scholarly journals New family with HSPB8-associated autosomal dominant rimmed vacuolar myopathy

2019 ◽  
Vol 5 (4) ◽  
pp. e349 ◽  
Author(s):  
Sejad Al-Tahan ◽  
Lan Weiss ◽  
Howard Yu ◽  
Sha Tang ◽  
Mario Saporta ◽  
...  
Keyword(s):  
Western Blot ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Autosomal Dominant ◽  
Stop Codon ◽  
Whole Genome ◽  
Muscle Mri ◽  
New Family ◽  
Vacuolar Myopathy ◽  
Rimmed Vacuolar Myopathy

ObjectiveWe clinically and molecularly characterize a new family with autosomal dominant rimmed vacuolar myopathy (RVM) caused by mutations in the HSPB8 gene.MethodsWe performed whole-exome and whole-genome sequencing in the family. Western blot and immunocytochemistry were used to analyze 3 patient fibroblasts, and findings were compared with their age- and sex-matched controls.ResultsAffected patients have distal and proximal myopathy, with muscle biopsy showing rimmed vacuoles, muscle fiber atrophy, and endomysial fibrosis typical of RVM. Muscle MRI showed severe relatively symmetric multifocal fatty degenerative changes of the lower extremities. We identified a duplication of C at position 515 of the HSPB8 gene (c.515dupC) by whole-genome sequencing, which caused a frameshift with a predicted alternate stop codon p.P173SFS*43 in all affected individuals, resulting in an elongated protein product. Western blot and immunocytochemistry studies revealed reduced expression of heat shock protein beta 8 in patient fibroblasts compared with control fibroblasts, in addition to disrupted autophagy pathology.ConclusionsWe report a novel family with autosomal dominant RVM caused by the c.515dupC mutation of the HSPB8 gene, causing a translational frameshift that results in an elongated protein. Understanding the mechanism for the RVM pathology caused by mutated chaperone will permit novel targeted strategies to alter the natural history progression. As next-generation sequencing becomes more available, additional myopathic families will be identified with HSPB8 mutations.

scholarly journals Effective variant filtering and expected candidate variant yield in studies of rare human disease

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Brent S. Pedersen ◽  
Joe M. Brown ◽  
Harriet Dashnow ◽  
Amelia D. Wallace ◽  
Matt Velinder ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Rare Disease ◽  
Genome Sequencing ◽  
Autosomal Dominant ◽  
De Novo ◽  
Autosomal Dominant Inheritance ◽  
Compound Heterozygous ◽  
Whole Genome ◽  
Dominant Inheritance ◽  
Family Based

AbstractIn studies of families with rare disease, it is common to screen for de novo mutations, as well as recessive or dominant variants that explain the phenotype. However, the filtering strategies and software used to prioritize high-confidence variants vary from study to study. In an effort to establish recommendations for rare disease research, we explore effective guidelines for variant (SNP and INDEL) filtering and report the expected number of candidates for de novo dominant, recessive, and autosomal dominant modes of inheritance. We derived these guidelines using two large family-based cohorts that underwent whole-genome sequencing, as well as two family cohorts with whole-exome sequencing. The filters are applied to common attributes, including genotype-quality, sequencing depth, allele balance, and population allele frequency. The resulting guidelines yield ~10 candidate SNP and INDEL variants per exome, and 18 per genome for recessive and de novo dominant modes of inheritance, with substantially more candidates for autosomal dominant inheritance. For family-based, whole-genome sequencing studies, this number includes an average of three de novo, ten compound heterozygous, one autosomal recessive, four X-linked variants, and roughly 100 candidate variants following autosomal dominant inheritance. The slivar software we developed to establish and rapidly apply these filters to VCF files is available at https://github.com/brentp/slivar under an MIT license, and includes documentation and recommendations for best practices for rare disease analysis.

scholarly journals Genetic Mapping Reveals that Sinefungin Resistance in Toxoplasma gondii Is Controlled by a Putative Amino Acid Transporter Locus That Can Be Used as a Negative Selectable Marker

2014 ◽  
Vol 14 (2) ◽  
pp. 140-148 ◽  
Author(s):  
Michael S. Behnke ◽  
Asis Khan ◽  
L. David Sibley
Keyword(s):  
Toxoplasma Gondii ◽  
Genetic Mapping ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Genetic Map ◽  
Selectable Marker ◽  
Stop Codon ◽  
Whole Genome ◽  
Content Type ◽  
Negative Selectable Marker

ABSTRACTQuantitative trait locus (QTL) mapping studies have been integral in identifying and understanding virulence mechanisms in the parasiteToxoplasma gondii. In this study, we interrogated a different phenotype by mapping sinefungin (SNF) resistance in the genetic cross between type 2 ME49-FUDRrand type 10 VAND-SNFr. The genetic map of this cross was generated by whole-genome sequencing of the progeny and subsequent identification of single nucleotide polymorphisms (SNPs) inherited from the parents. Based on this high-density genetic map, we were able to pinpoint the sinefungin resistance phenotype to one significant locus on chromosome IX. Within this locus, a single nonsynonymous SNP (nsSNP) resulting in an early stop codon in the TGVAND_290860 gene was identified, occurring only in the sinefungin-resistant progeny. Using CRISPR/CAS9, we were able to confirm that targeted disruption of TGVAND_290860 renders parasites sinefungin resistant. Because disruption of theSNR1gene confers resistance, we also show that it can be used as a negative selectable marker to insert either a positive drug selection cassette or a heterologous reporter. These data demonstrate the power of combining classical genetic mapping, whole-genome sequencing, and CRISPR-mediated gene disruption for combined forward and reverse genetic strategies inT. gondii.

scholarly journals WGS and RNA Studies Diagnose Noncoding DMD Variants in Males With High Creatine Kinase

2021 ◽  
Vol 7 (1) ◽  
pp. e554
Author(s):  
Leigh B. Waddell ◽  
Samantha J. Bryen ◽  
Beryl B. Cummings ◽  
Adam Bournazos ◽  
Frances J. Evesson ◽  
...  
Keyword(s):  
Creatine Kinase ◽  
Western Blot ◽  
Whole Genome Sequencing ◽  
Rna Sequencing ◽  
Genome Sequencing ◽  
Premature Stop Codon ◽  
Clinical Severity ◽  
Whole Genome ◽  
Wild Type ◽  
Quantitative Western Blot

ObjectiveTo describe the diagnostic utility of whole-genome sequencing and RNA studies in boys with suspected dystrophinopathy, for whom multiplex ligation-dependent probe amplification and exomic parallel sequencing failed to yield a genetic diagnosis, and to use remnant normal DMD splicing in 3 families to define critical levels of wild-type dystrophin bridging clinical spectrums of Duchenne to myalgia.MethodsExome, genome, and/or muscle RNA sequencing was performed for 7 males with elevated creatine kinase. PCR of muscle-derived complementary DNA (cDNA) studied consequences for DMD premessenger RNA (pre-mRNA) splicing. Quantitative Western blot was used to determine levels of dystrophin, relative to control muscle.ResultsSplice-altering intronic single nucleotide variants or structural rearrangements in DMD were identified in all 7 families. Four individuals, with abnormal splicing causing a premature stop codon and nonsense-mediated decay, expressed remnant levels of normally spliced DMD mRNA. Quantitative Western blot enabled correlation of wild-type dystrophin and clinical severity, with 0%–5% dystrophin conferring a Duchenne phenotype, 10% ± 2% a Becker phenotype, and 15% ± 2% dystrophin associated with myalgia without manifesting weakness.ConclusionsWhole-genome sequencing relied heavily on RNA studies to identify DMD splice-altering variants. Short-read RNA sequencing was regularly confounded by the effectiveness of nonsense-mediated mRNA decay and low read depth of the giant DMD mRNA. PCR of muscle cDNA provided a simple, yet informative approach. Highly relevant to genetic therapies for dystrophinopathies, our data align strongly with previous studies of mutant dystrophin in Becker muscular dystrophy, with the collective conclusion that a fractional increase in levels of normal dystrophin between 5% and 20% is clinically significant.

scholarly journals Whole-genome sequencing overcomes pseudogene homology to diagnose autosomal dominant polycystic kidney disease

2016 ◽  
Vol 24 (11) ◽  
pp. 1584-1590 ◽  
Author(s):  
Amali C Mallawaarachchi ◽  
Yvonne Hort ◽  
Mark J Cowley ◽  
Mark J McCabe ◽  
André Minoche ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Whole Genome Sequencing ◽  
Polycystic Kidney Disease ◽  
Genome Sequencing ◽  
Autosomal Dominant ◽  
Whole Genome ◽  
Polycystic Kidney ◽  
Autosomal Dominant Polycystic Kidney

scholarly journals Whole-genome sequencing reveals a recurrent missense mutation in the Connexin 46 (GJA3) gene causing autosomal-dominant lamellar cataract

Eye ◽  
2018 ◽  
Vol 32 (10) ◽  
pp. 1661-1668 ◽  
Author(s):  
Vanita Berry ◽  
Alexander C. W. Ionides ◽  
Nikolas Pontikos ◽  
Ismail Moghul ◽  
Anthony T. Moore ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Missense Mutation ◽  
Genome Sequencing ◽  
Autosomal Dominant ◽  
Whole Genome

scholarly journals Whole genome sequencing and phylogenetic analysis of human metapneumovirus strains from Kenya and Zambia

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Everlyn Kamau ◽  
John W. Oketch ◽  
Zaydah R. de Laurent ◽  
My V. T. Phan ◽  
Charles N. Agoti ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Stop Codon ◽  
Amino Acid Level ◽  
Respiratory Illness ◽  
Human Metapneumovirus ◽  
Clinical Samples ◽  
Whole Genome ◽  
Individual Gene

Abstract Background Human metapneumovirus (HMPV) is an important cause of acute respiratory illness in young children. Whole genome sequencing enables better identification of transmission events and outbreaks, which is not always possible with sub-genomic sequences. Results We report a 2-reaction amplicon-based next generation sequencing method to determine the complete genome sequences of five HMPV strains, representing three subgroups (A2, B1 and B2), directly from clinical samples. In addition to reporting five novel HMPV genomes from Africa we examined genetic diversity and sequence patterns of publicly available HMPV genomes. We found that the overall nucleotide sequence identity was 71.3 and 80% for HMPV group A and B, respectively, the diversity between HMPV groups was greater at amino acid level for SH and G surface protein genes, and multiple subgroups co-circulated in various countries. Comparison of sequences between HMPV groups revealed variability in G protein length (219 to 241 amino acids) due to changes in the stop codon position. Genome-wide phylogenetic analysis showed congruence with the individual gene sequence sets except for F and M2 genes. Conclusion This is the first genomic characterization of HMPV genomes from African patients.

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