion and plasma membrane specialization.</p

Quantifying unobserved protein-coding variants in human populations provides a roadmap for large-scale sequencing projects

As new proposals aim to sequence ever larger collection of humans, it is critical to have a quantitative framework to evaluate the statistical power of these projects. We developed a new algorithm, UnseenEst, and applied it to the exomes of 60,706 individuals to estimate the frequency distribution of all protein-coding variants, including rare variants that have not been observed yet in the current cohorts. Our results quantified the number of new variants that we expect to identify as sequencing cohorts reach hundreds of thousands of individuals. With 500K individuals, we find that we expect to capture 7.5% of all possible loss-of-function variants and 12% of all possible missense variants. We also estimate that 2,900 genes have loss-of-function frequency of <0.00001 in healthy humans, consistent with very strong intolerance to gene inactivation.United States. National Institutes of Health (U54DK105566)United States. National Institutes of Health (R01GM104371

Allelic Expression of Deleterious Protein-Coding Variants across Human Tissues

Personal exome and genome sequencing provides access to loss-of-function and rare deleterious alleles whose interpretation is expected to provide insight into individual disease burden. However, for each allele, accurate interpretation of its effect will depend on both its penetrance and the trait's expressivity. In this regard, an important factor that can modify the effect of a pathogenic coding allele is its level of expression; a factor which itself characteristically changes across tissues. To better inform the degree to which pathogenic alleles can be modified by expression level across multiple tissues, we have conducted exome, RNA and deep, targeted allele-specific expression (ASE) sequencing in ten tissues obtained from a single individual. By combining such data, we report the impact of rare and common loss-of-function variants on allelic expression exposing stronger allelic bias for rare stop-gain variants and informing the extent to which rare deleterious coding alleles are consistently expressed across tissues. This study demonstrates the potential importance of transcriptome data to the interpretation of pathogenic protein-coding variants

A novel compound heterozygous mutation in the POMK gene causing limb-girdle muscular dystrophy-dystroglycanopathy in a sib pair

We describe two Finnish siblings in whom an incidentally detected elevated creatine kinase activity eventually led to a diagnosis of limb-girdle muscular dystrophy-dystroglycanopathy (Type C12; MDDGC12). When diagnosed at age 10 and 13 years, they were mildly affected with a slow or non-progressive disease course. The main symptoms comprised infrequent hip cramps triggered by flexion, neck cramps triggered by yawning, transient growing pains, calf hypertrophy and mild proximal muscle weakness. Their cognitive and motor developments were unremarkable and they were physically active. Whole-exome sequencing revealed compound heterozygous mutations, both of which were novel, in the protein O-mannosyl kinase (POMK) gene in both siblings; a missense mutation, p.Pro322Leu (c.965C > T), and a nonsense mutation, p.Arg46Ter (c.136C > T). The results were confirmed by Sanger sequencing, showing that the parents were heterozygous carriers of one mutation each. This report adds to the literature by providing phenotype and genotype data on this ultra-rare POMK-related dystroglycanopathy. (C) 2018 Elsevier B.V. All rights reserved.Peer reviewe

Biallelic Variants in TTLL5, Encoding a Tubulin Glutamylase, Cause Retinal Dystrophy

In a subset of inherited retinal degenerations (including cone, cone-rod, and macular dystrophies), cone photoreceptors are more severely affected than rods; ABCA4 mutations are the most common cause of this heterogeneous class of disorders. To identify retinal-disease-associated genes, we performed exome sequencing in 28 individuals with “cone-first” retinal disease and clinical features atypical for ABCA4 retinopathy. We then conducted a gene-based case-control association study with an internal exome data set as the control group. TTLL5, encoding a tubulin glutamylase, was highlighted as the most likely disease-associated gene; 2 of 28 affected subjects harbored presumed loss-of-function variants: c.[1586_1589delAGAG];[1586_1589delAGAG], p.[Glu529Valfs∗2];[Glu529Valfs∗2], and c.[401delT(;)3354G>A], p.[Leu134Argfs∗45(;)Trp1118∗]. We then inspected previously collected exome sequence data from individuals with related phenotypes and found two siblings with homozygous nonsense variant c.1627G>T (p.Glu543∗) in TTLL5. Subsequently, we tested a panel of 55 probands with retinal dystrophy for TTLL5 mutations; one proband had a homozygous missense change (c.1627G>A [p.Glu543Lys]). The retinal phenotype was highly similar in three of four families; the sibling pair had a more severe, early-onset disease. In human and murine retinae, TTLL5 localized to the centrioles at the base of the connecting cilium. TTLL5 has been previously reported to be essential for the correct function of sperm flagella in mice and play a role in polyglutamylation of primary cilia in vitro. Notably, genes involved in the polyglutamylation and deglutamylation of tubulin have been associated with photoreceptor degeneration in mice. The electrophysiological and fundus autofluorescence imaging presented here should facilitate the molecular diagnosis in further families

Deleterious Heteroplasmic Mitochondrial Mutations are associated With an increased Risk of Overall and Cancer-Specific Mortality

Mitochondria carry their own circular genome and disruption of the mitochondrial genome is associated with various aging-related diseases. Unlike the nuclear genome, mitochondrial DNA (mtDNA) can be present at 1000 s to 10,000 s copies in somatic cells and variants may exist in a state of heteroplasmy, where only a fraction of the DNA molecules harbors a particular variant. We quantify mtDNA heteroplasmy in 194,871 participants in the UK Biobank and find that heteroplasmy is associated with a 1.5-fold increased risk of all-cause mortality. Additionally, we functionally characterize mtDNA single nucleotide variants (SNVs) using a constraint-based score, mitochondrial local constraint score sum (MSS) and find it associated with all-cause mortality, and with the prevalence and incidence of cancer and cancer-related mortality, particularly leukemia. These results indicate that mitochondria may have a functional role in certain cancers, and mitochondrial heteroplasmic SNVs may serve as a prognostic marker for cancer, especially for leukemia

Centers For Mendelian Genomics: a Decade of Facilitating Gene Discovery

PURPOSE: Mendelian disease genomic research has undergone a massive transformation over the past decade. With increasing availability of exome and genome sequencing, the role of Mendelian research has expanded beyond data collection, sequencing, and analysis to worldwide data sharing and collaboration. METHODS: Over the past 10 years, the National Institutes of Health-supported Centers for Mendelian Genomics (CMGs) have played a major role in this research and clinical evolution. RESULTS: We highlight the cumulative gene discoveries facilitated by the program, biomedical research leveraged by the approach, and the larger impact on the research community. Beyond generating a list of gene-phenotype relationships and participating in widespread data sharing, the CMGs have created resources, tools, and training for the larger community to foster understanding of genes and genome variation. The CMGs have participated in a wide range of data sharing activities, including deposition of all eligible CMG data into the Analysis, Visualization, and Informatics Lab-space (AnVIL), sharing candidate genes through the Matchmaker Exchange and the CMG website, and sharing variants in Genotypes to Mendelian Phenotypes (Geno2MP) and VariantMatcher. CONCLUSION: The work is far from complete; strengthening communication between research and clinical realms, continued development and sharing of knowledge and tools, and improving access to richly characterized data sets are all required to diagnose the remaining molecularly undiagnosed patients

Detection of variants in dystroglycanopathy-associated genes through the application of targeted whole-exome sequencing analysis to a large cohort of patients with unexplained limb-girdle muscle weakness

Background: Dystroglycanopathies are a clinically and genetically heterogeneous group of disorders that are typically characterised by limb-girdle muscle weakness. Mutations in 18 different genes have been associated with dystroglycanopathies, the encoded proteins of which typically modulate the binding of alpha-dystroglycan to extracellular matrix ligands by altering its glycosylation. This results in a disruption of the structural integrity of the myocyte, ultimately leading to muscle degeneration. Methods: Deep phenotypic information was gathered using the PhenoTips online software for 1001 patients with unexplained limb-girdle muscle weakness from 43 different centres across 21 European and Middle Eastern countries. Whole-exome sequencing with at least 250 ng DNA was completed using an Illumina exome capture and a 38 Mb baited target. Genes known to be associated with dystroglycanopathies were analysed for disease-causing variants. Results: Suspected pathogenic variants were detected in DPM3, ISPD, POMT1 and FKTN in one patient each, in POMK in two patients, in GMPPB in three patients, in FKRP in eight patients and in POMT2 in ten patients. This indicated a frequency of 2.7% for the disease group within the cohort of 1001 patients with unexplained limb-girdle muscle weakness. The phenotypes of the 27 patients were highly variable, yet with a fundamental presentation of proximal muscle weakness and elevated serum creatine kinase. Conclusions: Overall, we have identified 27 patients with suspected pathogenic variants in dystroglycanopathy-associated genes. We present evidence for the genetic and phenotypic diversity of the dystroglycanopathies as a disease group, while also highlighting the advantage of incorporating next-generation sequencing into the diagnostic pathway of rare diseases.Peer reviewe

Health and population effects of rare gene knockouts in adult humans with related parents.

Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3222 British adults of Pakistani heritage with high parental relatedness, discovering 1111 rare-variant homozygous genotypes with predicted loss of function (knockouts) in 781 genes. We observed 13.7% fewer homozygous knockout genotypes than we expected, implying an average load of 1.6 recessive-lethal-equivalent loss-of-function (LOF) variants per adult. When genetic data were linked to the individuals' lifelong health records, we observed no significant relationship between gene knockouts and clinical consultation or prescription rate. In this data set, we identified a healthy PRDM9-knockout mother and performed phased genome sequencing on her, her child, and control individuals. Our results show that meiotic recombination sites are localized away from PRDM9-dependent hotspots. Thus, natural LOF variants inform on essential genetic loci and demonstrate PRDM9 redundancy in humans.The study was funded by the Wellcome Trust (WT102627 and WT098051), Barts Charity (845/1796), Medical Research Council (MR/M009017/1). This paper presents independent research funded by the National Institute for Health Research (NIHR) under its Collaboration for Applied Health Research and Care (CLAHRC) for Yorkshire and Humber. Core support for Born in Bradford is also provided by the Wellcome Trust (WT101597). V.N. was supported by the Wellcome Trust PhD Studentship (WT099769). D.G.M. and K.K. were supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number R01GM104371. E.R.M. is funded by NIHR Cambridge Biomedical Research Centre. H.H. is supported by awards to establish the Farr Institute of Health Informatics Research, London, from the Medical Research Council, Arthritis Research UK, British Heart Foundation, Cancer Research UK, Chief Scientist Office, Economic and Social Research Council, Engineering and Physical Sciences Research Council, NIHR, National Institute for Social Care and Health Research, and Wellcome Trust.This is the author accepted manuscript. The final version is available from the American Association for the Advancement of Science via https://doi.org/10.1126/science.aac862