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1.
Dig Dis Sci ; 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31489563

RESUMO

BACKGROUND: The leucine-rich repeat kinase 2 (LRRK2) gene was confirmed to be associated with a variety of diseases, while the physiological function of LRRK2 remains poorly understood. Intrahepatic cholangiocarcinoma (ICC) has over the last 10 years become the focus of increasing concern largely. Despite recent progress in the standard of care and management options for ICC, the prognosis for this devastating cancer remains dismal. METHODS: A total of 57 consecutive ICC patients who underwent curative hepatectomy in our institution were included in our study. We conduct a retrospective study to evaluate the prognostic value of LRRK2 in ICC after resection. The mechanism of LRRK2 in ICC development was also investigated in vitro. RESULTS: All patients were divided into two groups according to the content of LRRK2 in the tissue microarray blocks via immunohistochemistry: low-LRRK2 group (n = 33) and high-LRRK2 group (n = 24). The recurrence-free survival rate of high-LRRK2 group was significantly poorer than that of low-LRRK2 group (P = 0.010). Multivariate analysis showed high-LRRK2 was the prognostic factor for recurrence-free survival after hepatectomy. We demonstrated that downregulation of LRRK2 depressed the proliferation and metastasis of ICC cells in vitro. CONCLUSION: We provide evidence that LRRK2 was an independent prognostic factor for ICC in humans by participating in the proliferation and metastasis of ICC cells.

2.
Am J Hum Genet ; 105(1): 132-150, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31230720

RESUMO

Arthrogryposis is a clinical finding that is present either as a feature of a neuromuscular condition or as part of a systemic disease in over 400 Mendelian conditions. The underlying molecular etiology remains largely unknown because of genetic and phenotypic heterogeneity. We applied exome sequencing (ES) in a cohort of 89 families with the clinical sign of arthrogryposis. Additional molecular techniques including array comparative genomic hybridization (aCGH) and Droplet Digital PCR (ddPCR) were performed on individuals who were found to have pathogenic copy number variants (CNVs) and mosaicism, respectively. A molecular diagnosis was established in 65.2% (58/89) of families. Eleven out of 58 families (19.0%) showed evidence for potential involvement of pathogenic variation at more than one locus, probably driven by absence of heterozygosity (AOH) burden due to identity-by-descent (IBD). RYR3, MYOM2, ERGIC1, SPTBN4, and ABCA7 represent genes, identified in two or more families, for which mutations are probably causative for arthrogryposis. We also provide evidence for the involvement of CNVs in the etiology of arthrogryposis and for the idea that both mono-allelic and bi-allelic variants in the same gene cause either similar or distinct syndromes. We were able to identify the molecular etiology in nine out of 20 families who underwent reanalysis. In summary, our data from family-based ES further delineate the molecular etiology of arthrogryposis, yielded several candidate disease-associated genes, and provide evidence for mutational burden in a biological pathway or network. Our study also highlights the importance of reanalysis of individuals with unsolved diagnoses in conjunction with sequencing extended family members.

3.
Genome Med ; 11(1): 30, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101064

RESUMO

BACKGROUND: Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. METHODS: In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. RESULTS: The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. CONCLUSIONS: Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

6.
Eur J Hum Genet ; 27(4): 563-573, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30622330

RESUMO

Aberrant left-right patterning in the developing human embryo can lead to a broad spectrum of congenital malformations. The causes of most laterality defects are not known, with variants in established genes accounting for <20% of cases. We sought to characterize the genetic spectrum of these conditions by performing whole-exome sequencing of 323 unrelated laterality cases. We investigated the role of rare, predicted-damaging variation in 1726 putative laterality candidate genes derived from model organisms, pathway analyses, and human phenotypes. We also evaluated the contribution of homo/hemizygous exon deletions and gene-based burden of rare variation. A total of 28 candidate variants (26 rare predicted-damaging variants and 2 hemizygous deletions) were identified, including variants in genes known to cause heterotaxy and primary ciliary dyskinesia (ACVR2B, NODAL, ZIC3, DNAI1, DNAH5, HYDIN, MMP21), and genes without a human phenotype association, but with prior evidence for a role in embryonic laterality or cardiac development. Sanger validation of the latter variants in probands and their parents revealed no de novo variants, but apparent transmitted heterozygous (ROCK2, ISL1, SMAD2), and hemizygous (RAI2, RIPPLY1) variant patterns. Collectively, these variants account for 7.1% of our study subjects. We also observe evidence for an excess burden of rare, predicted loss-of-function variation in PXDNL and BMS1- two genes relevant to the broader laterality phenotype. These findings highlight potential new genes in the development of laterality defects, and suggest extensive locus heterogeneity and complex genetic models in this class of birth defects.

7.
Prenat Diagn ; 38(11): 858-865, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30094853

RESUMO

OBJECTIVE: This study aims to establish the incidence and implications of confined placental mosaicism (CPM) in the context of prenatal chromosomal microarray analysis (CMA). METHODS: We retrospectively reviewed prenatal array data on 1382 consecutive chorionic villus sampling (CVS) specimens spanning the past 6 years, focusing on those for which whole CVS biopsy (both cytotrophoblast and mesenchymal cells) was used for CMA and cultured cells (primarily mesenchyme) was also analyzed or amniotic fluid (AF)/newborn blood was used for confirmation, to determine the frequency of mosaic abnormal findings that were the result of CPM. RESULTS: Out of a total of 1382 consecutive CVS cases, we identified 42 (42/1382 = 3.0%) cases with abnormal array findings suggestive of mosaicism. Among them, 10 cases were unequivocally interpreted as CPM based on a normal AF/newborn blood confirmatory result. In addition, another 10 cases were interpreted as provisional CPM based on normal results on cultured cells. Notably, 40% (8/20) of the cases revealed complex findings, including multiple mosaic aneuploidies, mosaic submicroscopic copy number variation (CNV), and mosaic aneuploidy plus mosaic CNV. CONCLUSION: Abnormal CMA results from CVS specimens should be interpreted with caution when mosaicism is evident or suspected. Furthermore, confirmatory testing on amniotic fluid, which contains cells derived from the fetus, is recommended in these cases.

8.
Genome Res ; 28(8): 1228-1242, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29907612

RESUMO

Alu elements, the short interspersed element numbering more than 1 million copies per human genome, can mediate the formation of copy number variants (CNVs) between substrate pairs. These Alu/Alu-mediated rearrangements (AAMRs) can result in pathogenic variants that cause diseases. To investigate the impact of AAMR on gene variation and human health, we first characterized Alus that are involved in mediating CNVs (CNV-Alus) and observed that these Alus tend to be evolutionarily younger. We then computationally generated, with the assistance of a supercomputer, a test data set consisting of 78 million Alu pairs and predicted ∼18% of them are potentially susceptible to AAMR. We further determined the relative risk of AAMR in 12,074 OMIM genes using the count of predicted CNV-Alu pairs and experimentally validated the predictions with 89 samples selected by correlating predicted hotspots with a database of CNVs identified by clinical chromosomal microarrays (CMAs) on the genomes of approximately 54,000 subjects. We fine-mapped 47 duplications, 40 deletions, and two complex rearrangements and examined a total of 52 breakpoint junctions of simple CNVs. Overall, 94% of the candidate breakpoints were at least partially Alu mediated. We successfully predicted all (100%) of Alu pairs that mediated deletions (n = 21) and achieved an 87% positive predictive value overall when including AAMR-generated deletions and duplications. We provided a tool, AluAluCNVpredictor, for assessing AAMR hotspots and their role in human disease. These results demonstrate the utility of our predictive model and provide insights into the genomic features and molecular mechanisms underlying AAMR.


Assuntos
Elementos Alu/genética , Variações do Número de Cópias de DNA/genética , Instabilidade Genômica/genética , Duplicação Gênica/genética , Genoma Humano/genética , Humanos , Deleção de Sequência
9.
Sci Rep ; 8(1): 9630, 2018 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-29941943

RESUMO

MicroRNAs (miRNAs) are known to be crucial players in governing the differentiation of human induced pluripotent stem cells (hiPSCs). Despite their utter importance, identifying key lineage specifiers among the myriads of expressed miRNAs remains challenging. We believe that the current practice in mining miRNA specifiers via delineating dynamic fold-changes only is inadequate. Our study, therefore, provides evidence to pronounce "lineage specificity" as another important attribute to qualify for these lineage specifiers. Adopted hiPSCs were differentiated into representative lineages (hepatic, nephric and neuronal) over all three germ layers whilst the depicted miRNA expression changes compiled into an integrated atlas. We demonstrated inter-lineage analysis shall aid in the identification of key miRNAs with lineage-specificity, while these shortlisted candidates were collectively known as "lineage-specific miRNAs". Subsequently, we followed through the fold-changes along differentiation via computational analysis to identify miR-192 and miR-372-3p, respectively, as representative candidate key miRNAs for the hepatic and nephric lineages. Indeed, functional characterization validated that miR-192 and miR-372-3p regulate lineage differentiation via modulation of the expressions of lineage-specific genes. In summary, our presented miRNA atlas is a resourceful ore for the mining of key miRNAs responsible for lineage specification.

10.
Hum Mutat ; 39(7): 939-946, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29696747

RESUMO

Small supernumerary marker chromosomes (sSMC) are chromosomal fragments difficult to characterize genomically. Here, we detail a proband with schizoaffective disorder and a mother with bipolar disorder with psychotic features who present with a marker chromosome that segregates with disease. We explored the architecture of this marker and investigated its temporal origin. Array comparative genomic hybridization (aCGH) analysis revealed three duplications and three triplications that spanned the short arm of chromosome 9, suggestive of a chromoanasynthesis-like event. Segregation of marker genotypes, phased using sSMC mosaicism in the mother, provided evidence that it was generated during a germline-level event in the proband's maternal grandmother. Whole-genome sequencing (WGS) was performed to resolve the structure and junctions of the chromosomal fragments, revealing further complexities. While structural variations have been previously associated with neuropsychiatric disorders and marker chromosomes, here we detail the precise architecture, human life-cycle genesis, and propose a DNA replicative/repair mechanism underlying formation.

11.
Oncotarget ; 8(37): 61203-61214, 2017 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-28977857

RESUMO

OBJECTIVES: The aim of this study was to investigate the prognostic value and preoperative predictors of microvascular invasion (MVI) in solitary hepatocellular carcinoma (HCC) ≤ 5 cm without macrovascular invasion. METHODS: A total of 233 consecutive HCC patients underwent curative hepatectomy were included in our study. Independent risk factors influencing the prognosis were identified, and preoperative predictors for MVI were determined. RESULTS: Multivariate regression analysis identified ICG-R15, BCLC staging and MVI as independent risk factors for the overall survival rate. Type of resection and MVI were independent risk factors for the recurrence-free survival rate. Kaplan-Meier analysis showed the overall survival and recurrence-free survival rates in patients with MVI were significantly poorer than those in patients without MVI (P = 0.002 and P = 0.001). Anatomical resection obviously improved the overall survival and recurrence-free survival rates in patients with MVI compared with non-anatomical resection (P = 0.017 and P = 0.009). A prediction scoring system for MVI was built up according to the three independent predictors (tumor size > 3.5 cm, AFP > 200 ng/mL and GGT > 53 U/L). The prevalence of MVI in HCC patients with predictive score ≥ 2 was 58.3%, which was obviously higher than patients with predictive score < 2 (20.8%). CONCLUSIONS: MVI is associated with a poor prognosis in solitary HCC ≤ 5 cm after hepatectomy. Anatomical resection could improve the prognosis of HCC patients with MVI. The preoperative prediction scoring model has practical value for the prediction of MVI.

12.
Stem Cell Reports ; 8(3): 519-528, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28216146

RESUMO

In the process of generating presumably clonal human induced pluripotent stem cells (hiPSCs) from two carriers of a complex structural rearrangement, each having a psychotic disorder, we also serendipitously generated isogenic non-carrier control hiPSCs, finding that the rearrangement occurs as an extrachromosomal marker (mar) element. All confirmed carrier hiPSCs and differentiated neural progenitor cell lines were found to be mosaic. We caution that mar elements may be difficult to functionally evaluate in hiPSC cultures using currently available methods, as it is difficult to distinguish cells with and without mar elements in live mosaic cultures.


Assuntos
Cromossomos Humanos , Marcadores Genéticos , Células-Tronco Pluripotentes Induzidas/metabolismo , Transtornos Psicóticos/genética , Duplicação Cromossômica , Cromossomos Humanos Par 9 , Hibridização Genômica Comparativa , Estudos de Associação Genética , Predisposição Genética para Doença , Heterozigoto , Humanos , Regiões de Interação com a Matriz/genética , Mosaicismo , Trissomia
13.
Dig Dis Sci ; 62(2): 407-417, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28058595

RESUMO

BACKGROUND: Gene data on infiltrative hepatocellular carcinoma (iHCC) are still unknown. AIMS: This study aims to identify the gene expression signature of iHCC compared with single nodular (SN)-type HCC according to the gross classification. METHODS: The whole-exome sequencing was performed in six matched HCC tumor/normal pairs (three infiltrative type and three single nodular type) from six patients who received curative hepatectomy. Subsequent validation using Sanger sequencing and real-time PCR was performed in 30 HCC tumor samples (15 infiltrative type and 15 single nodular type). RESULTS: Following whole-exome sequencing, Sanger sequencing, and bioinformatics analysis, it revealed significant difference of iHCC from SN-type HCC in gene patterns. Particularly, a typical growth factor receptor tyrosine kinase FGFR3 was predominantly mutated in iHCC. One nonsynonymous variant c.G285T (p.Q95H) and five additional mutations (c.G938A:p.G313D, c.G1291A:p.A431T, c.C1355G:p.T452R, c.C1377T:p.L459L, and c.A1445T:p.E482V) were investigated by whole-exome and Sanger sequencing, respectively. Immunohistochemical studies confirmed the specific expression of FGFR3 in iHCC samples. CONCLUSION: Our studies indicated that FGFR3 may be a candidate oncogene in tumor progression and a promising therapeutic target in iHCC patients who had early recurrence.


Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/cirurgia , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Hepatectomia , Humanos , Imuno-Histoquímica , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/cirurgia , Mutação , Reação em Cadeia da Polimerase em Tempo Real , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Análise de Sequência de DNA
14.
Genet Med ; 19(4): 412-420, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27657687

RESUMO

PURPOSE: To investigate the utility of whole-exome sequencing (WES) to define a molecular diagnosis for patients clinically diagnosed with congenital anomalies of kidney and urinary tract (CAKUT). METHODS: WES was performed in 62 families with CAKUT. WES data were analyzed for single-nucleotide variants (SNVs) in 35 known CAKUT genes, putatively deleterious sequence changes in new candidate genes, and potentially disease-associated copy-number variants (CNVs). RESULTS: In approximately 5% of families, pathogenic SNVs were identified in PAX2, HNF1B, and EYA1. Observed phenotypes in these families expand the current understanding about the role of these genes in CAKUT. Four pathogenic CNVs were also identified using two CNV detection tools. In addition, we found one deleterious de novo SNV in FOXP1 among the 62 families with CAKUT. The clinical database of the Baylor Miraca Genetics laboratory was queried and seven additional unrelated individuals with novel de novo SNVs in FOXP1 were identified. Six of these eight individuals with FOXP1 SNVs have syndromic urinary tract defects, implicating this gene in urinary tract development. CONCLUSION: We conclude that WES can be used to identify molecular etiology (SNVs, CNVs) in a subset of individuals with CAKUT. WES can also help identify novel CAKUT genes.Genet Med 19 4, 412-420.


Assuntos
Variações do Número de Cópias de DNA , Predisposição Genética para Doença/genética , Anormalidades Urogenitais/diagnóstico , Refluxo Vesicoureteral/diagnóstico , Sequenciamento Completo do Exoma/métodos , Adolescente , Criança , Pré-Escolar , Feminino , Fatores de Transcrição Forkhead/genética , Fator 1-beta Nuclear de Hepatócito/genética , Humanos , Lactente , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas Nucleares/genética , Fator de Transcrição PAX2/genética , Linhagem , Polimorfismo de Nucleotídeo Único , Proteínas Tirosina Fosfatases/genética , Proteínas Repressoras/genética , Anormalidades Urogenitais/genética , Refluxo Vesicoureteral/genética , Adulto Jovem
15.
J Gastroenterol Hepatol ; 32(4): 870-878, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27671209

RESUMO

BACKGROUND AND AIM: The superiority of anatomical resection (AR) in patients with hepatocellular carcinoma compared with non-anatomical resection (NAR) remains controversial. We aimed to investigate the prognostic outcomes of AR and NAR for solitary hepatocellular carcinoma (HCC) patients without macroscopic vascular invasion, using a propensity score matching (PSM) analysis. METHODS: A total of 305 consecutive HCC patients without macroscopic vascular invasion who underwent curative hepatectomy were included in our study. PSM was performed in order to eliminate possible selection bias. RESULTS: By PSM, the patients were divided into propensity-matched anatomical resection (PS-AR) (n = 114) and propensity-matched non-anatomical resection (PS-NAR) (n = 114) groups. The 1-year, 3-year, and 5-year overall survival rates were 90.4%, 77.7%, and 65.7% in PS-AR and 88.6%, 70.7%, and 52.2% in PS-NAR (P = 0.053), respectively. The 1-year, 3-year, and 5-year recurrence-free survival (RFS) rates were 84.1%, 64.9%, and 45.1% in PS-AR and 75.4%, 48.1%, and 31.0% in PS-NAR (P = 0.005), respectively. Multivariate analysis showed that ICG-R15 (P = 0.022); the Barcelona clinic liver cancer staging (P = 0.044) and microvascular invasion (MVI; P = 0.005) were independent risk factors for the overall survival rate, while type of resection (P = 0.027), surgical margin (P = 0.039), and MVI (P = 0.024) were independent risk factors for the RFS rate. Patients who underwent NAR were prone to early recurrence and marginal recurrence. Subgroup analysis indicated that the RFS rate was significantly better in PS-AR than that in PS-NAR (surgical margin ≥ 1 cm) (P = 0.025). Better RFS rate was observed in PS-AR with MVI compared with PS-NAR (P = 0.016). CONCLUSIONS: Anatomical resection contributed to improve the RFS rate in solitary HCC patients without macroscopic vascular invasion using PSM analysis, especially in patients with MVI.


Assuntos
Carcinoma Hepatocelular/cirurgia , Hepatectomia/métodos , Neoplasias Hepáticas/cirurgia , Pontuação de Propensão , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Intervalo Livre de Doença , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Recidiva Local de Neoplasia , Estadiamento de Neoplasias , Fatores de Risco , Taxa de Sobrevida , Fatores de Tempo , Adulto Jovem
16.
J Allergy Clin Immunol ; 139(1): 232-245, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27577878

RESUMO

BACKGROUND: Primary immunodeficiency diseases (PIDDs) are clinically and genetically heterogeneous disorders thus far associated with mutations in more than 300 genes. The clinical phenotypes derived from distinct genotypes can overlap. Genetic etiology can be a prognostic indicator of disease severity and can influence treatment decisions. OBJECTIVE: We sought to investigate the ability of whole-exome screening methods to detect disease-causing variants in patients with PIDDs. METHODS: Patients with PIDDs from 278 families from 22 countries were investigated by using whole-exome sequencing. Computational copy number variant (CNV) prediction pipelines and an exome-tiling chromosomal microarray were also applied to identify intragenic CNVs. Analytic approaches initially focused on 475 known or candidate PIDD genes but were nonexclusive and further tailored based on clinical data, family history, and immunophenotyping. RESULTS: A likely molecular diagnosis was achieved in 110 (40%) unrelated probands. Clinical diagnosis was revised in about half (60/110) and management was directly altered in nearly a quarter (26/110) of families based on molecular findings. Twelve PIDD-causing CNVs were detected, including 7 smaller than 30 Kb that would not have been detected with conventional diagnostic CNV arrays. CONCLUSION: This high-throughput genomic approach enabled detection of disease-related variants in unexpected genes; permitted detection of low-grade constitutional, somatic, and revertant mosaicism; and provided evidence of a mutational burden in mixed PIDD immunophenotypes.


Assuntos
Síndromes de Imunodeficiência/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Feminino , Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Adulto Jovem
17.
Nucleic Acids Res ; 45(4): 1633-1648, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-27980096

RESUMO

We developed an algorithm, HMZDelFinder, that uses whole exome sequencing (WES) data to identify rare and intragenic homozygous and hemizygous (HMZ) deletions that may represent complete loss-of-function of the indicated gene. HMZDelFinder was applied to 4866 samples in the Baylor-Hopkins Center for Mendelian Genomics (BHCMG) cohort and detected 773 HMZ deletion calls (567 homozygous or 206 hemizygous) with an estimated sensitivity of 86.5% (82% for single-exonic and 88% for multi-exonic calls) and precision of 78% (53% single-exonic and 96% for multi-exonic calls). Out of 773 HMZDelFinder-detected deletion calls, 82 were subjected to array comparative genomic hybridization (aCGH) and/or breakpoint PCR and 64 were confirmed. These include 18 single-exon deletions out of which 8 were exclusively detected by HMZDelFinder and not by any of seven other CNV detection tools examined. Further investigation of the 64 validated deletion calls revealed at least 15 pathogenic HMZ deletions. Of those, 7 accounted for 17-50% of pathogenic CNVs in different disease cohorts where 7.1-11% of the molecular diagnosis solved rate was attributed to CNVs. In summary, we present an algorithm to detect rare, intragenic, single-exon deletion CNVs using WES data; this tool can be useful for disease gene discovery efforts and clinical WES analyses.


Assuntos
Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Exoma , Doenças Genéticas Inatas/genética , Hemizigoto , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Algoritmos , Processamento Alternativo , Estudos de Coortes , Consanguinidade , Conjuntos de Dados como Assunto , Doenças Genéticas Inatas/diagnóstico , Humanos , Padrões de Herança , Modelos Genéticos , Linhagem , Reprodutibilidade dos Testes , Deleção de Sequência , Fluxo de Trabalho
18.
PLoS Genet ; 12(11): e1006446, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27880765

RESUMO

Chromosomal insertions are genomic rearrangements with a chromosome segment inserted into a non-homologous chromosome or a non-adjacent locus on the same chromosome or the other homologue, constituting ~2% of nonrecurrent copy-number gains. Little is known about the molecular mechanisms of their formation. We identified 16 individuals with complex insertions among 56,000 individuals tested at Baylor Genetics using clinical array comparative genomic hybridization (aCGH) and fluorescence in situ hybridization (FISH). Custom high-density aCGH was performed on 10 individuals with available DNA, and breakpoint junctions were fine-mapped at nucleotide resolution by long-range PCR and DNA sequencing in 6 individuals to glean insights into potential mechanisms of formation. We observed microhomologies and templated insertions at the breakpoint junctions, resembling the breakpoint junction signatures found in complex genomic rearrangements generated by replication-based mechanism(s) with iterative template switches. In addition, we analyzed 5 families with apparently balanced insertion in one parent detected by FISH analysis and found that 3 parents had additional small copy-number variants (CNVs) at one or both sides of the inserting fragments as well as at the inserted sites. We propose that replicative repair can result in interchromosomal complex insertions generated through chromothripsis-like chromoanasynthesis involving two or three chromosomes, and cause a significant fraction of apparently balanced insertions harboring small flanking CNVs.


Assuntos
Aberrações Cromossômicas , Inversão Cromossômica/genética , Replicação do DNA/genética , Duplicação Gênica/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA/genética , Feminino , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Masculino , Análise de Sequência de DNA , Translocação Genética
19.
Genome Med ; 8(1): 106, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27799064

RESUMO

BACKGROUND: Mitochondrial presequence proteases perform fundamental functions as they process about 70 % of all mitochondrial preproteins that are encoded in the nucleus and imported posttranslationally. The mitochondrial intermediate presequence protease MIP/Oct1, which carries out precursor processing, has not yet been established to have a role in human disease. METHODS: Whole exome sequencing was performed on four unrelated probands with left ventricular non-compaction (LVNC), developmental delay (DD), seizures, and severe hypotonia. Proposed pathogenic variants were confirmed by Sanger sequencing or array comparative genomic hybridization. Functional analysis of the identified MIP variants was performed using the model organism Saccharomyces cerevisiae as the protein and its functions are highly conserved from yeast to human. RESULTS: Biallelic single nucleotide variants (SNVs) or copy number variants (CNVs) in MIPEP, which encodes MIP, were present in all four probands, three of whom had infantile/childhood death. Two patients had compound heterozygous SNVs (p.L582R/p.L71Q and p.E602*/p.L306F) and one patient from a consanguineous family had a homozygous SNV (p.K343E). The fourth patient, identified through the GeneMatcher tool, a part of the Matchmaker Exchange Project, was found to have inherited a paternal SNV (p.H512D) and a maternal CNV (1.4-Mb deletion of 13q12.12) that includes MIPEP. All amino acids affected in the patients' missense variants are highly conserved from yeast to human and therefore S. cerevisiae was employed for functional analysis (for p.L71Q, p.L306F, and p.K343E). The mutations p.L339F (human p.L306F) and p.K376E (human p.K343E) resulted in a severe decrease of Oct1 protease activity and accumulation of non-processed Oct1 substrates and consequently impaired viability under respiratory growth conditions. The p.L83Q (human p.L71Q) failed to localize to the mitochondria. CONCLUSIONS: Our findings reveal for the first time the role of the mitochondrial intermediate peptidase in human disease. Loss of MIP function results in a syndrome which consists of LVNC, DD, seizures, hypotonia, and cataracts. Our approach highlights the power of data exchange and the importance of an interrelationship between clinical and research efforts for disease gene discovery.


Assuntos
Genes Recessivos/genética , Cardiopatias Congênitas/etiologia , Metaloendopeptidases/genética , Hipotonia Muscular/etiologia , Morte Súbita do Lactente/etiologia , Adulto , Sequência de Aminoácidos , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Fenótipo , Homologia de Sequência de Aminoácidos , Síndrome
20.
Genome Med ; 8(1): 105, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27799067

RESUMO

BACKGROUND: Smith-Magenis syndrome (SMS) is a developmental disability/multiple congenital anomaly disorder resulting from haploinsufficiency of RAI1. It is characterized by distinctive facial features, brachydactyly, sleep disturbances, and stereotypic behaviors. METHODS: We investigated a cohort of 15 individuals with a clinical suspicion of SMS who showed neither deletion in the SMS critical region nor damaging variants in RAI1 using whole exome sequencing. A combination of network analysis (co-expression and biomedical text mining), transcriptomics, and circularized chromatin conformation capture (4C-seq) was applied to verify whether modified genes are part of the same disease network as known SMS-causing genes. RESULTS: Potentially deleterious variants were identified in nine of these individuals using whole-exome sequencing. Eight of these changes affect KMT2D, ZEB2, MAP2K2, GLDC, CASK, MECP2, KDM5C, and POGZ, known to be associated with Kabuki syndrome 1, Mowat-Wilson syndrome, cardiofaciocutaneous syndrome, glycine encephalopathy, mental retardation and microcephaly with pontine and cerebellar hypoplasia, X-linked mental retardation 13, X-linked mental retardation Claes-Jensen type, and White-Sutton syndrome, respectively. The ninth individual carries a de novo variant in JAKMIP1, a regulator of neuronal translation that was recently found deleted in a patient with autism spectrum disorder. Analyses of co-expression and biomedical text mining suggest that these pathologies and SMS are part of the same disease network. Further support for this hypothesis was obtained from transcriptome profiling that showed that the expression levels of both Zeb2 and Map2k2 are perturbed in Rai1 -/- mice. As an orthogonal approach to potentially contributory disease gene variants, we used chromatin conformation capture to reveal chromatin contacts between RAI1 and the loci flanking ZEB2 and GLDC, as well as between RAI1 and human orthologs of the genes that show perturbed expression in our Rai1 -/- mouse model. CONCLUSIONS: These holistic studies of RAI1 and its interactions allow insights into SMS and other disorders associated with intellectual disability and behavioral abnormalities. Our findings support a pan-genomic approach to the molecular diagnosis of a distinctive disorder.


Assuntos
Exoma/genética , Redes Reguladoras de Genes , Genômica/métodos , Mutação/genética , Síndrome de Smith-Magenis/genética , Fatores de Transcrição/fisiologia , Transcriptoma , Animais , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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