RESUMO
Recent technological advancements have permitted high-throughput measurement of the human genome, epigenome, metabolome, transcriptome, and proteome at the population level. We hypothesized that subsets of genes identified from omic studies might have closely related biological functions and thus might interact directly at the network level. Therefore, we conducted an integrative analysis of multi-omic datasets of non-small cell lung cancer (NSCLC) to search for association patterns beyond the genome and transcriptome. A large, complex, and robust gene network containing well-known lung cancer-related genes, including EGFR and TERT, was identified from combined gene lists for lung adenocarcinoma. Members of the hypoxia-inducible factor (HIF) gene family were at the center of this network. Subsequent sequencing of network hub genes within a subset of samples from the Transdisciplinary Research in Cancer of the Lung-International Lung Cancer Consortium (TRICL-ILCCO) consortium revealed a SNP (rs12614710) in EPAS1 associated with NSCLC that reached genome-wide significance (ORâ¯=â¯1.50; 95% CI: 1.31-1.72; pâ¯=â¯7.75â¯×â¯10-9). Using imputed data, we found that this SNP remained significant in the entire TRICL-ILCCO consortium (pâ¯=â¯.03). Additional functional studies are warranted to better understand interrelationships among genetic polymorphisms, DNA methylation status, and EPAS1 expression.
Assuntos
Adenocarcinoma/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Idoso , Carcinoma Pulmonar de Células não Pequenas/patologia , Metilação de DNA/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo ÚnicoRESUMO
This unit describes a technique for generating exome-enriched sequencing libraries using DNA extracted from formalin-fixed paraffin-embedded (FFPE) samples. Utilizing commercially available kits, we present a low-input FFPE workflow starting with 50 ng of DNA. This procedure includes a repair step to address damage caused by FFPE preservation that improves sequence quality. Subsequently, libraries undergo an in-solution-targeted selection for exons, followed by sequencing using the Illumina next-generation short-read sequencing platform. © 2017 by John Wiley & Sons, Inc.
Assuntos
DNA/genética , Sequenciamento do Exoma , Exoma/genética , Formaldeído , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Inclusão em Parafina , Fixação de Tecidos , Humanos , ParafinaRESUMO
IMPORTANCE: Microphthalmias are rare disorders whose genetic bases are not fully understood. HMGB3 is a new candidate gene for X-linked forms of this disease. OBJECTIVE: To identify the causative gene in a pedigree with an X-linked colobomatous microphthalmos phenotype. DESIGN, SETTING, AND PARTICIPANTS: Whole-genome sequencing and chromosome X-exome-targeted sequencing were performed at the High Throughput Sequencing Laboratory of the Genetic Resources Core Facility at the Johns Hopkins University School of Medicine on the DNA of the male proband and informatically filtered to identify rare variants. Polymerase chain reaction and Sanger sequencing were used to confirm the variant in the proband and the carrier status of his mother. Thirteen unrelated male patients with a similar phenotype were also screened. MAIN OUTCOMES AND MEASURES: Whole-genome and X-exome sequencing to identify a frameshift variant in HMGB3. RESULTS: A 2-base pair frameshift insertion (c.477_478insTA, coding for p.Lys161Ilefs*54) in the HGMB3 gene was found in the proband and his carrier mother but not in the unrelated patients. The mutation, confirmed by 3 orthogonal methods, alters an evolutionarily conserved region of the HMGB3 protein from a negatively charged polyglutamic acid tract to a positively charged arginine-rich motif that is likely to interfere with normal protein function. CONCLUSIONS AND RELEVANCE: In this family, microphthalmia, microcephaly, intellectual disability, and short stature are associated with a mutation on the X chromosome in the HMGB3 gene. HMGB3 should be considered when performing genetic studies of patients with similar phenotypes.
Assuntos
Coloboma/genética , Mutação da Fase de Leitura/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Proteína HMGB3/genética , Microftalmia/genética , Criança , Cromossomos Humanos X/genética , Exoma/genética , Genoma Humano/genética , Transtornos do Crescimento/genética , Humanos , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Linhagem , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.