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1.
Nat Rev Genet ; 24(12): 801-802, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37723349
2.
Hum Genet ; 142(1): 33-43, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36048236

RESUMO

Genetic variants in GJB2 are the most frequent cause of congenital and childhood hearing loss worldwide. The purpose of this study was to delineate the genetic and phenotypic landscape of GJB2 SNV variants. All possible single-nucleotide substitution variants of the coding region of GJB2 (N = 2043) were manually curated following the ACMG/AMP hearing loss guidelines. As a result, 60 (2.9%), 177 (8.7%), 1499 (73.4%), 301 (14.7%) and 6 (0.3%) of the variants were classified as pathogenic, likely pathogenic, variant of uncertain significance, likely benign, and benign, respectively. 53% (84/158) of the pathogenic/likely pathogenic missense variants were not present in ClinVar. The second transmembrane domain and the 310 helix were highly enriched for pathogenic missense variants, while the intracellular loops were tolerant to variation. The N-terminal tail and the extracellular loop showed high clustering of variants that are associated with syndromic or dominant non-syndromic hearing loss. In conclusion, our study interpreted all possible single-nucleotide substitution coding variants, characterized novel clinically significant variants in GJB2, and revealed significant genotype-phenotype correlations at this common hearing loss locus. Our work provides a prototype for other genes with similarly high genetic and phenotypic heterogeneity.


Assuntos
Surdez , Perda Auditiva , Humanos , Conexinas/genética , Conexina 26/genética , Perda Auditiva/genética , Surdez/genética , Mutação de Sentido Incorreto , Mutação
3.
Genome Res ; 29(7): 1144-1151, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31235655

RESUMO

Recent advances in DNA sequencing have expanded our understanding of the molecular basis of genetic disorders and increased the utilization of clinical genomic tests. Given the paucity of evidence to accurately classify each variant and the difficulty of experimentally evaluating its clinical significance, a large number of variants generated by clinical tests are reported as variants of unknown clinical significance. Population-scale variant databases can improve clinical interpretation. Specifically, pathogenicity prediction for novel missense variants can use features describing regional variant constraint. Constrained genomic regions are those that have an unusually low variant count in the general population. Computational methods have been introduced to capture these regions and incorporate them into pathogenicity classifiers, but these methods have yet to be compared on an independent clinical variant data set. Here, we introduce one variant data set derived from clinical sequencing panels and use it to compare the ability of different genomic constraint metrics to determine missense variant pathogenicity. This data set is compiled from 17,071 patients surveyed with clinical genomic sequencing for cardiomyopathy, epilepsy, or RASopathies. We further use this data set to demonstrate the necessity of disease-specific classifiers and to train PathoPredictor, a disease-specific ensemble classifier of pathogenicity based on regional constraint and variant-level features. PathoPredictor achieves an average precision >90% for variants from all 99 tested disease genes while approaching 100% accuracy for some genes. The accumulation of larger clinical variant training data sets can significantly enhance their performance in a disease- and gene-specific manner.


Assuntos
Cardiomiopatias/genética , Conjuntos de Dados como Assunto , Epilepsia/genética , Variação Genética , Proteínas ras/genética , Humanos , Mutação de Sentido Incorreto
4.
Genet Med ; 24(4): 924-930, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34955381

RESUMO

PURPOSE: According to the American College of Medical Genetics and Genomics/Association of Medical Pathology (ACMG/AMP) guidelines, in silico evidence is applied at the supporting strength level for pathogenic (PP3) and benign (BP4) evidence. Although PP3 is commonly used, less is known about the effect of these criteria on variant classification outcomes. METHODS: A total of 727 missense variants curated by Clinical Genome Resource expert groups were analyzed to determine how often PP3 and BP4 were applied and their impact on variant classification. The ACMG/AMP categorical system of variant classification was compared with a quantitative point-based system. The pathogenicity likelihood ratios of REVEL, VEST, FATHMM, and MPC were calibrated using a gold standard set of 237 pathogenic and benign variants (classified independent of the PP3/BP4 criteria). RESULTS: The PP3 and BP4 criteria were applied by Variant Curation Expert Panels to 55% of missense variants. Application of those criteria changed the classification of 15% of missense variants for which either criterion was applied. The point-based system resolved borderline classifications. REVEL and VEST performed best at a strength level consistent with moderate evidence. CONCLUSION: We show that in silico criteria are commonly applied and often affect the final variant classifications. When appropriate thresholds for in silico predictors are established, our results show that PP3 and BP4 can be used at a moderate strength.


Assuntos
Variação Genética , Genoma Humano , Humanos , Testes Genéticos/métodos , Variação Genética/genética , Genômica/métodos
5.
Hum Mutat ; 40(3): 243-257, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30582250

RESUMO

The PCDH19 gene consists of six exons encoding a 1,148 amino acid transmembrane protein, Protocadherin 19, which is involved in brain development. Heterozygous pathogenic variants in this gene are inherited in an unusual X-linked dominant pattern in which heterozygous females are affected, while hemizygous males are typically unaffected, although they pass on the pathogenic variant to each affected daughter. PCDH19-related disorder is known to cause early-onset epilepsy in females characterized by seizure clusters exacerbated by fever and in most cases, onset is within the first year of life. This condition was initially described in 1971 and in 2008 PCDH19 was identified as the underlying genetic etiology. This condition is the result of pathogenic loss-of-function variants that may be de novo or inherited from an affected mother or unaffected father and cellular interference has been hypothesized to be the culprit. Heterozygous females are symptomatic because of the presence of both wild-type and mutant cells that interfere with one another due to the production of different surface proteins, whereas nonmosaic hemizygous males produce a homogenous population of cells. Here, we review novel pathogenic variants in the PCDH19 gene since 2012 to date, and summarize any genotype-phenotype correlations.


Assuntos
Caderinas/genética , Epilepsia/genética , Mutação/genética , Idade de Início , Éxons/genética , Feminino , Estudos de Associação Genética , Humanos , Masculino , Protocaderinas
6.
Genet Med ; 21(11): 2442-2452, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31160754

RESUMO

PURPOSE: Pathogenic variants in GJB2 are the most common cause of autosomal recessive sensorineural hearing loss. The classification of c.101T>C/p.Met34Thr and c.109G>A/p.Val37Ile in GJB2 are controversial. Therefore, an expert consensus is required for the interpretation of these two variants. METHODS: The ClinGen Hearing Loss Expert Panel collected published data and shared unpublished information from contributing laboratories and clinics regarding the two variants. Functional, computational, allelic, and segregation data were also obtained. Case-control statistical analyses were performed. RESULTS: The panel reviewed the synthesized information, and classified the p.Met34Thr and p.Val37Ile variants utilizing professional variant interpretation guidelines and professional judgment. We found that p.Met34Thr and p.Val37Ile are significantly overrepresented in hearing loss patients, compared with population controls. Individuals homozygous or compound heterozygous for p.Met34Thr or p.Val37Ile typically manifest mild to moderate hearing loss. Several other types of evidence also support pathogenic roles for these two variants. CONCLUSION: Resolving controversies in variant classification requires coordinated effort among a panel of international multi-institutional experts to share data, standardize classification guidelines, review evidence, and reach a consensus. We concluded that p.Met34Thr and p.Val37Ile variants in GJB2 are pathogenic for autosomal recessive nonsyndromic hearing loss with variable expressivity and incomplete penetrance.


Assuntos
Conexinas/genética , Perda Auditiva/genética , Alelos , Estudos de Casos e Controles , Conexina 26/genética , Conexinas/metabolismo , Surdez/genética , Feminino , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Mutação , Polimorfismo de Nucleotídeo Único/genética
7.
Hum Mutat ; 39(11): 1517-1524, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30192042

RESUMO

The 2015 ACMG/AMP sequence variant interpretation guideline provided a framework for classifying variants based on several benign and pathogenic evidence criteria, including a pathogenic criterion (PVS1) for predicted loss of function variants. However, the guideline did not elaborate on specific considerations for the different types of loss of function variants, nor did it provide decision-making pathways assimilating information about variant type, its location, or any additional evidence for the likelihood of a true null effect. Furthermore, this guideline did not take into account the relative strengths for each evidence type and the final outcome of their combinations with respect to PVS1 strength. Finally, criteria specifying the genes for which PVS1 can be applied are still missing. Here, as part of the ClinGen Sequence Variant Interpretation (SVI) Workgroup's goal of refining ACMG/AMP criteria, we provide recommendations for applying the PVS1 criterion using detailed guidance addressing the above-mentioned gaps. Evaluation of the refined criterion by seven disease-specific groups using heterogeneous types of loss of function variants (n = 56) showed 89% agreement with the new recommendation, while discrepancies in six variants (11%) were appropriately due to disease-specific refinements. Our recommendations will facilitate consistent and accurate interpretation of predicted loss of function variants.


Assuntos
Genoma Humano/genética , Sociedades Médicas/normas , Biologia Computacional/métodos , Éxons/genética , Testes Genéticos/métodos , Testes Genéticos/normas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas , Estados Unidos
8.
Hum Mutat ; 39(11): 1593-1613, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30311386

RESUMO

Due to the high genetic heterogeneity of hearing loss (HL), current clinical testing includes sequencing large numbers of genes, which often yields a significant number of novel variants. Therefore, the standardization of variant interpretation is crucial to provide consistent and accurate diagnoses. The Hearing Loss Variant Curation Expert Panel was created within the Clinical Genome Resource to provide expert guidance for standardized genomic interpretation in the context of HL. As one of its major tasks, our Expert Panel has adapted the American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines for the interpretation of sequence variants in HL genes. Here, we provide a comprehensive illustration of the newly specified ACMG/AMP HL rules. Three rules remained unchanged, four rules were removed, and the remaining 21 rules were specified. These rules were further validated and refined using a pilot set of 51 variants assessed by curators and disease experts. Of the 51 variants evaluated in the pilot, 37% (19/51) changed category based upon application of the expert panel specified rules and/or aggregation of evidence across laboratories. These HL-specific ACMG/AMP rules will help standardize variant interpretation, ultimately leading to better care for individuals with HL.


Assuntos
Testes Genéticos/métodos , Genoma Humano/genética , Perda Auditiva/genética , Frequência do Gene/genética , Variação Genética/genética , Genômica/métodos , Humanos , Mutação/genética , Análise de Sequência de DNA , Sociedades Médicas , Estados Unidos
9.
Genet Med ; 20(12): 1600-1608, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29595809

RESUMO

PURPOSE: Hereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes. METHODS: A tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes. RESULTS: ES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis. CONCLUSION: The tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.


Assuntos
Predisposição Genética para Doença , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Patologia Molecular , Exoma/genética , Feminino , Perda Auditiva Neurossensorial/fisiopatologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA , Sequenciamento do Exoma
10.
Clin Chem ; 64(4): 705-714, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29339441

RESUMO

BACKGROUND: Copy number variants (CNVs) can substantially contribute to the pathogenic variant spectrum in several disease genes. The detection of this type of variant is complicated in genes with high homology to other genomic sequences, yet such genomics regions are more likely to lead to CNVs, making it critical to address detection in these settings. METHODS: We developed a copy number analysis approach for high homology genes/regions that consisted of next-generation sequencing (NGS)-based dosage analysis accompanied by allele-specific droplet digital PCR (ddPCR) confirmatory testing. We applied this approach to copy number analysis in STRC, a gene with 98.9% homology to a nonfunctional pseudogene, pSTRC, and characterized its accuracy in detecting different copy number states by use of known samples. RESULTS: Using a cohort of 517 patients with hearing loss, we prospectively demonstrated the clinical utility of the approach, which contributed 30 of the 122 total positives (6%) to the diagnostic yield, increasing the overall yield from 17.6% to 23.6%. Positive STRC genotypes included homozygous (n = 15) or compound heterozygous (n = 8) deletions, or heterozygous deletions in trans with pathogenic sequence variants (n = 7). Finally, this approach limited ddPCR testing to cases with NGS copy number findings, thus markedly reducing the number of costly and laborious, albeit specific, ddPCR tests. CONCLUSIONS: NGS-based CNV detection followed by allele-specific ddPCR confirmatory testing is a reliable and affordable approach for copy number analysis in medically relevant genes with homology issues.


Assuntos
Algoritmos , Alelos , Variações do Número de Cópias de DNA , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase/métodos , Estudos de Casos e Controles , Perda Auditiva/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Estudo de Prova de Conceito
11.
Prenat Diagn ; 38(1): 26-32, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28345240

RESUMO

Clinical diagnostic laboratories are producing next-generation sequencing-based test results that are becoming increasingly incorporated into patient care. Whole genome and exome sequencing on fetal material derived from amniocytes, chorionic villi, or products of conception is starting to be offered clinically in specialized centers, but it has not yet become routine practice. The technical, interpretation, and ethical challenges are greatest in the area of prenatal medicine because the fetus has a limited health history, and the physical examination is only indirectly available via prenatal sonography. Here, we provide an overview of these challenges and highlight the clinical utility, reporting, and counseling issues associated with prenatal DNA sequencing. Future considerations are also discussed. © 2017 John Wiley & Sons, Ltd.


Assuntos
Diagnóstico Pré-Natal , Sequenciamento Completo do Genoma , Técnicas de Laboratório Clínico , Feminino , Aconselhamento Genético , Humanos , Gravidez
12.
Genet Med ; 19(5): 496-504, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27657688

RESUMO

PURPOSE: Classification of novel variants is a major challenge facing the widespread adoption of comprehensive clinical genomic sequencing and the field of personalized medicine in general. This is largely because most novel variants do not have functional, genetic, or population data to support their clinical classification. METHODS: To improve variant interpretation, we leveraged the Exome Aggregation Consortium (ExAC) data set (N = ~60,000) as well as 7,000 clinically curated variants in 132 genes identified in more than 11,000 probands clinically tested for cardiomyopathies, rasopathies, hearing loss, or connective tissue disorders to perform a systematic evaluation of domain level disease associations. RESULTS: We statistically identify regions that are most sensitive to functional variation in the general population and also most commonly impacted in symptomatic individuals. Our data show that a significant number of exons and domains in genes strongly associated with disease can be defined as disease-sensitive or disease-tolerant, leading to potential reclassification of at least 26% (450 out of 1,742) of variants of uncertain clinical significance in the 132 genes. CONCLUSION: This approach leverages domain functional annotation and associated disease in each gene to prioritize candidate disease variants, increasing the sensitivity and specificity of novel variant assessment within these genes.Genet Med advance online publication 22 September 2016.


Assuntos
Predisposição Genética para Doença , Variação Genética , Análise de Sequência de DNA/métodos , Cardiomiopatias/genética , Doenças do Tecido Conjuntivo/genética , Bases de Dados Genéticas , Estudos de Associação Genética , Perda Auditiva/genética , Humanos
13.
Genet Med ; 18(6): 545-53, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-26562227

RESUMO

PURPOSE: With next generation sequencing technology improvement and cost reductions, it has become technically feasible to sequence a large number of genes in one diagnostic test. This is especially relevant for diseases with large genetic and/or phenotypic heterogeneity, such as hearing loss. However, variant interpretation remains the major bottleneck. This is further exacerbated by the lack in the clinical genetics community of consensus criteria for defining the evidence necessary to include genes on targeted disease panels or in genomic reports, and the consequent risk of reporting variants in genes with no relevance to disease. METHODS: We describe a systematic evidence-based approach for assessing gene-disease associations and for curating relevant genes for different disease aspects, including mode of inheritance, phenotypic severity, and mutation spectrum. RESULTS: By applying this approach to clinically available hearing loss gene panels with a total of 163 genes, we show that a significant number (45%) of genes lack sufficient evidence of association with disease and thus are expected to increase uncertainty and patient anxiety, in addition to intensifying the interpretation burden. Information about all curated genes is summarized. Our retrospective analysis of 539 hearing loss cases tested by our previous OtoGenomeV2 panel demonstrates the impact of including genes with weak disease association in laboratory wet-bench and interpretation processes. CONCLUSION: Our study is, to our knowledge, the first to highlight the urgent need for defining the clinical validity of gene-disease relationships for more efficient and accurate clinical testing and reporting.Genet Med 18 6, 545-553.


Assuntos
Heterogeneidade Genética , Predisposição Genética para Doença , Testes Genéticos , Perda Auditiva/diagnóstico , Bases de Dados Genéticas , Genômica , Perda Auditiva/genética , Perda Auditiva/patologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação
14.
Exp Mol Pathol ; 98(2): 214-8, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25595915

RESUMO

INTRODUCTION: For developing countries, sexually transmitted infections (STIs) and their complications are ranked in the top 5 disease categories for which adults seek medical treatment. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) are the three most common STIs worldwide, with TV accounting for over half of the cases. In developing countries, traditional methods for diagnosing STIs are laborious, often not very sensitive, and have a long turnaround time with most recent commercially available diagnostic tests targeting one or, at most, two of these STIs at a time. Here, we describe the development of a highly sensitive, rapid and affordable sample-to-answer multiplex PCR-based assay for the simultaneous detection of Trichomonas vaginalis, Neisseria gonorrhoeae, and Chlamydia trachomatis. MATERIALS AND METHODS: We designed a multiplex PCR assay for the detection of 4 targets (CT, TV, NG, and process/PCR control) using melt curve analysis. To establish the limit of detection (LOD) for each pathogen, we used previously extracted and quantified TV, NG, and CT genomic DNA (Vircell, Spain). For each target, the LOD was determined by lowering its copy number while increasing the other two STI loads in a stepwise fashion. The process/PCR control remained constant in the optimized assay and was spiked into each sample before extraction. For a concordance study, we tested urine, vaginal and rectal swab specimens from 26 patients positive for one or more of the tested STIs. In addition, 56 liquid cytology specimens (Thinprep) were used to assess specificity. RESULTS: This assay has a turnaround time of less than 2h and has a limit of detection as low as 7-31 copies for each STI in the presence of the other 2 targets. Our assay also demonstrated 100% concordance with 26 known clinical samples from urine, vaginal and rectal swab specimens. TV, NG, CT, and our process/PCR control were consistently identified at 78°C, 82.3°C, 85.7°C, and ~92°C, respectively. When applied to DNA extracted from residual Thinprep specimens, the assay was negative in 54/56 samples. Two samples were found to be co-infected with CT. CONCLUSIONS: Our multiplex assay combines a rapid and cost-effective approach to molecular diagnostics with the versatility required for use within a variety of laboratory settings. These performance characteristics make this multiplex STI assay highly suitable for use in a clinical laboratory.


Assuntos
Infecções por Chlamydia/diagnóstico , Gonorreia/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Vaginite por Trichomonas/diagnóstico , Chlamydia trachomatis/genética , DNA Bacteriano/análise , DNA de Protozoário/análise , Feminino , Humanos , Limite de Detecção , Neisseria gonorrhoeae/genética , Trichomonas vaginalis/genética , Urinálise , Esfregaço Vaginal
15.
Exp Mol Pathol ; 97(1): 111-5, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24945443

RESUMO

INTRODUCTION: The CDC estimates that there are currently over 1million people living with human immunodeficiency virus (HIV-1) in the United States, with new cases increasing by approximately 50,000 each year. HIV-1 consists of four distinct groups: the major M group, and the rare N, O, and P groups, each comprising of various subtypes. Without proper care, HIV-1 can lead to cardiovascular, kidney, and liver diseases, cancer, and rapid progression into acquired immune deficiency syndrome (AIDS). Here, we describe a novel, rapid, and highly sensitive assay for the detection of HIV-1 using intercalating dye based RT-PCR and melt curve analysis. MATERIALS AND METHODS: We designed an RT-PCR assay for the detection of the major M subtypes in addition to the rare (O, N, and P) HIV-1 groups, as well as an extraction/RT-PCR control, using melt curve analysis. Viral RNA was extracted using the automated Qiagen EZ1 robotic system (Qiagen, Valencia, CA). To establish the limit of detection (LOD) for this assay, we diluted the AcroMetrix HIV-1 panel (LifeTechnologies, Grand Island, NY) to concentrations ranging from 25 to 500 copies/ml. Armored RNA BCR/ABL b3/a2 (Asuragen, Austin, Texas) was used as our extraction and RT-PCR control. Specificity and accuracy were assessed by testing plasma specimens from 48 anonymized patients negative for HIV-1. RESULTS: This assay has a turnaround time of less than 2.5h and has a limit of detection of 50 copies/ml of plasma. Our assay also demonstrated 100% concordance with 53 previously quantified plasma patient specimens, including 48 negative samples and 5 positive samples. HIV-1 and our extraction/RT-PCR control were consistently identified at 79 °C and 82.5 °C, respectively. CONCLUSIONS: We developed a comprehensive, easy to use assay for the detection of HIV-1 in human plasma. Our assay combines a rapid and cost-effective method for molecular diagnostics with the versatility necessary for widespread laboratory use. These performance characteristics make this HIV-1 detection assay highly suitable for use in a clinical laboratory.


Assuntos
Infecções por HIV/diagnóstico , HIV-1/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Primers do DNA , Infecções por HIV/virologia , Humanos , Limite de Detecção , Reação em Cadeia da Polimerase em Tempo Real/métodos , Sensibilidade e Especificidade
16.
Clin Chem ; 59(10): 1481-8, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23775370

RESUMO

BACKGROUND: Cystic fibrosis is a life-threatening genetic disorder that has been associated with mutations in the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene. Hundreds of CFTR mutations have been detected to date. Current CFTR genotyping assays target a subset of these mutations, particularly a mutation panel recommended by the American College of Medical Genetics for carrier screening of the general population. Fast sequencing of the entire coding sequence in a scalable manner could expand the detection of CFTR mutations and facilitate management of costs and turnaround times in the clinical laboratory. METHODS: We describe a proof-of-concept CFTR assay that uses PCR target enrichment and next-generation sequencing on the Ion Torrent Personal Genome Machine™ (PGM™) platform. RESULTS: The scalability of the assay was demonstrated, with an average mean depth of coverage ranging from 500× to 3500×, depending on the number of multiplexed patient samples and the Ion Torrent chip used. In a blinded study of 79 previously genotyped patient DNA samples and cell lines, our assay detected most of the mutations, including single-nucleotide variants, small insertions and deletions, and large copy-number variants. The reproducibility was 100% for detecting mutations in independent runs. Our assay demonstrated high specificity, with only 2 false-positive calls (at 2184delA) found in 2 samples caused by a sequencing error in a homopolymer stretch of sequence. The detection rate for variants of unknown significance was very low in the targeted region. CONCLUSIONS: With continued optimization and system refinements, PGM sequencing promises to be a powerful, rapid, and scalable means of clinical diagnostic sequencing.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/sangue , Dosagem de Genes , Humanos , Mutação , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Análise de Sequência de DNA
17.
Genome Med ; 15(1): 5, 2023 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-36703223

RESUMO

BACKGROUND: Rare diseases collectively impose a significant burden on healthcare systems, especially in underserved regions, like the Middle East, which lack access to genomic diagnostic services and the associated personalized management plans. METHODS: We established a clinical genomics and genetic counseling facility, within a multidisciplinary tertiary pediatric center, in the United Arab Emirates to locally diagnose and manage patients with rare diseases. Clinical genomic investigations included exome-based sequencing, chromosomal microarrays, and/or targeted testing. We assessed the diagnostic yield and implications for clinical management among this population. Variables were compared using the Fisher exact test. Tests were 2-tailed, and P < .05 was considered statistically significant. RESULTS: We present data on 1000 patients with rare diseases (46.2% females; average age, 4.6 years) representing 47 countries primarily from the Arabian Peninsula, the Levant, Africa, and Asia. The cumulative diagnostic yield was 32.5% (95% CI, 29.7-35.5%) and was higher for genomic sequencing-based testing than chromosomal microarrays (37.9% versus 17.2%, P = 0.0001) across all indications, consistent with the higher burden of single gene disorders. Of the 221 Mendelian disorders identified in this cohort, the majority (N = 184) were encountered only once, and those with recessive inheritance accounted for ~ 62% of sequencing diagnoses. Of patients with positive genetic findings (N = 325), 67.7% were less than 5 years of age, and 60% were offered modified management and/or intervention plans. Interestingly, 24% of patients with positive genetic findings received delayed diagnoses (average age, 12.4 years; range 7-37 years), most likely due to a lack of access to genomic investigations in this region. One such genetic finding ended a 15-year-long diagnostic odyssey, leading to a life-threatening diagnosis in one patient, who was then successfully treated using an experimental allogenic bone marrow transplant. Finally, we present cases with candidate genes within regions of homozygosity, likely underlying novel recessive disorders. CONCLUSIONS: Early access to genomic diagnostics for patients with suspected rare disorders in the Middle East is likely to improve clinical outcomes while driving gene discovery in this genetically underrepresented population.


Assuntos
Testes Genéticos , Doenças Raras , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Exoma , Genômica , Oriente Médio , Doenças Raras/diagnóstico , Doenças Raras/genética , Doenças Raras/terapia , Adolescente , Adulto Jovem , Adulto
18.
J Neurosci ; 31(39): 13897-910, 2011 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-21957252

RESUMO

The contribution of the SK (small-conductance calcium-activated potassium) channel to neuronal functions in complex circuits underlying sensory processing and behavior is largely unknown in the absence of suitable animal models. Here, we generated a Drosophila line that lacks the single highly conserved SK gene in its genome (dSK). In R1-R6 photoreceptors, dSK encodes a slow Ca²âº-activated K(+) current similar to its mammalian counterparts. Compared with wild-type, dSK(-) photoreceptors and interneurons showed accelerated oscillatory responses and adaptation. These enhanced kinetics were accompanied with more depolarized dSK(-) photoreceptors axons, assigning a role for dSK in network gain control during light-to-dark transitions. However, compensatory network adaptation, through increasing activity between synaptic neighbors, overcame many detriments of missing dSK current enabling dSK(-) photoreceptors to maintain normal information transfer rates to naturalistic stimuli. While demonstrating important functional roles for dSK channel in the visual circuitry, these results also clarify how homeostatically balanced network functions can compensate missing or faulty ion channels.


Assuntos
Proteínas de Drosophila/fisiologia , Rede Nervosa/fisiologia , Células Fotorreceptoras/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/fisiologia , Vias Visuais/fisiologia , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Masculino , Dados de Sequência Molecular , Canais de Potássio Ativados por Cálcio de Condutância Baixa/genética
19.
Exp Mol Pathol ; 93(2): 261-3, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22579954

RESUMO

There is an increasing need for affordable, thermostable PCR reagents that can be used for diagnostic testing in resource limited settings. The development of point-of-care devices in such settings is highly dependent on the availability and efficacy of thermostable reagents. Here, we assess the thermostability of commercial, intercalating dye-based real-time PCR master mixes. We show that several of these master mixes have thermostability and robust performance at 20°C, 40°C, and 45°C for 6, 4 and 2 weeks, respectively. However, none of the master mixes that we evaluated was able to withstand more than 1 month at 45°C incubation. Our findings have implications for clinical diagnostics in the developing world where cold-chain delivery of diagnostic assays is difficult to guarantee.


Assuntos
DNA/análise , Temperatura Alta , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/métodos , Países em Desenvolvimento , Estabilidade de Medicamentos , Humanos , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas
20.
Front Cell Dev Biol ; 10: 943924, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36147741

RESUMO

Neuroblastoma is a pediatric tumour that accounts for more than 15% of cancer-related deaths in children. High-risk tumours are often difficult to treat, and patients' survival chances are less than 50%. Retinoic acid treatment is part of the maintenance therapy given to neuroblastoma patients; however, not all tumours differentiate in response to retinoic acid. Within neuroblastoma tumors, two phenotypically distinct cell types have been identified based on their super-enhancer landscape and transcriptional core regulatory circuitries: adrenergic (ADRN) and mesenchymal (MES). We hypothesized that the distinct super-enhancers in these different tumour cells mediate differential response to retinoic acid. To this end, three different neuroblastoma cell lines, ADRN (MYCN amplified and non-amplified) and MES cells, were treated with retinoic acid, and changes in the super-enhancer landscape upon treatment and after subsequent removal of retinoic acid was studied. Using ChIP-seq for the active histone mark H3K27ac, paired with RNA-seq, we compared the super-enhancer landscape in cells that undergo neuronal differentiation in response to retinoic acid versus those that fail to differentiate and identified unique super-enhancers associated with neuronal differentiation. Among the ADRN cells that respond to treatment, MYCN-amplified cells remain differentiated upon removal of retinoic acid, whereas MYCN non-amplified cells revert to an undifferentiated state, allowing for the identification of super-enhancers responsible for maintaining differentiation. This study identifies key super-enhancers that are crucial for retinoic acid-mediated differentiation.

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