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UNLABELLED: Cordova is an out-of-the-box solution for building and maintaining an online database of genetic variations integrated with pathogenicity prediction results from popular algorithms. Our primary motivation for developing this system is to aid researchers and clinician-scientists in determining the clinical significance of genetic variations. To achieve this goal, Cordova provides an interface to review and manually or computationally curate genetic variation data as well as share it for clinical diagnostics and the advancement of research. AVAILABILITY AND IMPLEMENTATION: Cordova is open source under the MIT license and is freely available for download at https://github.com/clcg/cordova.
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Bases de Dados de Ácidos Nucleicos , Variação Genética , Algoritmos , Humanos , Internet , SoftwareRESUMO
Deafness is the most frequent sensory disorder. With over 90 genes and 110 loci causally implicated in non-syndromic hearing loss, it is phenotypically and genetically heterogeneous. Here, we investigate the genetic etiology of deafness in four families of Iranian origin segregating autosomal recessive non-syndromic hearing loss (ARNSHL). We used a combination of linkage analysis, homozygosity mapping, and a targeted genomic enrichment platform to simultaneously screen 90 known deafness-causing genes for pathogenic variants. Variant segregation was confirmed by Sanger sequencing. Linkage analysis and homozygosity mapping showed segregation with the DFNB57 locus on chromosome 10 in two families. Targeted genomic enrichment with massively parallel sequencing identified causal variants in PDZD7: a homozygous missense variant (p.Gly103Arg) in one family and compound heterozygosity for missense (p.Met285Arg) and nonsense (p.Tyr500Ter) variants in the second family. Screening of two additional families identified two more variants: (p.Gly228Arg) and (p.Gln526Ter). Variant segregation with the hearing loss phenotype was confirmed in all families by Sanger sequencing. The missense variants are predicted to be deleterious, and the two nonsense mutations produce null alleles. This report is the first to show that mutations in PDZD7 cause ARNSHL, a finding that offers addition insight into the USH2 interactome. We also describe a novel likely disease-causing mutation in CIB2 and illustrate the complexity associated with gene identification in diseases that exhibit large genetic and phenotypic heterogeneity.
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Proteínas de Transporte/genética , Surdez/genética , Perda Auditiva/genética , Mutação , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cromossomos Humanos Par 10 , Feminino , Genes Recessivos , Heterogeneidade Genética , Ligação Genética , Haplótipos , Heterozigoto , Homozigoto , Humanos , Masculino , Modelos Moleculares , LinhagemRESUMO
OBJECTIVE: We present 2 patients who were identified with mutations in the GPR98 gene that causes Usher syndrome type 2 (USH2). METHODS: One hundred ninety-four (194) Japanese subjects from unrelated families were enrolled in the study. Targeted genomic enrichment and massively parallel sequencing of all known nonsyndromic hearing loss genes were used to identify the genetic causes of hearing loss. RESULTS: We identified causative mutations in the GPR98 gene in 1 family (2 siblings). The patients had moderate sloping hearing loss, and no progression was observed over a period of 10 years. Fundus examinations were normal. However, electroretinograms revealed impaired responses in both patients. CONCLUSION: Early diagnosis of Usher syndrome has many advantages for patients and their families. This study supports the use of comprehensive genetic diagnosis for Usher syndrome, especially prior to the onset of visual symptoms, to provide the highest chance of diagnostic success in early life stages.
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Sequenciamento de Nucleotídeos em Larga Escala , Receptores Acoplados a Proteínas G/genética , Síndromes de Usher/genética , Adolescente , Povo Asiático/genética , Eletrorretinografia , Feminino , Humanos , Mutação , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVES: We present a family with a mitochondrial DNA 3243A>G mutation resulting in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), of which some members have hearing loss in which a novel mutation in the P2RX2 gene was identified. METHODS: One hundred ninety-four (194) Japanese subjects from unrelated families were enrolled in the study. Targeted genomic enrichment and massively parallel sequencing of all known nonsyndromic hearing loss genes were performed to identify the genetic causes of hearing loss. RESULTS: A novel mutation in the P2RX2 gene that corresponded to c.601G>A (p.Asp201Tyr) was identified. Two patients carried the mutation and had severe sensorineural hearing loss, while other members with MELAS (who did not carry the P2RX2 mutation) had normal hearing. CONCLUSION: This is the first case report of a diagnosis of hearing loss caused by P2RX2 mutation in patients with MELAS. A potential explanation is that a decrease in adenosine triphosphate (ATP) production due to MELAS with a mitochondrial 3243A>G mutation might suppress activation of P2X2 receptors. We also suggest that hearing loss caused by the P2RX2 mutation might be influenced by the decrease in ATP production due to MELAS.
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Perda Auditiva Neurossensorial/genética , Síndrome MELAS/genética , Mitocôndrias/genética , Receptores Purinérgicos P2X2/genética , Trifosfato de Adenosina/metabolismo , Surdez/genética , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Síndrome MELAS/metabolismo , Pessoa de Meia-Idade , Linhagem , Análise de Sequência de DNA/métodosRESUMO
OBJECTIVES: We present 3 patients with congenital sensorineural hearing loss (SNHL) caused by novel PTPRQ mutations, including clinical manifestations and phenotypic features. METHODS: Two hundred twenty (220) Japanese subjects with SNHL from unrelated and nonconsanguineous families were enrolled in the study. Targeted genomic enrichment with massively parallel DNA sequencing of all known nonsyndromic hearing loss genes was performed to identify the genetic cause of hearing loss. RESULTS: Four novel causative PTPRQ mutations were identified in 3 cases. Case 1 had progressive profound SNHL with a homozygous nonsense mutation. Case 2 had nonprogressive profound SNHL with a compound heterozygous mutation (nonsense and missense mutation). Case 3 had nonprogressive moderate SNHL with a compound heterozygous mutation (missense and splice site mutation). Caloric test and vestibular evoked myogenic potential (VEMP) test showed vestibular dysfunction in Case 1. CONCLUSION: Hearing loss levels and progression among the present cases were varied, and there seem to be no obvious correlations between genotypes and the phenotypic features of their hearing loss. The PTPRQ mutations appeared to be responsible for vestibular dysfunction.
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Códon sem Sentido , Perda Auditiva Neurossensorial/genética , Mutação de Sentido Incorreto , Proteínas Tirosina Fosfatases Classe 3 Semelhantes a Receptores/genética , Povo Asiático/genética , Audiometria de Tons Puros , Análise Mutacional de DNA/métodos , Surdez/genética , Potenciais Evocados Auditivos , Perda Auditiva Neurossensorial/congênito , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , LinhagemRESUMO
OBJECTIVES: In this report, we present a male patient with no family history of hearing loss, in whom we identified a novel de novo mutation in the POU3F4 gene. METHODS: One hundred ninety-four (194) Japanese subjects from unrelated and nonconsanguineous families were enrolled in this study. We used targeted genomic enrichment and massively parallel sequencing of all known nonsyndromic hearing loss genes for identifying the genetic causes of hearing loss. RESULTS: A novel de novo frameshift mutation of POU3F4 to c.727_728insA (p.N244KfsX26) was identified. The patient was a 7-year-old male with congenital progressive hearing loss and inner ear deformity. Although the patient had received a cochlear implant, auditory skills were still limited. The patient also exhibited developmental delays similar to those previously associated with POU3F4 mutation. CONCLUSION: This is the first report of a mutation in POU3F4 causing hearing loss in a Japanese patient without a family history of hearing loss. This study underscores the importance of comprehensive genetic testing of patients with hearing loss for providing accurate prognostic information and guiding the optimal management of patient rehabilitation.
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Mutação da Fase de Leitura , Fatores do Domínio POU/genética , Povo Asiático/genética , Criança , Análise Mutacional de DNA , Surdez/genética , Deficiências do Desenvolvimento/genética , Potenciais Evocados Auditivos do Tronco Encefálico , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , MasculinoRESUMO
BACKGROUND: Non-syndromic hearing loss (NSHL) is the most common sensory impairment in humans. Until recently its extreme genetic heterogeneity precluded comprehensive genetic testing. Using a platform that couples targeted genomic enrichment (TGE) and massively parallel sequencing (MPS) to sequence all exons of all genes implicated in NSHL, we tested 100 persons with presumed genetic NSHL and in so doing established sequencing requirements for maximum sensitivity and defined MPS quality score metrics that obviate Sanger validation of variants. METHODS: We examined DNA from 100 sequentially collected probands with presumed genetic NSHL without exclusions due to inheritance, previous genetic testing, or type of hearing loss. We performed TGE using post-capture multiplexing in variable pool sizes followed by Illumina sequencing. We developed a local Galaxy installation on a high performance computing cluster for bioinformatics analysis. RESULTS: To obtain maximum variant sensitivity with this platform 3.2-6.3 million total mapped sequencing reads per sample were required. Quality score analysis showed that Sanger validation was not required for 95% of variants. Our overall diagnostic rate was 42%, but this varied by clinical features from 0% for persons with asymmetric hearing loss to 56% for persons with bilateral autosomal recessive NSHL. CONCLUSIONS: These findings will direct the use of TGE and MPS strategies for genetic diagnosis for NSHL. Our diagnostic rate highlights the need for further research on genetic deafness focused on novel gene identification and an improved understanding of the role of non-exonic mutations. The unsolved families we have identified provide a valuable resource to address these areas.
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Surdez/genética , Testes Genéticos/métodos , Genômica/métodos , Adolescente , Adulto , Feminino , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Reprodutibilidade dos Testes , Análise de Sequência de DNARESUMO
Autosomal dominant nonsyndromic hearing loss (ADNSHL) is a common and often progressive sensory deficit. ADNSHL displays a high degree of genetic heterogeneity and varying rates of progression. Accurate, comprehensive, and cost-effective genetic testing facilitates genetic counseling and provides valuable prognostic information to affected individuals. In this article, we describe the algorithm underlying AudioGene, a software system employing machine-learning techniques that utilizes phenotypic information derived from audiograms to predict the genetic cause of hearing loss in persons segregating ADNSHL. Our data show that AudioGene has an accuracy of 68% in predicting the causative gene within its top three predictions, as compared with 44% for a majority classifier. We also show that AudioGene remains effective for audiograms with high levels of clinical measurement noise. We identify audiometric outliers for each genetic locus and hypothesize that outliers may reflect modifying genetic effects. As personalized genomic medicine becomes more common, AudioGene will be increasingly useful as a phenotypic filter to assess pathogenicity of variants identified by massively parallel sequencing.
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Perda Auditiva/diagnóstico , Perda Auditiva/genética , Software , Algoritmos , Audiometria , Testes Genéticos , Genótipo , Humanos , Internet , Fenótipo , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: To present audiometric data in 3 dimensions by considering age as an addition dimension. METHODS: Audioprofile surfaces (APSs) were fitted to a set of audiograms by plotting each measurement of an audiogram as an independent point in 3 dimensions with the x, y, and z axes representing frequency, hearing loss in dB, and age, respectively. RESULTS: Using the Java-based APS viewer as a standalone application, APSs were pre-computed for 34 loci. By selecting APSs for the appropriate genetic locus, a clinician can compare this APS-generated average surface to a specific patient's audiogram. CONCLUSION: Audioprofile surfaces provide an easily interpreted visual representation of a person's hearing acuity relative to others with the same genetic cause of hearing loss. Audioprofile surfaces will support the generation and testing of sophisticated hypotheses to further refine our understanding of the biology of hearing.
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Audiometria de Tons Puros/tendências , Limiar Auditivo/fisiologia , Perda Auditiva Neurossensorial/diagnóstico , Audição/fisiologia , Software , Perda Auditiva Neurossensorial/fisiopatologia , Humanos , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Copy number variants (CNVs) are a well-recognized cause of genetic disease; however, methods for their identification are often gene-specific, excluded as 'routine' in screens of genetically heterogeneous disorders, and not implemented in most next-generation sequencing pipelines. For this reason, the contribution of CNVs to non-syndromic hearing loss (NSHL) is most likely under-recognized. We aimed to incorporate a method for CNV identification as part of our standard analysis pipeline and to determine the contribution of CNVs to genetic hearing loss. METHODS: We used targeted genomic enrichment and massively parallel sequencing to isolate and sequence all exons of all genes known to cause NSHL. We completed testing on 686 patients with hearing loss with no exclusions based on type of hearing loss or any other clinical features. For analysis we used an integrated method for detection of single nucleotide changes, indels and CNVs. CNVs were identified using a previously published method that utilizes median read-depth ratios and a sliding-window approach. RESULTS: Of 686 patients tested, 15.2% (104) carried at least one CNV within a known deafness gene. Of the 38.9% (267) of individuals for whom we were able to determine a genetic cause of hearing loss, a CNV was implicated in 18.7% (50). We identified CNVs in 16 different genes including 7 genes for which no CNVs have been previously reported. CNVs of STRC were most common (73% of CNVs identified) followed by CNVs of OTOA (13% of CNVs identified). CONCLUSION: CNVs are an important cause of NSHL and their detection must be included in comprehensive genetic testing for hearing loss.
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RhoA, RhoB and RhoC GTPases are over 85% identical at the amino acid level, with RhoA and RhoC differing at only one residue (43) across the initial two-thirds of their sequences. A putative regulatory distinction between the molecules is their capacity to be uniquely activated by guanine nucleotide exchange factors (GEFs). We hypothesize that variation of amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) impacts GEF activity. Direct participation of residue 43 in GEF-catalyzed exchange was confirmed by the observation that mutation of this position to a threonine reduced GEF-catalyzed nucleotide exchange activity in vitro (Vav2, XPLN, GEFT, Dbl and Dbs) and greatly depressed RhoA and RhoC GTP-loading profiles in cell lysates. Using a residue swap approach, substitution of RhoA Val 43 with an Ile was found to significantly promote basal nucleotide exchange activity and enhance GTP-loading in cells. Substitution of Val 43 with an Ile in RhoB negatively affected nucleotide exchange in vitro. Substitution of RhoC Ile 43 with a Val increased GEF-catalyzed exchange in vitro. In addition, RhoC-I43V was more efficacious at driving ovarian cancer cell invasion through matrigrel than wild-type RhoC, RhoC-I43T, wild-type RhoA, RhoA-V43I or RhoA-V43T GTPases. These findings suggest that a divergence between RhoA/B and RhoC at residue 43 impacts basal and GEF-stimulated nucleotide exchange activity.
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Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoB de Ligação ao GTP/metabolismo , Animais , Linhagem Celular Tumoral , Guanosina Trifosfato/metabolismo , Humanos , Camundongos , Células NIH 3T3 , Mutação Puntual , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas rho de Ligação ao GTP/química , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/química , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoB de Ligação ao GTP/química , Proteína rhoB de Ligação ao GTP/genética , Proteína de Ligação a GTP rhoCRESUMO
Our understanding of hereditary hearing loss has greatly improved since the discovery of the first human deafness gene. These discoveries have only accelerated due to the great strides in DNA sequencing technology since the completion of the human genome project. Here, we review the immense impact that these developments have had in both deafness research and clinical arenas. We review commonly used genomic technologies as well as the application of these technologies to the genetic diagnosis of hereditary hearing loss and to the discovery of novel deafness genes.