Phevor integrates phenotype, gene function, and disease information with personal genomic data for improved power to identify disease-causing alleles. Phevor works by combining knowledge resident in ...multiple biomedical ontologies with the outputs of variant-prioritization tools. It does so by using an algorithm that propagates information across and between ontologies. This process enables Phevor to accurately reprioritize potentially damaging alleles identified by variant-prioritization tools in light of gene function, disease, and phenotype knowledge. Phevor is especially useful for single-exome and family-trio-based diagnostic analyses, the most commonly occurring clinical scenarios and ones for which existing personal genome diagnostic tools are most inaccurate and underpowered. Here, we present a series of benchmark analyses illustrating Phevor’s performance characteristics. Also presented are three recent Utah Genome Project case studies in which Phevor was used to identify disease-causing alleles. Collectively, these results show that Phevor improves diagnostic accuracy not only for individuals presenting with established disease phenotypes but also for those with previously undescribed and atypical disease presentations. Importantly, Phevor is not limited to known diseases or known disease-causing alleles. As we demonstrate, Phevor can also use latent information in ontologies to discover genes and disease-causing alleles not previously associated with disease.
Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular ...cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.
High-throughput sequencing of related individuals has become an important tool for studying human disease. However, owing to technical complexity and lack of available tools, most pedigree-based ...sequencing studies rely on an ad hoc combination of suboptimal analyses. Here we present pedigree-VAAST (pVAAST), a disease-gene identification tool designed for high-throughput sequence data in pedigrees. pVAAST uses a sequence-based model to perform variant and gene-based linkage analysis. Linkage information is then combined with functional prediction and rare variant case-control association information in a unified statistical framework. pVAAST outperformed linkage and rare-variant association tests in simulations and identified disease-causing genes from whole-genome sequence data in three human pedigrees with dominant, recessive and de novo inheritance patterns. The approach is robust to incomplete penetrance and locus heterogeneity and is applicable to a wide variety of genetic traits. pVAAST maintains high power across studies of monogenic, high-penetrance phenotypes in a single pedigree to highly polygenic, common phenotypes involving hundreds of pedigrees.
Although reported gene variants in the RET oncogene have been directly associated with multiple endocrine neoplasia type 2 and hereditary medullary thyroid carcinoma, other mutations are classified ...as variants of uncertain significance (VUS) until the associated clinical phenotype is made clear. Currently, some 46 non-synonymous VUS entries exist in curated archives. In the absence of a gold standard method for predicting phenotype outcomes, this follow up study applies feature selected amino acid physical and chemical properties feeding a Bayes classifier to predict disease association of uncertain gene variants into categories of benign and pathogenic. Algorithm performance and VUS predictions were compared to established phylogenetic based mutation prediction algorithms. Curated outcomes and unpublished RET gene variants with known disease association were used to benchmark predictor performance. Reliable classification of RET uncertain gene variants will augment current clinical information of RET mutations and assist in improving prediction algorithms as knowledge increases.
Next-generation sequencing is being implemented in the clinical laboratory environment for the purposes of candidate causal variant discovery in patients affected with a variety of genetic disorders. ...The successful implementation of this technology for diagnosing genetic disorders requires a rapid, user-friendly method to annotate variants and generate short lists of clinically relevant variants of interest. This report describes Omicia's Opal platform, a new software tool designed for variant discovery and interpretation in a clinical laboratory environment. The software allows clinical scientists to process, analyze, interpret and report on personal genome files.
To demonstrate the software, the authors describe the interactive use of the system for the rapid discovery of disease-causing variants using three cases.
Here, the authors show the features of the Opal system and their use in uncovering variants of clinical significance.
The Multiple Endocrine Neoplasia type 2 (MEN2) RET proto‐oncogene database, originally published in 2008, is a comprehensive repository of all publicly available RET gene variations associated with ...MEN2 syndromes. The variant‐specific genotype/phenotype information, age of earliest reported medullary thyroid carcinoma (MTC) onset, and relevant references with a brief summary of findings are cataloged. The ACMG/AMP 2015 consensus statement on variant classification was modified specifically for MEN2 syndromes and RET variants using ClinGen sequence variant interpretation working group recommendations and ClinGen expert panel manuscripts, as well as manuscripts from the American Thyroid Association Guidelines Task Force on Medullary Thyroid Carcinoma and other MEN2 RET literature. The classifications for the 166 single unique variants in the MEN2 RET database were reanalyzed using the MEN2 RET specifically modified ACMG/AMP classification guidelines (version 1). Applying these guidelines added two new variant classifications to the database (likely benign and likely pathogenic) and resulted in clinically significant classification changes (e.g., from pathogenic to uncertain) in 15.7% (26/166) of the original variants. Of those clinically significant changes, the highest percentage of changes, 46.2% (12/26), were changes from uncertain to benign or likely benign. The modified ACMG/AMP criteria with MEN2 RET specifications will optimize and standardize RET variant classifications.
Friedreich ataxia is a rare autosomal recessive, neuromuscular degenerative disease caused by an expansion of a trinucleotide guanine-adenine-adenine (GAA) repeat in intron 1 of the FXN gene. It is ...common in the White population, characterized by progressive gait and limb ataxia, lack of tendon reflexes in the legs, loss of position sense, and hypertrophic cardiomyopathy. Detection and genotyping of the trinucleotide repeat length is important for the diagnosis and prognosis of the disease. A two-tier genotyping assay with an improved triple-repeat primed PCR (TR-PCR) for alleles <200 GAA repeats (±1 to 5 repeats) and an agarose gel-based, long-range PCR (LR-PCR) assay to genotype expanded alleles >200 GAA repeats (±50 repeats) is described. Of the 1236 DNA samples tested using TR-PCR, 31 were identified to have expanded alleles >200 repeats and were reflexed to the LR-PCR procedure for confirmation and quantification. The TR-PCR assay described herein is a diagnostic genotyping assay that reduces the need for further testing. The LR-PCR component is a confirmatory test for true homozygous and heterozygous samples with normal and expanded alleles, as indicated by the TR-PCR assay. The use of this two-tier method offers a comprehensive evaluation to detect and genotype the smallest and largest number of GAA repeats, improving the classification of FXN alleles as normal, mutable normal, borderline, and expanded alleles.
The observed-to-expected ratio describes the ratio of the number of observed loss-of-function to a mathematically modeled expected number of loss-of-function variants in the gene, with the lower ...value suggestive of sequence variants not being tolerated in the gene.1 CXXC5 belongs to the CXXC-zinc finger family of epigenetic regulators.2 The N-terminus of the protein does not have a functionally annotated domain, but the C-terminus of the protein has a DNA-binding zinc finger domain. The C-to-T change at position 814 in the CXXC5 gene (NM_001317199), which causes substitution of cysteine for arginine residue at position 272 in the protein (NP_001304128.1) is in the evolutionarily conserved CXXC domain of the CXXC5 protein (Fig E1, B). Because the CXXC domain is the DNA-binding domain of the CXXC5 protein,3,4 we compared the binding of CXXC5 wild-type and CXXC5R272C proteins to the DNA probes by electrophoretic mobility shift assay. Additionally, CXXC5R272C protein was partially localized in the cytoplasm during transient expression experiments, whereas wild-type CXXC5 was exclusively nuclear (Fig E1, D). ...in vitro DNA binding assays and the cellular localization experiments suggested a partial loss of function in the CXXC5 protein by substitution of cysteine for arginine at position 272 of the protein. Compared with the percentage of wild-type Cxxc5-infected LSK cells, a greater percentage of the cells were in the S phase and a lesser percentage of the cells were in the G0/G1 phase in Cxxc5R267C LSK cells (Fig 1, C). ...there were lower percentages of annexin V–positive cells after Cxxc5R267C infection than after the wild-type Cxxc5 infection of LSK cells (Fig 1, D).
An unlabeled probe assay relies on a double-stranded DNA-binding dye to detect and verify target based on amplicon and probe melting. During the development and application of unlabeled probe assays, ...aberrant melting peaks are sometimes observed that may interfere with assay interpretation. In this report, we investigated the origin of aberrant melting profiles observed in an unlabeled probe assay for exon 10 of the RET gene. It was determined that incomplete 3′ blocking of the unlabeled probe allowed polymerase-mediated probe extension resulting in extension products that generated the aberrant melting profiles. This report further examined the blocking ability of the 3′ modifications C3 spacer, amino-modified C6, phosphate, inverted dT, and single 3′ nucleotide mismatches in unlabeled probe experiments. Although no 3′ blocking modifications in these experiments were 100% effective, the amino-modified C6, inverted dT, and C3 spacer provided the best blocking efficiencies (1% or less unblocked), phosphate was not as effective of a block (up to 2% unblocked), and single nucleotide mismatches should be avoided as a 3′ blocking modification.
NF1 Somatic Mutation in Dystrophic Scoliosis Margraf, Rebecca L.; VanSant-Webb, Chad; Mao, Rong ...
Journal of molecular neuroscience,
05/2019, Letnik:
68, Številka:
1
Journal Article
Recenzirano
Scoliosis is a common manifestation of neurofibromatosis type 1, causing significant morbidity. The etiology of dystrophic scoliosis in neurofibromatosis type 1 is not fully understood and therapies ...are lacking. Somatic mutations in
NF1
have been shown in tibial pseudarthrosis providing rationale for similar processes in neurofibromatosis type 1–associated dystrophic scoliosis. Spinal samples from surgical procedures with matched peripheral blood of two individuals with neurofibromatosis type 1 and dystrophic scoliosis were obtained and DNA extracted. Next generation sequencing of various spinal sections as well as the germline/blood sample were performed using a RASopathy gene panel (includes the
NF1
gene). Variants were compared between the spinal tissue samples and the germline data. In addition, the next generation sequencing allele frequency data were used to detect somatic loss of heterozygosity. All samples had a detected potentially inactivating
NF1
germline mutation. Both individuals demonstrated an allelic imbalance inclusive of
NF1
in the next generation sequencing data. In addition, for the same two individuals, there was an increase in the % variant reads for the germline mutation in some of the surgical spinal samples corresponding to the allelic imbalance. Contra analysis did not show any deletion in Chromosome 17 next generation sequencing data. Microarray analysis verified somatic copy neutral loss of heterozygosity for these two individuals for the majority of the chromosome 17 q-arm, inclusive of the
NF1
gene. These results suggest that the cause of dystrophic scoliosis is multifactorial and that a somatic
NF1
mutation contributes to the etiology.
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