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1.
Gigascience ; 8(8)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31378808

RESUMO

BACKGROUND: Depth of coverage calculation is an important and computationally intensive preprocessing step in a variety of next-generation sequencing pipelines, including the analysis of RNA-sequencing data, detection of copy number variants, or quality control procedures. RESULTS: Building upon big data technologies, we have developed SeQuiLa-cov, an extension to the recently released SeQuiLa platform, which provides efficient depth of coverage calculations, reaching >100× speedup over the state-of-the-art tools. The performance and scalability of our solution allow for exome and genome-wide calculations running locally or on a cluster while hiding the complexity of the distributed computing with Structured Query Language Application Programming Interface. CONCLUSIONS: SeQuiLa-cov provides significant performance gain in depth of coverage calculations streamlining the widely used bioinformatic processing pipelines.

2.
Genome Med ; 11(1): 48, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349857

RESUMO

BACKGROUND: Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. METHODS: To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. RESULTS: We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. CONCLUSIONS: In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31189067

RESUMO

Lethal lung developmental disorders (LLDD) are rare but an important group of pediatric diffuse lung disease (chILD) presenting with neonatal respiratory failure. Based on histopathological appearance at lung biopsy or autopsy, they have been termed: alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV), acinar dysplasia (AcDys), congenital alveolar dysplasia (CAD), and other unspecified primary pulmonary hypoplasias. However, the histopathological continuum in these lethal developmental disorders has made accurate diagnosis challenging, which has implications for recurrence risk. Over the past decade, genetic studies in infants with ACDMPV have revealed the causative role of the dosage-sensitive FOXF1 gene and its non-coding regulatory variants in the distant lung-specific enhancer at chromosome 16q24.1. In contrast, the molecular bases of AcDys and CAD have remained poorly understood. Most recently, disruption of the TBX4-FGF10-FGFR2 epithelial-mesenchymal signaling pathway has been reported in patients with these lethal pulmonary dysplasias. Application of next generation sequencing techniques, including exome sequencing and whole genome sequencing (WGS) has demonstrated their complex compound inheritance. These data indicate that non-coding regulatory elements play a critical role in lung development in humans. We propose that for more precise LLDD diagnosis, a diagnostic pathway including WGS should be implemented.

4.
BMC Bioinformatics ; 20(1): 266, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138108

RESUMO

BACKGROUND: There are over 25 tools dedicated for the detection of Copy Number Variants (CNVs) using Whole Exome Sequencing (WES) data based on read depth analysis. The tools reported consist of several steps, including: (i) calculation of read depth for each sequencing target, (ii) normalization, (iii) segmentation and (iv) actual CNV calling. The essential aspect of the entire process is the normalization stage, in which systematic errors and biases are removed and the reference sample set is used to increase the signal-to-noise ratio. Although some CNV calling tools use dedicated algorithms to obtain the optimal reference sample set, most of the advanced CNV callers do not include this feature. To our knowledge, this work is the first attempt to assess the impact of reference sample set selection on CNV detection performance. METHODS: We used WES data from the 1000 Genomes project to evaluate the impact of various methods of reference sample set selection on CNV calling performance of three chosen state-of-the-art tools: CODEX, CNVkit and exomeCopy. Two naive solutions (all samples as reference set and random selection) as well as two clustering methods (k-means and k nearest neighbours (kNN) with a variable number of clusters or group sizes) have been evaluated to discover the best performing sample selection method. RESULTS AND CONCLUSIONS: The performed experiments have shown that the appropriate selection of the reference sample set may greatly improve the CNV detection rate. In particular, we found that smart reduction of reference sample size may significantly increase the algorithms' precision while having negligible negative effect on sensitivity. We observed that a complete CNV calling process with the k-means algorithm as the selection method has significantly better time complexity than kNN-based solution.


Assuntos
Algoritmos , Variações do Número de Cópias de DNA/genética , Benchmarking , Bases de Dados Genéticas , Feminino , Humanos , Masculino , Padrões de Referência , Tamanho da Amostra
6.
Am J Hum Genet ; 104(2): 213-228, 2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30639323

RESUMO

Primary defects in lung branching morphogenesis, resulting in neonatal lethal pulmonary hypoplasias, are incompletely understood. To elucidate the pathogenetics of human lung development, we studied a unique collection of samples obtained from deceased individuals with clinically and histopathologically diagnosed interstitial neonatal lung disorders: acinar dysplasia (n = 14), congenital alveolar dysplasia (n = 2), and other lethal lung hypoplasias (n = 10). We identified rare heterozygous copy-number variant deletions or single-nucleotide variants (SNVs) involving TBX4 (n = 8 and n = 2, respectively) or FGF10 (n = 2 and n = 2, respectively) in 16/26 (61%) individuals. In addition to TBX4, the overlapping ∼2 Mb recurrent and nonrecurrent deletions at 17q23.1q23.2 identified in seven individuals with lung hypoplasia also remove a lung-specific enhancer region. Individuals with coding variants involving either TBX4 or FGF10 also harbored at least one non-coding SNV in the predicted lung-specific enhancer region, which was absent in 13 control individuals with the overlapping deletions but without any structural lung anomalies. The occurrence of rare coding variants involving TBX4 or FGF10 with the putative hypomorphic non-coding SNVs implies a complex compound inheritance of these pulmonary hypoplasias. Moreover, they support the importance of TBX4-FGF10-FGFR2 epithelial-mesenchymal signaling in human lung organogenesis and help to explain the histopathological continuum observed in these rare lethal developmental disorders of the lung.

7.
J Clin Res Pediatr Endocrinol ; 11(3): 319-326, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-30563316

RESUMO

Primary polyneuropathy in the context of Seip-Berardinelli type 1 seipinopathy, or congenital generalized lipodystrophy type 1 (CGL1) has not been previously reported. We report the case history of a 27 year old female CGL1 patient presenting with an unusual additional development of non-diabetic peripheral neuropathy and learning disabilities in early adolescence. Whole exome sequencing (WES) of the patient genome identified a novel variant, homozygous for a 52 bp intronic deletion in the AGPAT2 locus, coding for 1-acylglycerol-3-phosphate O-acyltransferase 2, which is uniquely associated with CGL1 seipinopathies, with no molecular evidence for dual diagnosis. Functional studies using RNA isolated from patient peripheral blood leucocytes showed abnormal RNA splicing resulting in the loss of 25 amino acids from the patient AGPAT2 protein coding sequence. Stability and transcription levels for the misspliced AGPAT2 mRNA in our patient nonetheless remained normal. Any AGPAT2 protein produced in our patient is therefore likely to be dysfunctional. However, formal linkage of this deletion to the neuropathy observed remains to be shown. The classical clinical presentation of a patient with AGPAT2-associated lipodystrophy shows normal cognition and no development of polyneuropathy. Cognitive disabilities and polyneuropathy are features associated exclusively with clinical CGL type 2 arising from seipin (BSCL2) gene mutations. This case study suggests that in some genetic contexts, AGPAT2 mutations can also produce phenotypes with primary polyneuropathy.

8.
Bioinformatics ; 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30428005

RESUMO

Summary: Efficient processing of large-scale genomic datasets has recently become possible due to the application of 'big data' technologies in bioinformatics pipelines.We present SeQuiLa - a distributed, ANSI SQL compliant solution for speedy querying and processing of genomic intervals that is available as an Apache Spark package. Proposed range join strategy is significantly (~22x) faster than the default Apache Spark implementation and outperforms other state-of-the-art tools for genomic intervals processing. Availability and implementation: The project is available at http://biodatageeks.org/sequila/. Supplementary Information: Supplementary data (SD) are available at Bioinformatics online.

10.
Am J Hum Genet ; 103(2): 171-187, 2018 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-30032986

RESUMO

Premature termination codon (PTC)-bearing transcripts are often degraded by nonsense-mediated decay (NMD) resulting in loss-of-function (LoF) alleles. However, not all PTCs result in LoF mutations, i.e., some such transcripts escape NMD and are translated to truncated peptide products that result in disease due to gain-of-function (GoF) effects. Since the location of the PTC is a major factor determining transcript fate, we hypothesized that depletion of protein-truncating variants (PTVs) within the gene region predicted to escape NMD in control databases could provide a rank for genic susceptibility for disease through GoF versus LoF. We developed an NMD escape intolerance score to rank genes based on the depletion of PTVs that would render them able to escape NMD using the Atherosclerosis Risk in Communities Study (ARIC) and the Exome Aggregation Consortium (ExAC) control databases, which was further used to screen the Baylor-Center for Mendelian Genomics disease database. This analysis revealed 1,996 genes significantly depleted for PTVs that are predicted to escape from NMD, i.e., PTVesc; further studies provided evidence that revealed a subset as candidate genes underlying Mendelian phenotypes. Importantly, these genes have characteristically low pLI scores, which can cause them to be overlooked as candidates for dominant diseases. Collectively, we demonstrate that this NMD escape intolerance score is an effective and efficient tool for gene discovery in Mendelian diseases due to production of truncated or altered proteins. More importantly, we provide a complementary analytical tool to aid identification of genes associated with dominant traits through a mechanism distinct from LoF.

11.
Eur J Hum Genet ; 26(8): 1121-1131, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29706646

RESUMO

Malformations of cortical development (MCDs) manifest with structural brain anomalies that lead to neurologic sequelae, including epilepsy, cerebral palsy, developmental delay, and intellectual disability. To investigate the underlying genetic architecture of patients with disorders of cerebral cortical development, a cohort of 54 patients demonstrating neuroradiologic signs of MCDs was investigated. Individual genomes were interrogated for single-nucleotide variants (SNV) and copy number variants (CNV) with whole-exome sequencing and chromosomal microarray studies. Variation affecting known MCDs-associated genes was found in 16/54 cases, including 11 patients with SNV, 2 patients with CNV, and 3 patients with both CNV and SNV, at distinct loci. Diagnostic pathogenic SNV and potentially damaging variants of unknown significance (VUS) were identified in two groups of seven individuals each. We demonstrated that de novo variants are important among patients with MCDs as they were identified in 10/16 individuals with a molecular diagnosis. Three patients showed changes in known MCDs genes  and a clinical phenotype beyond the usual characteristics observed, i.e., phenotypic expansion, for a particular known disease gene clinical entity. We also discovered 2 likely candidate genes, CDH4, and ASTN1, with human and animal studies supporting their roles in brain development, and 5 potential candidate genes. Our findings emphasize genetic heterogeneity of MCDs disorders and postulate potential novel candidate genes involved in cerebral cortical development.

12.
Hum Mol Genet ; 27(12): 2064-2075, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29618029

RESUMO

Congenital diaphragmatic hernia (CDH) has been reported twice in individuals with a clinical diagnosis of Fraser syndrome, a genetic disorder that can be caused by recessive mutations affecting FREM2 and FRAS1. In the extracellular matrix, FREM2 and FRAS1 form a self-stabilizing complex with FREM1, a protein whose deficiency causes sac CDH in humans and mice. By sequencing FREM2 and FRAS1 in a CDH cohort, and searching online databases, we identified five individuals who carried recessive or double heterozygous, putatively deleterious variants in these genes which may represent susceptibility alleles. Three of these alleles were significantly enriched in our CDH cohort compared with ethnically matched controls. We subsequently demonstrated that 8% of Frem2ne/ne and 1% of Fras1Q1263*/Q1263* mice develop the same type of anterior sac CDH seen in FREM1-deficient mice. We went on to show that development of sac hernias in FREM1-deficient mice is preceded by failure of anterior mesothelial fold progression resulting in the persistence of an amuscular, poorly vascularized anterior diaphragm that is abnormally adherent to the underlying liver. Herniation occurs in the perinatal period when the expanding liver protrudes through this amuscular region of the anterior diaphragm that is juxtaposed to areas of muscular diaphragm. Based on these data, we conclude that deficiency of FREM2, and possibly FRAS1, are associated with an increased risk of developing CDH and that loss of the FREM1/FREM2/FRAS1 complex, or its function, leads to anterior sac CDH development through its effects on mesothelial fold progression.

14.
Eur J Hum Genet ; 26(3): 330-339, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29343805

RESUMO

Kinesin proteins are critical for various cellular functions such as intracellular transport and cell division, and many members of the family have been linked to monogenic disorders and cancer. We report eight individuals with intellectual disability and microcephaly from four unrelated families with parental consanguinity. In the affected individuals of each family, homozygosity for likely pathogenic variants in KIF14 were detected; two loss-of-function (p.Asn83Ilefs*3 and p.Ser1478fs), and two missense substitutions (p.Ser841Phe and p.Gly459Arg). KIF14 is a mitotic motor protein that is required for spindle localization of the mitotic citron rho-interacting kinase, CIT, also mutated in microcephaly. Our results demonstrate the involvement of KIF14 in development and reveal a wide phenotypic variability ranging from fetal lethality to moderate developmental delay and microcephaly.

16.
Genome Med ; 9(1): 83, 2017 09 21.
Artigo em Inglês | MEDLINE | ID: mdl-28934986

RESUMO

BACKGROUND: Exon-targeted microarrays can detect small (<1000 bp) intragenic copy number variants (CNVs), including those that affect only a single exon. This genome-wide high-sensitivity approach increases the molecular diagnosis for conditions with known disease-associated genes, enables better genotype-phenotype correlations, and facilitates variant allele detection allowing novel disease gene discovery. METHODS: We retrospectively analyzed data from 63,127 patients referred for clinical chromosomal microarray analysis (CMA) at Baylor Genetics laboratories, including 46,755 individuals tested using exon-targeted arrays, from 2007 to 2017. Small CNVs harboring a single gene or two to five non-disease-associated genes were identified; the genes involved were evaluated for a potential disease association. RESULTS: In this clinical population, among rare CNVs involving any single gene reported in 7200 patients (11%), we identified 145 de novo autosomal CNVs (117 losses and 28 intragenic gains), 257 X-linked deletion CNVs in males, and 1049 inherited autosomal CNVs (878 losses and 171 intragenic gains); 111 known disease genes were potentially disrupted by de novo autosomal or X-linked (in males) single-gene CNVs. Ninety-one genes, either recently proposed as candidate disease genes or not yet associated with diseases, were disrupted by 147 single-gene CNVs, including 37 de novo deletions and ten de novo intragenic duplications on autosomes and 100 X-linked CNVs in males. Clinical features in individuals with de novo or X-linked CNVs encompassing at most five genes (224 bp to 1.6 Mb in size) were compared to those in individuals with larger-sized deletions (up to 5 Mb in size) in the internal CMA database or loss-of-function single nucleotide variants (SNVs) detected by clinical or research whole-exome sequencing (WES). This enabled the identification of recently published genes (BPTF, NONO, PSMD12, TANGO2, and TRIP12), novel candidate disease genes (ARGLU1 and STK3), and further confirmation of disease association for two recently proposed disease genes (MEIS2 and PTCHD1). Notably, exon-targeted CMA detected several pathogenic single-exon CNVs missed by clinical WES analyses. CONCLUSIONS: Together, these data document the efficacy of exon-targeted CMA for detection of genic and exonic CNVs, complementing and extending WES in clinical diagnostics, and the potential for discovery of novel disease genes by genome-wide assay.


Assuntos
Variações do Número de Cópias de DNA , Éxons , Doenças Genéticas Inatas , Estudos de Coortes , Genoma Humano , Proteínas de Homeodomínio/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Transtornos do Neurodesenvolvimento/genética , Proteínas Serina-Treonina Quinases/genética , Estudos Retrospectivos , Fatores de Transcrição/genética , Sequenciamento Completo do Genoma
18.
Sci Rep ; 7(1): 2543, 2017 05 31.
Artigo em Inglês | MEDLINE | ID: mdl-28566687

RESUMO

Autosomal recessive diseases (ARD) are typically caused by a limited number of mutations whose identification is challenged by their low prevalence. Our purpose was to develop a novel approach allowing an efficient search for mutations causing ARD and evaluation of their pathogenicity without a control group. We developed Iterative Sequencing and Variant Screening (ISVS) approach based on iterative cycles of gene sequencing and mutation screening, and ISVS Simulator software ( http://zsibio.ii.pw.edu.pl/shiny/isvs/ ) for assessment of detected variants' significance. As shown by simulations, ISVS efficiently identifies and correctly classifies pathogenic mutations except for cases where the gene of interest has extremely high number of low frequency nonpathogenic variants. By applying ISVS, we found 4 known and 9 novel (p.C73Y, p.S124L, p.C194Mfs*17, c.782 + 2 T > A, c.953-5 A > G, p.L325Q, p.D334Mfs*24, p.R436G, p.M448T) TMPRSS3 variants among deaf patients. For 3 known and 5 novel variants the disease association was supported by ISVS Simulator odds >90:1. Pathogenicity of 6 novel mutations has been supported by in-silico predictions of variants' deleteriousness. By directly comparing variant prevalence in patients and controls, disease association was demonstrated only for two variants and it was relatively weak (P < 0.05). Summarizing, ISVS strategy and ISVS Simulator are useful for detection of genetic variants causing AR diseases.

20.
Genome Med ; 9(1): 26, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28327206

RESUMO

BACKGROUND: Given the rarity of most single-gene Mendelian disorders, concerted efforts of data exchange between clinical and scientific communities are critical to optimize molecular diagnosis and novel disease gene discovery. METHODS: We designed and implemented protocols for the study of cases for which a plausible molecular diagnosis was not achieved in a clinical genomics diagnostic laboratory (i.e. unsolved clinical exomes). Such cases were recruited to a research laboratory for further analyses, in order to potentially: (1) accelerate novel disease gene discovery; (2) increase the molecular diagnostic yield of whole exome sequencing (WES); and (3) gain insight into the genetic mechanisms of disease. Pilot project data included 74 families, consisting mostly of parent-offspring trios. Analyses performed on a research basis employed both WES from additional family members and complementary bioinformatics approaches and protocols. RESULTS: Analysis of all possible modes of Mendelian inheritance, focusing on both single nucleotide variants (SNV) and copy number variant (CNV) alleles, yielded a likely contributory variant in 36% (27/74) of cases. If one includes candidate genes with variants identified within a single family, a potential contributory variant was identified in a total of ~51% (38/74) of cases enrolled in this pilot study. The molecular diagnosis was achieved in 30/63 trios (47.6%). Besides this, the analysis workflow yielded evidence for pathogenic variants in disease-associated genes in 4/6 singleton cases (66.6%), 1/1 multiplex family involving three affected siblings, and 3/4 (75%) quartet families. Both the analytical pipeline and the collaborative efforts between the diagnostic and research laboratories provided insights that allowed recent disease gene discoveries (PURA, TANGO2, EMC1, GNB5, ATAD3A, and MIPEP) and increased the number of novel genes, defined in this study as genes identified in more than one family (DHX30 and EBF3). CONCLUSION: An efficient genomics pipeline in which clinical sequencing in a diagnostic laboratory is followed by the detailed reanalysis of unsolved cases in a research environment, supplemented with WES data from additional family members, and subject to adjuvant bioinformatics analyses including relaxed variant filtering parameters in informatics pipelines, can enhance the molecular diagnostic yield and provide mechanistic insights into Mendelian disorders. Implementing these approaches requires collaborative clinical molecular diagnostic and research efforts.


Assuntos
Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Doenças Genéticas Inatas/diagnóstico , Genômica/métodos , Polimorfismo de Nucleotídeo Único , Análise de Sequência de DNA/métodos , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Proteínas de Ligação a DNA/genética , Exoma , Feminino , Subunidades beta da Proteína de Ligação ao GTP/genética , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Metaloendopeptidases/genética , Proteínas Mitocondriais/genética , Projetos Piloto , Fatores de Transcrição/genética
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