RESUMO
BACKGROUND: Several techniques have been tailored to the quantification of microRNA expression, including hybridization arrays, quantitative PCR (qPCR), and high-throughput sequencing. Each of these has certain strengths and limitations depending both on the technology itself and the algorithm used to convert raw data into expression estimates. Reliable quantification of microRNA expression is challenging in part due to the relatively low abundance and short length of the miRNAs. While substantial research has been devoted to the development of methods to quantify mRNA expression, relatively little effort has been spent on microRNA expression. RESULTS: In this work, we focus on the Life Technologies TaqMan OpenArray(â) system, a qPCR-based platform to measure microRNA expression. Several algorithms currently exist to estimate expression from the raw amplification data produced by qPCR-based technologies. To assess and compare the performance of these methods, we performed a set of dilution/mixture experiments to create a benchmark data set. We also developed a suite of statistical assessments that evaluate many different aspects of performance: accuracy, precision, titration response, number of complete features, limit of detection, and data quality. The benchmark data and software are freely available via two R/Bioconductor packages, miRcomp and miRcompData. Finally, we demonstrate use of our software by comparing two widely used algorithms and providing assessments for four other algorithms. CONCLUSIONS: Benchmark data sets and software are crucial tools for the assessment and comparison of competing algorithms. We believe that the miRcomp and miRcompData packages will facilitate the development of new methodology for microRNA expression estimation.
Assuntos
MicroRNAs/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Software , Algoritmos , Benchmarking , Humanos , Limite de Detecção , MicroRNAs/metabolismoRESUMO
This study examined the association between 49 markers in the Runt-related transcription factor 2 (RUNX2) gene and nonsyndromic cleft lip with/without cleft palate (CL/P) among 326 Chinese case-parent trios, while considering gene-environment (GxE) interaction and parent-of-origin effects. Five single-nucleotide polymorphisms (SNPs) showed significant evidence of linkage and association with CL/P and these results were replicated in an independent European sample of 825 case-parent trios. We also report compelling evidence for interaction between markers in RUNX2 and environmental tobacco smoke (ETS). Although most marginal SNP effects (i.e., ignoring maternal exposures) were not statistically significant, eight SNPs were significant when considering possible interaction with ETS when testing for gene (G) and GxE interaction simultaneously or when considering GxE alone. Independent samples from European populations showed consistent evidence of significant GxETS interaction at two SNPs (rs6904353 and rs7748231). Our results suggest genetic variation in RUNX2 may influence susceptibility to CL/P through interacting with ETS.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Interação Gene-Ambiente , Polimorfismo de Nucleotídeo Único , Poluição por Fumaça de Tabaco/efeitos adversos , Povo Asiático/genética , China , Fenda Labial/etnologia , Fissura Palatina/etnologia , Europa (Continente) , Feminino , Ligação Genética , Predisposição Genética para Doença , Genótipo , Humanos , Masculino , Exposição Materna , Gravidez , Efeitos Tardios da Exposição Pré-Natal , População Branca/genéticaRESUMO
BACKGROUND: The myeloproliferative neoplasms, essential thrombocytosis, polycythemia vera and primary myelofibrosis, share the same acquired genetic lesion, but the concept of JAK2 V617F serving as the sole lesion responsible for these neoplasms is under question, and there has been interest in identifying additional mutations that may contribute to disease pathogenesis. Because ASXL1 lesions have been increasingly identified in myeloid neoplasms, we examined the relationships of ASXL1 mutation or deletion to both clinical phenotype and associated molecular features in 166 patients with myeloproliferative neoplasms. DESIGN AND METHODS: Exon 12 of ASXL1 was amplified from neutrophil genomic DNA and bidirectionally sequenced in 77 patients with myelofibrosis (including patients with primary and post-essential thrombocytosis or post-polycythemia myelofibrosis), 42 patients with polycythemia vera, 41 with essential thrombocytosis and 6 with post-myelofibrosis acute myeloid leukemia. Pyrosequencing assays were designed to determine the allele percentages of JAK2 V617F (G5073770T), ASXL1 2475dupA, and ASXL1 2846_2847del in neutrophil genomic DNA samples. Clinical and laboratory characteristics of patients with wild-type and ASXL1 mutations were then compared. RESULTS: We identified nonsense mutations or hemizygous deletion of ASXL1 in 36% of the patients with myelofibrosis, but very rarely among those with polycythemia vera or essential thrombocytosis. Among the patients with myelofibrosis, those with ASXL1 lesions were not distinguished from their wild-type counterparts with regard to JAK2 V617F status, exposure to chemotherapy or evolution to leukemia. Myelofibrosis patients with ASXL1 lesions were more likely to have received anemia-directed therapy compared to those without lesions [15/26 (58%) versus 11/39 (23%); P=0.02]. Using serial banked samples and quantitative ASXL1 mutant allele burden assays, we observed the acquisition and accumulation of ASXL1 mutations over time in two patients with post-essential thrombocytosis myelofibrosis. CONCLUSIONS: ASXL1 haploinsufficiency is associated with a myelofibrosis phenotype in the context of other known and unknown lesions, and disruption of ASXL1 function may contribute to the disease pathogenesis of myelofibrosis.
Assuntos
Mutação , Policitemia Vera/genética , Mielofibrose Primária/genética , Proteínas Repressoras/genética , Trombocitose/genética , Adolescente , Adulto , Alelos , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Genótipo , Humanos , Lactente , Recém-Nascido , Cariotipagem , Masculino , Transtornos Mieloproliferativos/genética , Fenótipo , Polimorfismo de Nucleotídeo Único , Mielofibrose Primária/diagnóstico , Trombocitemia Essencial/genética , Adulto JovemRESUMO
Although multiple genes have been identified as genetic risk factors for isolated, non-syndromic cleft lip with/without cleft palate (CL/P), a complex and heterogeneous birth defect, interferon regulatory factor 6 gene (IRF6) is one of the best documented genetic risk factors. In this study, we tested for association between markers in IRF6 and CL/P in 326 Chinese case-parent trios, considering gene-environment interaction for two common maternal exposures, and parent-of-origin effects. CL/P case-parent trios from three sites in mainland China and Taiwan were genotyped for 22 single nucleotide polymorphisms (SNPs) in IRF6. The transmission disequilibrium test was used to test for marginal effects of individual SNPs. We used PBAT to screen the SNPs and haplotypes for gene-environment (G×E) interaction and conditional logistic regression models to quantify effect sizes for SNP-environment interaction. After Bonferroni correction, 14 SNPs showed statistically significant association with CL/P. Evidence of G×E interaction was found for both maternal exposures, multivitamin supplementation and environmental tobacco smoke (ETS). Two SNPs showed evidence of interaction with multivitamin supplementation in conditional logistic regression models (rs2076153 nominal P=0.019, rs17015218 nominal P=0.012). In addition, rs1044516 yielded evidence for interaction with maternal ETS (nominal P=0.041). Haplotype analysis using PBAT also suggested interaction between SNPs in IRF6 and both multivitamin supplementation and ETS. However, no evidence for maternal genotypic effects or significant parent-of-origin effects was seen in these data. These results suggest IRF6 gene may influence risk of CL/P through interaction with multivitamin supplementation and ETS in the Chinese population.
Assuntos
Fenda Labial/genética , Fatores Reguladores de Interferon/genética , Polimorfismo de Nucleotídeo Único , Poluição por Fumaça de Tabaco/efeitos adversos , Vitaminas/efeitos adversos , Adulto , Povo Asiático/genética , China , Fenda Labial/etnologia , Fenda Labial/etiologia , Fissura Palatina/etnologia , Fissura Palatina/etiologia , Fissura Palatina/genética , Suplementos Nutricionais , Feminino , Predisposição Genética para Doença , Genótipo , Haplótipos , Humanos , Lactente , Recém-Nascido , Desequilíbrio de Ligação , Modelos Logísticos , Masculino , Exposição Materna/efeitos adversos , Núcleo Familiar , Gravidez , Efeitos Tardios da Exposição Pré-Natal/genética , Fatores de Risco , Taiwan , Vitaminas/administração & dosagemRESUMO
BACKGROUND: Idiopathic pulmonary fibrosis is progressive and often fatal; causes of familial clustering of the disease are unknown. Germ-line mutations in the genes hTERT and hTR, encoding telomerase reverse transcriptase and telomerase RNA, respectively, cause autosomal dominant dyskeratosis congenita, a rare hereditary disorder associated with premature death from aplastic anemia and pulmonary fibrosis. METHODS: To test the hypothesis that familial idiopathic pulmonary fibrosis may be caused by short telomeres, we screened 73 probands from the Vanderbilt Familial Pulmonary Fibrosis Registry for mutations in hTERT and hTR. RESULTS: Six probands (8%) had heterozygous mutations in hTERT or hTR; mutant telomerase resulted in short telomeres. Asymptomatic subjects with mutant telomerase also had short telomeres, suggesting that they may be at risk for the disease. We did not identify any of the classic features of dyskeratosis congenita in five of the six families. CONCLUSIONS: Mutations in the genes encoding telomerase components can appear as familial idiopathic pulmonary fibrosis. Our findings support the idea that pathways leading to telomere shortening are involved in the pathogenesis of this disease.
Assuntos
Mutação , Fibrose Pulmonar/genética , RNA/genética , Telomerase/genética , Telômero/patologia , Feminino , Genes Dominantes , Heterozigoto , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Fibrose Pulmonar/diagnóstico por imagem , Radiografia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Telomerase/metabolismo , Telômero/enzimologia , Telômero/genéticaRESUMO
Craniosynostosis (CS), the premature fusion of one or more cranial sutures, is a relatively common congenital anomaly, occurring in 3-5 per 10,000 live births. Nonsyndromic CS (NCS) accounts for up to 80% of all CS cases, yet the genetic factors contributing to the disorder remain largely unknown. The RUNX2 gene, encoding a transcription factor critical for bone and skull development, is a well known CS candidate gene, as copy number variations of this gene locus have been found in patients with syndromic craniosynostosis. In the present study, we aimed to characterize RUNX2 to better understand its role in the genetic etiology and in the molecular mechanisms underlying midline suture ossification in NCS. We report four nonsynonymous variants, one intronic variant and one 18 bp in-frame deletion in RUNX2 not found in our study control population. Significant difference in allele frequency (AF) for the deletion variant RUNX2 p.Ala84-Ala89del (ClinVar 257,095; dbSNP rs11498192) was observed in our sagittal NCS cohort when compared to the general population (P = 1.28 × 10-6), suggesting a possible role in the etiology of NCS. Dual-luciferase assays showed that three of four tested RUNX2 variants conferred a gain-of-function effect on RUNX2, further suggesting their putative pathogenicity in the tested NCS cases. Downregulation of RUNX2 expression was observed in prematurely ossified midline sutures. Metopic sites showed significant downregulation of promoter 1-specific isoforms compared to sagittal sites. Suture-derived mesenchymal stromal cells showed an increased expression of RUNX2 over matched unfused suture derived cells. This demonstrates that RUNX2, and particularly the distal promoter 1-isoform group, are overexpressed in the osteogenic precursors within the pathological suture sites.
Assuntos
Subunidade alfa 1 de Fator de Ligação ao Core , Craniossinostoses , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Suturas Cranianas , Craniossinostoses/genética , Variações do Número de Cópias de DNA , Mutação com Ganho de Função , HumanosRESUMO
Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence around 1 in 700 live births. The Runt-related transcription factor 2 (RUNX2) gene has been suggested as a candidate gene for CL/P based largely on mouse models; however, no human studies have focused on RUNX2 as a risk factor for CL/P. This study examines the association between markers in RUNX2 and isolated, nonsyndromic CL/P using a case-parent trio design, while considering parent-of-origin effects. Case-parent trios from four populations (77 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 24 single nucleotide polymorphisms (SNPs) in the RUNX2 gene. We performed the transmission disequilibrium test on individual SNPs. Parent-of-origin effects were assessed using the transmission asymmetry test and the parent-of-origin likelihood ratio test (PO-LRT). When all trios were combined, the transmission asymmetry test revealed a block of 11 SNPs showing excess maternal transmission significant at the P<0.01 level, plus one SNP (rs1934328) showing excess paternal transmission (P=0.002). For the 11 SNPs showing excess maternal transmission, odds ratios of being transmitted to the case from the mother ranged between 3.00 and 4.00. The parent-of-origin likelihood ratio tests for equality of maternal and paternal transmission were significant for three individual SNPs (rs910586, rs2819861, and rs1934328). Thus, RUNX2 appears to influence risk of CL/P through a parent-of-origin effect with excess maternal transmission.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Impressão Genômica , Feminino , Marcadores Genéticos , Predisposição Genética para Doença , Genótipo , Humanos , Padrões de Herança , Coreia (Geográfico) , Funções Verossimilhança , Desequilíbrio de Ligação , Masculino , Maryland , Polimorfismo de Nucleotídeo Único , Singapura , TaiwanRESUMO
This study examined the association between markers in transforming growth factor alpha (TGFA) and isolated, non-syndromic cleft lip with/without palate (CL/P) using a case-parent trio design, considering parent-of-origin effects. We also tested for gene-environmental interaction with common maternal exposures, and for gene-gene interaction using markers in TGFA and another recognized causal gene, IRF6. CL/P case-parent trios from four populations (76 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 17 single nucleotide polymorphisms (SNPs) in TGFA. The transmission disequilibrium test was used to test individual SNPs, and the parent-of-origin likelihood ratio test (PO-LRT) was used to assess parent-of-origin effects. We also screened for possible gene-environment interaction using PBAT, and tested for gene-gene interaction using conditional logistic regression models. When all trios were combined, four SNPs showed significant excess maternal transmission, two of which gave significant PO-LRT values [rs3821261: P = 0.004 and OR(imprinting) = 4.17; and rs3771475: P = 0.027 and OR(imprinting) = 2.44]. Haplotype analysis of these two SNPS also supported excess maternal transmission. We saw intriguing but suggestive evidence of G x E interaction for several SNPs in TGFA when either individual SNPs or haplotypes of adjacent SNPs were considered. Thus, TGFA appears to influence risk of CL/P through unconventional means with an apparent parent-of-origin effect (excess maternal transmission) and possible interaction with maternal exposures.
Assuntos
Fenda Labial/complicações , Fenda Labial/genética , Fissura Palatina/complicações , Fissura Palatina/genética , Fator de Crescimento Transformador alfa/genética , Feminino , Genótipo , Humanos , Fatores Reguladores de Interferon/genética , Desequilíbrio de Ligação , Masculino , Exposição Materna , Modelos Genéticos , Pais , Polimorfismo de Nucleotídeo Único , Mapeamento de Interação de Proteínas , SingapuraRESUMO
Neurofibromatosis type I (NF1) is an autosomal dominant genetic condition characterized by peripheral nervous system tumors (PNSTs), including plexiform neurofibromas (pNFs) that cause nerve dysfunction, deformity, pain damage to adjacent structures, and can undergo malignant transformation. There are no effective therapies to prevent or treat pNFs. Drug discovery efforts are slowed by the 'benign' nature of the Schwann cells that are the progenitor cells of pNF. In this work we characterize a set of pNF-derived cell lines at the genomic level (via SNP Arrays, RNAseq, and Whole Exome- Sequencing), and carry out dose response-based quantitative high-throughput screening (qHTS) with a collection of 1,912 oncology-focused compounds in a 1536-well microplate cell proliferation assays. Through the characterization and screening of NF1-/-, NF1+/+ and NF1+/- Schwann cell lines, this resource introduces novel therapeutic avenues for the development for NF1 associated pNF as well as all solid tumors with NF1 somatic mutations. The integrated data sets are openly available for further analysis at http://www.synapse.org/pnfCellCulture.
Assuntos
Perfilação da Expressão Gênica , Neurofibromatose 1 , Células de Schwann , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Humanos , Neurofibroma Plexiforme/genética , Neurofibroma Plexiforme/patologia , Neurofibroma Plexiforme/terapia , Neurofibromatose 1/genética , Neurofibromatose 1/patologia , Neurofibromatose 1/terapiaRESUMO
Importance: The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants. Objective: We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM. Design: This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh. Main Outcomes and Measures: The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations. Results: Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation. Conclusions and Relevance: This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Reguladoras de Apoptose/genética , Negro ou Afro-Americano/genética , Cardiomiopatia Dilatada/etnologia , Mutação , População Branca/genética , Animais , Cardiomiopatia Dilatada/genética , Estudos de Casos e Controles , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Camundongos , Prevalência , Prognóstico , Análise de Sequência de DNA , Análise de SobrevidaRESUMO
PURPOSE: To evaluate the effect of naturally occurring variants in genes encoding the cytochrome P450 (CYP) isoforms CYP3A4 and CYP3A5 in patients with cancer receiving midazolam as a phenotyping probe. EXPERIMENTAL DESIGN: Five variants in CYP3A4 and CYP3A5 were evaluated in 58 patients (21 women and 37 men) receiving a short i.v. bolus of midazolam (dose, 0.0145 or 0.025 mg/kg). Midazolam concentrations in plasma were determined using liquid chromatography-mass spectrometry, and pharmacokinetic variables were calculated using noncompartmental analysis. Genomic DNA was characterized for the variants by PCR-RFLP, and all genotypes were confirmed by direct nucleotide sequencing. RESULTS: The mean clearance of midazolam was 24.4 +/- 9.12 L/h, and phenotypic CYP3A activity varied about 4-fold in this population (range, 10.8-44.3 L/h). There were six carriers of the CYP3A4*1B allele (allele frequency, 0.061). No variant alleles for CYP3A4*17, CYP3A4*18A, or CYP3A5*6 were identified. Forty-eight of the 58 patients were homozygous variant for CYP3A5*3C, eight were heterozygous, and two were homozygous wild type (allele frequency, 0.897). No associations were noted between any of the studied genotypes and the phenotypic measures (P > or = 0.16). Likewise, a common variant in exon 26 in the gene encoding P-glycoprotein [i.e., ABCB1 (MDR1) 3435C>T] that was previously reported to be linked to CYP3A4 mRNA levels was unrelated to any of the studied phenotypic measures (P > or = 0.49). CONCLUSIONS: The studied genetic variants in CYP3A4 and CYP3A5 are unlikely to have an important functional significance to phenotypic CYP3A activity in patients with cancer.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Midazolam/farmacocinética , Neoplasias/metabolismo , Polimorfismo de Nucleotídeo Único , Transportadores de Cassetes de Ligação de ATP/genética , Adulto , Idoso , Alelos , Área Sob a Curva , Citocromo P-450 CYP3A , Feminino , Frequência do Gene , Genótipo , Humanos , Desequilíbrio de Ligação , Masculino , Midazolam/sangue , Pessoa de Meia-Idade , Neoplasias/genética , FenótipoRESUMO
STUDY OBJECTIVES: Marked variability exists in coronary artery collaterals in patients with ischemic heart disease. Although multiple factors are thought to play a role in collateral development, the contribution of genetic factors is largely unknown. Hypoxia inducible factor 1 (HIF-1), a transcriptional activator that functions as a master regulator of oxygen homeostasis, is one possible genetic factor that could play an important role in modulating collateral development. DESIGN, SETTING, AND PARTICIPANTS: Collateral vessels were determined in 100 patients with > or = 70% narrowing of at least one coronary artery without acute myocardial infarction or prior revascularization. DNA was genotyped for the presence of a single nucleotide (C to T) polymorphism that changes residue 582 of HIF-1alpha from proline to serine. MEASUREMENTS AND RESULTS: The frequency of the T allele was significantly higher among patients without collaterals compared to patients with collaterals (0.188 vs 0.037, p < 0.001). In multivariate analyses, two variables affecting collateral formation were detected: two- or three-vessel coronary artery disease was a significant positive predictor (odds ratio [OR], 4.17; 95% confidence interval [CI], 1.61 to 10.8; p = 0.001), whereas the presence of HIF-1alpha genotype CT or TT was a negative predictor (OR, 0.19; 95% CI, 0.04 to 0.84; p = 0.03). CONCLUSIONS: These data suggest that variations in HIF-1alpha genotype may influence development of coronary artery collaterals in patients with significant coronary artery disease.
Assuntos
Circulação Colateral/genética , Isquemia Miocárdica/genética , Polimorfismo Genético , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia , Masculino , Pessoa de Meia-IdadeRESUMO
Anticipation has been frequently found in bipolar families ascertained for linkage studies. An association of polymorphic triplet repeats with the bipolar phenotype in some pedigrees has been proposed. We have previously found linkage to chromosome 18 in a set of families with evidence of anticipation. As part of a search for CAG/CTG motifs on chromosome 18, we screened a genomic chromosome 18 cosmid library and identified 65 loci with trinucleotide repeats. Eleven of 33 genotyped loci were polymorphic, though none of these showed any evidence of instability. We performed genetic analysis of six loci in the Hopkins/Dana bipolar pedigrees ascertained for a genetic linkage study of bipolar disorder and found that the CAG repeat within the AD4D2 clone on 18q21.1 showed nominally significant over-transmission of the rare CAG23 allele (P=0.034). We have characterized all 65 trinucleotide repeats and flanking sequences with GENSCAN analysis and find that 29 were predicted to be in coding regions. These 29 trinucleotide-repeat-containing genes may be involved in functional modulation of their respective proteins, and may be candidates for other diseases or disease mechanisms that map to this region.
Assuntos
Transtorno Bipolar/genética , Cromossomos Humanos Par 18 , Repetições de Trinucleotídeos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , HumanosRESUMO
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
Insulin secretion has a crucial role in glucose homeostasis, and failure to secrete sufficient insulin is a hallmark of type 2 diabetes. Genome-wide association studies (GWAS) have identified loci contributing to insulin processing and secretion; however, a substantial fraction of the genetic contribution remains undefined. To examine low-frequency (minor allele frequency (MAF) 0.5-5%) and rare (MAF < 0.5%) nonsynonymous variants, we analyzed exome array data in 8,229 nondiabetic Finnish males using the Illumina HumanExome Beadchip. We identified low-frequency coding variants associated with fasting proinsulin concentrations at the SGSM2 and MADD GWAS loci and three new genes with low-frequency variants associated with fasting proinsulin or insulinogenic index: TBC1D30, KANK1 and PAM. We also show that the interpretation of single-variant and gene-based tests needs to consider the effects of noncoding SNPs both nearby and megabases away. This study demonstrates that exome array genotyping is a valuable approach to identify low-frequency variants that contribute to complex traits.
Assuntos
Exoma/genética , Variação Genética , Insulina/genética , Insulina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Amidina-Liases/genética , Proteínas do Citoesqueleto , Proteínas Adaptadoras de Sinalização de Receptores de Domínio de Morte/genética , Jejum/sangue , Finlândia , Frequência do Gene , Genética Populacional , Genótipo , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Secreção de Insulina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Oxigenases de Função Mista/genética , Anotação de Sequência Molecular , Proinsulina/sangue , Proteínas Supressoras de Tumor/genéticaRESUMO
BACKGROUND: Congenital Cytomegalovirus (CMV) infection is an important medical problem that has yet no current solution. A clinical trial of CMV glycoprotein B (gB) vaccine in young women showed promising efficacy. Improved understanding of the basis for prevention of CMV infection is essential for developing improved vaccines. RESULTS: We genotyped 142 women previously vaccinated with three doses of CMV gB for single nucleotide polymorphisms (SNPs) in TLR 1-4, 6, 7, 9, and 10, and their associated intracellular signaling genes. SNPs in the platelet-derived growth factor receptor (PDGFRA) and integrins were also selected based on their role in binding gB. Specific SNPs in TLR7 and IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) were associated with antibody responses to gB vaccine. Homozygous carriers of the minor allele at four SNPs in TLR7 showed higher vaccination-induced antibody responses to gB compared to heterozygotes or homozygotes for the common allele. SNP rs1953090 in IKBKE was associated with changes in antibody level from second to third dose of vaccine; homozygotes for the minor allele exhibited lower antibody responses while homozygotes for the major allele showed increased responses over time. CONCLUSIONS: These data contribute to our understanding of the immunogenetic mechanisms underlying variations in the immune response to CMV vaccine.
Assuntos
Anticorpos Antivirais/biossíntese , Infecções por Citomegalovirus/prevenção & controle , Vacinas contra Citomegalovirus/imunologia , Polimorfismo de Nucleotídeo Único/imunologia , Receptores Toll-Like/imunologia , Vacinação , Adulto , Alelos , Anticorpos Antivirais/imunologia , Estudos de Coortes , Citomegalovirus/imunologia , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/genética , Infecções por Citomegalovirus/imunologia , Vacinas contra Citomegalovirus/administração & dosagem , Feminino , Genótipo , Heterozigoto , Homozigoto , Humanos , Quinase I-kappa B/genética , Quinase I-kappa B/imunologia , Integrinas/genética , Integrinas/imunologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Receptores Toll-Like/genética , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologiaRESUMO
5-Fluorouracil (5FU), a widely used chemotherapeutic drug, inhibits the DNA replicative enzyme, thymidylate synthase (Tyms). Prior studies implicated a VNTR (variable numbers of tandem repeats) polymorphism in the 5'-untranslated region (5'-UTR) of the TYMS gene as a determinant of Tyms expression in tumors and normal tissues and proposed that these VNTR genotypes could help decide fluoropyrimidine dosing. Clinical associations between 5FU-related toxicity and the TYMS VNTR were reported, however, results were inconsistent, suggesting that additional genetic variation in the TYMS gene might influence Tyms expression. We thus conducted a detailed genetic analysis of this region, defining new polymorphisms in this gene including mononucleotide (poly A:T) repeats and novel single nucleotide polymorphisms (SNPs) flanking the VNTR in the TYMS genetic region. Our haplotype analysis of this region used data from both established and novel genetic variants and found nine SNP haplotypes accounting for more than 90% of the studied population. We observed non-exclusive relationships between the VNTR and adjacent SNP haplotypes, such that each type of VNTR commonly occurred on several haplotype backgrounds. Our results confirmed the expectation that the VNTR alleles exhibit homoplasy and lack the common ancestry required for a reliable marker of a linked adjacent locus that might govern toxicity. We propose that it may be necessary in a clinical trial to assay multiple types of genetic polymorphisms in the TYMS region to meaningfully model linkage of genetic markers to 5FU-related toxicity. The presence of multiple long (up to 26 nt), polymorphic monothymidine repeats in the promoter region of the sole human thymidylate synthetic enzyme is intriguing.
Assuntos
Fluoruracila/uso terapêutico , Haplótipos/genética , Neoplasias Pancreáticas/genética , Farmacogenética , Polimorfismo Genético/efeitos dos fármacos , Sequências de Repetição em Tandem/genética , Timidilato Sintase/genética , Antimetabólitos Antineoplásicos/uso terapêutico , Sequência de Bases , Humanos , Repetições Minissatélites , Dados de Sequência Molecular , Pâncreas/efeitos dos fármacos , Pâncreas/enzimologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
Isolated cleft lip with or without cleft palate and cleft palate are among the most common human birth defects. Several candidate gene studies on MSX1 have shown significant association between markers in MSX1 and risk of oral clefts, and re-sequencing studies have identified multiple mutations in MSX1 in a small minority of cases, which may account for 1-2% of all isolated oral clefts cases. We explored the 2-Mb region around MSX1, using a marker map of 393 single nucleotide polymorphisms (SNPs) in 297 cleft lip, with or without cleft palate, case-parent trios and 84 cleft palate trios from Maryland, Taiwan, Singapore, and Korea. Both individual markers and haplotypes of two to five SNPs showed several regions yielding statistical evidence for linkage and disequilibrium. Two genes (STK32B and EVC) yielded consistent evidence from cleft lip, with or without cleft palate, trios in all four populations. These two genes plus EVC2 also yielded suggestive evidence for linkage and disequilibrium among cleft palate trios. This analysis suggests that several genes, not just MSX1, in this region may influence risk of oral clefts.
Assuntos
Cromossomos Humanos Par 4/genética , Fenda Labial/genética , Fissura Palatina/genética , Estudo de Associação Genômica Ampla , Feminino , Genes , Predisposição Genética para Doença , Genética Populacional , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Coreia (Geográfico) , Desequilíbrio de Ligação , Fator de Transcrição MSX1/genética , Masculino , Maryland , Proteínas de Membrana , Polimorfismo de Nucleotídeo Único , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Singapura , TaiwanRESUMO
The genetic structure of sheep reflects their domestication and subsequent formation into discrete breeds. Understanding genetic structure is essential for achieving genetic improvement through genome-wide association studies, genomic selection and the dissection of quantitative traits. After identifying the first genome-wide set of SNP for sheep, we report on levels of genetic variability both within and between a diverse sample of ovine populations. Then, using cluster analysis and the partitioning of genetic variation, we demonstrate sheep are characterised by weak phylogeographic structure, overlapping genetic similarity and generally low differentiation which is consistent with their short evolutionary history. The degree of population substructure was, however, sufficient to cluster individuals based on geographic origin and known breed history. Specifically, African and Asian populations clustered separately from breeds of European origin sampled from Australia, New Zealand, Europe and North America. Furthermore, we demonstrate the presence of stratification within some, but not all, ovine breeds. The results emphasize that careful documentation of genetic structure will be an essential prerequisite when mapping the genetic basis of complex traits. Furthermore, the identification of a subset of SNP able to assign individuals into broad groupings demonstrates even a small panel of markers may be suitable for applications such as traceability.
Assuntos
Estruturas Genéticas , Estudo de Associação Genômica Ampla , Genoma/genética , Polimorfismo de Nucleotídeo Único/genética , Carneiro Doméstico/genética , Animais , Cruzamento , Marcadores Genéticos , Genética PopulacionalRESUMO
Whole genome amplification (wga) of DNA is being widely implemented in many laboratories to extend the life of samples only available in limited quantities for genetic analysis. We determined the reliability of wgaDNA genotypes in three sets of replicates from the same individuals: (i) 23 pairs of genomic DNA (gDNA), (ii) 43 pairs gDNA versus wgaDNA, and (iii) 29 pairs of independently amplified wgaDNA. Amplification was performed using multiple displacement amplification (MDA). Genotyping was successful for both DNA types for 1268 out of 1534 SNPs from 164 cardiovascular candidate genes assayed in a single Illumina panel. Amplified DNA failed for 77 SNPs (6%) that were genotyped successfully with genomic material. Percent of successful SNP calls, and concordance between pairs and kappa statistics (kappa) were determined. A total of 54 110 genotypes from gDNA-wgaDNA pairs were available for concordance analysis. Mean kappa for gDNA-wgaDNA pairs was 0.99. Concordance between gDNA-wgaDNA pairs was higher than amongst wgaDNA pairs (mean kappa for the 29 independently amplified pairs of wgaDNA was 0.95; interquartile range: 0.93-1.00). A statistical analysis of those SNPs which failed to genotype from amplified DNA only revealed that those loci were more likely to be closer to the telomeres and in locally GC-rich sequences. In summary, the MDA method produces wgaDNA samples that can be genotyped using high-throughput technology with a very high reproducibility to the original DNA but with slightly lower call rates. DNA amplification methodologies provide a useful solution for current and future large-scale genetic analyses especially with limited quantities of samples and DNA.