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1.
Hum Mol Genet ; 28(17): 2937-2951, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31152168

RESUMO

KCNMA1 encodes the large-conductance Ca2+- and voltage-activated K+ (BK) potassium channel α-subunit, and pathogenic gain-of-function variants in this gene have been associated with a dominant form of generalized epilepsy and paroxysmal dyskinesia. Here, we genetically and functionally characterize eight novel loss-of-function (LoF) variants of KCNMA1. Genome or exome sequencing and the participation in the international Matchmaker Exchange effort allowed for the identification of novel KCNMA1 variants. Patch clamping was used to assess functionality of mutant BK channels. The KCNMA1 variants p.(Ser351Tyr), p.(Gly356Arg), p.(Gly375Arg), p.(Asn449fs) and p.(Ile663Val) abolished the BK current, whereas p.(Cys413Tyr) and p.(Pro805Leu) reduced the BK current amplitude and shifted the activation curves toward positive potentials. The p.(Asp984Asn) variant reduced the current amplitude without affecting kinetics. A phenotypic analysis of the patients carrying the recurrent p.(Gly375Arg) de novo missense LoF variant revealed a novel syndromic neurodevelopmental disorder associated with severe developmental delay, visceral and cardiac malformations, connective tissue presentations with arterial involvement, bone dysplasia and characteristic dysmorphic features. Patients with other LoF variants presented with neurological and developmental symptoms including developmental delay, intellectual disability, ataxia, axial hypotonia, cerebral atrophy and speech delay/apraxia/dysarthria. Therefore, LoF KCNMA1 variants are associated with a new syndrome characterized by a broad spectrum of neurological phenotypes and developmental disorders. LoF variants of KCNMA1 cause a new syndrome distinctly different from gain-of-function variants in the same gene.

2.
J Genet Couns ; 28(2): 438-448, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30964585

RESUMO

Advances in genomic knowledge and technology have increased the use of comprehensive clinical sequencing tests. Genome sequencing has established utility for diagnosing patients with rare, undiagnosed diseases as well as interest in an elective context, without a clinical indication for testing. The Smith Family Clinic for Genomic Medicine, LLC in Huntsville, AL is a private practice genomic medicine clinic caring for both diagnostic (79%) and elective (21%) patients. Diagnostic and elective patients are seen on a clinical basis and receive standard care. Genome sequencing is provided on a self-pay basis, with assistance available for diagnostic patients who have financial need. Here, we describe demographics and motivations of the distinct patient populations and our experiences engaging patients in online education. Diagnostic patients were motivated by the possibility of receiving an explanation for symptoms (96%) while elective patients were motivated by the chance to learn about future disease risk (57%). Elective patients were less likely to engage with online education, with only 28% reading all assigned topics compared to 54% of diagnostic patients. Understanding the needs, interests, and barriers unique to diagnostic and elective patients is critical to inform individualized and scalable best practices in patient education and engagement.

3.
J Med Genet ; 2019 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-31023718

RESUMO

Up to 350 million people worldwide suffer from a rare disease, and while the individual diseases are rare, in aggregate they represent a substantial challenge to global health systems. The majority of rare disorders are genetic in origin, with children under the age of five disproportionately affected. As these conditions are difficult to identify clinically, genetic and genomic testing have become the backbone of diagnostic testing in this population. In the last 10 years, next-generation sequencing technologies have enabled testing of multiple disease genes simultaneously, ranging from targeted gene panels to exome sequencing (ES) and genome sequencing (GS). GS is quickly becoming a practical first-tier test, as cost decreases and performance improves. A growing number of studies demonstrate that GS can detect an unparalleled range of pathogenic abnormalities in a single laboratory workflow. GS has the potential to deliver unbiased, rapid and accurate molecular diagnoses to patients across diverse clinical indications and complex presentations. In this paper, we discuss clinical indications for testing and historical testing paradigms. Evidence supporting GS as a diagnostic tool is supported by superior genomic coverage, types of pathogenic variants detected, simpler laboratory workflow enabling shorter turnaround times, diagnostic and reanalysis yield, and impact on healthcare.

4.
J Genet Couns ; 28(2): 213-228, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30964584

RESUMO

There are approximately 7,000 rare diseases affecting 25-30 million Americans, with 80% estimated to have a genetic basis. This presents a challenge for genetics practitioners to determine appropriate testing, make accurate diagnoses, and conduct up-to-date patient management. Exome sequencing (ES) is a comprehensive diagnostic approach, but only 25%-41% of the patients receive a molecular diagnosis. The remaining three-fifths to three-quarters of patients undergoing ES remain undiagnosed. The Stanford Center for Undiagnosed Diseases (CUD), a clinical site of the Undiagnosed Diseases Network, evaluates patients with undiagnosed and rare diseases using a combination of methods including ES. Frequently these patients have non-diagnostic ES results, but strategic follow-up techniques identify diagnoses in a subset. We present techniques used at the CUD that can be adopted by genetics providers in clinical follow-up of cases where ES is non-diagnostic. Solved case examples illustrate different types of non-diagnostic results and the additional techniques that led to a diagnosis. Frequent approaches include segregation analysis, data reanalysis, genome sequencing, additional variant identification, careful phenotype-disease correlation, confirmatory testing, and case matching. We also discuss prioritization of cases for additional analyses.

5.
J Mol Diagn ; 2018 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-30453057

RESUMO

The increasing quality and diminishing cost of next-generation sequencing has transformed our ability to interrogate large quantities of genetic information. This has led to a dramatic increase in the number of elective genomic tests performed. In this article, elective test denotes a test that a patient chooses to undertake without a clinical indication. The variety of elective genomic testing options is considerable. Because these offerings provide differing levels of sensitivity and specificity, it can be difficult to choose among them. A simple rubric to compare offerings is not readily available. We propose a framework designated completeness that evaluates both analytical and interpretative components of genomic tests. We then illustrate how this framework can be used to evaluate the expanding landscape of elective genomic testing.

6.
Nat Commun ; 9(1): 4065, 2018 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-30283131

RESUMO

Mitochondrial protein synthesis requires charging mt-tRNAs with their cognate amino acids by mitochondrial aminoacyl-tRNA synthetases, with the exception of glutaminyl mt-tRNA (mt-tRNAGln). mt-tRNAGln is indirectly charged by a transamidation reaction involving the GatCAB aminoacyl-tRNA amidotransferase complex. Defects involving the mitochondrial protein synthesis machinery cause a broad spectrum of disorders, with often fatal outcome. Here, we describe nine patients from five families with genetic defects in a GatCAB complex subunit, including QRSL1, GATB, and GATC, each showing a lethal metabolic cardiomyopathy syndrome. Functional studies reveal combined respiratory chain enzyme deficiencies and mitochondrial dysfunction. Aminoacylation of mt-tRNAGln and mitochondrial protein translation are deficient in patients' fibroblasts cultured in the absence of glutamine but restore in high glutamine. Lentiviral rescue experiments and modeling in S. cerevisiae homologs confirm pathogenicity. Our study completes a decade of investigations on mitochondrial aminoacylation disorders, starting with DARS2 and ending with the GatCAB complex.

7.
Hum Mol Genet ; 27(14): 2454-2465, 2018 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-29726930

RESUMO

The 17 genes of the T-box family are transcriptional regulators that are involved in all stages of embryonic development, including craniofacial, brain, heart, skeleton and immune system. Malformation syndromes have been linked to many of the T-box genes. For example, haploinsufficiency of TBX1 is responsible for many structural malformations in DiGeorge syndrome caused by a chromosome 22q11.2 deletion. We report four individuals with an overlapping spectrum of craniofacial dysmorphisms, cardiac anomalies, skeletal malformations, immune deficiency, endocrine abnormalities and developmental impairments, reminiscent of DiGeorge syndrome, who are heterozygotes for TBX2 variants. The p.R20Q variant is shared by three affected family members in an autosomal dominant manner; the fourth unrelated individual has a de novo p.R305H mutation. Bioinformatics analyses indicate that these variants are rare and predict them to be damaging. In vitro transcriptional assays in cultured cells show that both variants result in reduced transcriptional repressor activity of TBX2. We also show that the variants result in reduced protein levels of TBX2. Heterologous over-expression studies in Drosophila demonstrate that both p.R20Q and p.R305H function as partial loss-of-function alleles. Hence, these and other data suggest that TBX2 is a novel candidate gene for a new multisystem malformation disorder.

9.
J Pediatr Genet ; 7(1): 23-28, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29441218

RESUMO

We report a 19-year-old female patient with a history of short stature, primary ovarian insufficiency, sensorineural hearing loss, sacral teratoma, neurogenic bladder, and intellectual disability with underlying mosaicism for der(X)t(X;3)(q13.2;q25.33), a ring X chromosome, and monosomy X. Derivative X chromosomes from unbalanced X-autosomal translocations are preferentially silenced by the XIST gene (Xq13.2) located within the X-inactivation center. The unbalanced X-autosomal translocation in our case resulted in loss of the XIST gene thus precluding the inactivation of the derivative X chromosome. As a result, clinical features of functional disomy Xp, Turner's syndrome, and duplication 3q syndrome were observed. Importantly, indications of the derivative X chromosome were revealed by microarray analysis following an initial diagnosis of Turner's syndrome made by conventional cytogenetic studies approximately 18 months earlier. This case demonstrates the importance of utilizing microarray analysis as a first-line test in patients with clinical features beyond the scope of a well-defined genetic syndrome.

10.
J Assoc Genet Technol ; 43(2): 56-58, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28511170

RESUMO

To the Editor: Partial and whole duplications of the short arm of chromosome 9 have been commonly reported in the literature with characteristic phenotypic features and intellectual disabilities. The clinical features of 9p duplications are broad and can include growth retardation, developmental delay, intellectual disability, microbrachycephaly, deep set eyes, hypertelorism, downslanting palpebral fissures, prominent nasal root, bulbous nasal tip, low-set ears, short fingers and toes with hypoplastic nails, and delayed bone age (Bonaglia et al., 2002; Zou et al., 2009; Guilherme et al., 2014).

11.
J Pediatr Genet ; 6(2): 61-76, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28496993

RESUMO

A pilot program was initiated using whole genome sequencing (WGS) to diagnose suspected genetic disorders in the Genetics Clinic at Children's Hospital of Wisconsin. Twenty-two patients underwent WGS between 2010 and 2013. Initially, we obtained a 14% (3/22) diagnosis rate over 2 years; with subsequent reanalysis, this increased to 36% (8/22). Disease causing variants were identified in SKIV2L, CECR1, DGKE, PYCR2, RYR1, PDGFRB, EFTUD2, and BCS1L. In 75% (6/8) of diagnosed cases, the diagnosis affected treatment and/or medical surveillance. Additionally, one case demonstrated a homozygous A18V variant in VLDLR that appears to be associated with a previously undescribed phenotype.

12.
Genet Med ; 19(5): 575-582, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27811861

RESUMO

PURPOSE: While the diagnostic success of genomic sequencing expands, the complexity of this testing should not be overlooked. Numerous laboratory processes are required to support the identification, interpretation, and reporting of clinically significant variants. This study aimed to examine the workflow and reporting procedures among US laboratories to highlight shared practices and identify areas in need of standardization. METHODS: Surveys and follow-up interviews were conducted with laboratories offering exome and/or genome sequencing to support a research program or for routine clinical services. The 73-item survey elicited multiple choice and free-text responses that were later clarified with phone interviews. RESULTS: Twenty-one laboratories participated. Practices highly concordant across all groups included consent documentation, multiperson case review, and enabling patient opt-out of incidental or secondary findings analysis. Noted divergence included use of phenotypic data to inform case analysis and interpretation and reporting of case-specific quality metrics and methods. Few laboratory policies detailed procedures for data reanalysis, data sharing, or patient access to data. CONCLUSION: This study provides an overview of practices and policies of experienced exome and genome sequencing laboratories. The results enable broader consideration of which practices are becoming standard approaches, where divergence remains, and areas of development in best practice guidelines that may be helpful.Genet Med advance online publication 03 Novemeber 2016.


Assuntos
Testes Genéticos/métodos , Laboratórios/normas , Análise de Sequência de DNA/métodos , Revelação , Testes Genéticos/normas , Humanos , Achados Incidentais , Disseminação de Informação , Laboratórios/ética , Guias de Prática Clínica como Assunto , Relatório de Pesquisa , Tamanho da Amostra , Análise de Sequência de DNA/normas , Inquéritos e Questionários
15.
Mol Genet Metab ; 116(3): 139-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26385305

RESUMO

Very long chain acyl-coA dehydrogenase deficiency (VLCADD) is an autosomal recessive inborn error of fatty acid oxidation detected by newborn screening (NBS). Follow-up molecular analyses are often required to clarify VLCADD-suggestive NBS results, but to date the outcome of these studies are not well described for the general screen-positive population. In the following study, we report the molecular findings for 693 unrelated patients that sequentially received Sanger sequence analysis of ACADVL as a result of a positive NBS for VLCADD. Highlighting the variable molecular underpinnings of this disorder, we identified 94 different pathogenic ACADVL variants (40 novel), as well as 134 variants of unknown clinical significance (VUSs). Evidence for the pathogenicity of a subset of recurrent VUSs was provided using multiple in silico analyses. Surprisingly, the most frequent finding in our cohort was carrier status, 57% all individuals had a single pathogenic variant or VUS. This result was further supported by follow-up array and/or acylcarnitine analysis that failed to provide evidence of a second pathogenic allele. Notably, exon-targeted array analysis of 131 individuals screen positive for VLCADD failed to identify copy number changes in ACADVL thus suggesting this test has a low yield in the setting of NBS follow-up. While no genotype was common, the c.848T>C (p.V283A) pathogenic variant was clearly the most frequent; at least one copy was found in ~10% of all individuals with a positive NBS. Clinical and biochemical data for seven unrelated patients homozygous for the p.V283A allele suggests that it results in a mild phenotype that responds well to standard treatment, but hypoglycemia can occur. Collectively, our data illustrate the molecular heterogeneity of VLCADD and provide novel insight into the outcomes of NBS for this disorder.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Acil-CoA Desidrogenase de Cadeia Longa/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Triagem Neonatal , Alelos , Carnitina/análogos & derivados , Simulação por Computador , Éxons , Feminino , Triagem de Portadores Genéticos , Genótipo , Humanos , Hipoglicemia/etiologia , Recém-Nascido , Masculino , Mutação de Sentido Incorreto , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de DNA , Espectrometria de Massas em Tandem , Estados Unidos
16.
Hum Mutat ; 36(11): 1052-63, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26178382

RESUMO

Neurofibromatosis type 1 (NF1) is one of the most frequent genetic disorders, affecting 1:3,000 worldwide. Identification of genotype-phenotype correlations is challenging because of the wide range clinical variability, the progressive nature of the disorder, and extreme diversity of the mutational spectrum. We report 136 individuals with a distinct phenotype carrying one of five different NF1 missense mutations affecting p.Arg1809. Patients presented with multiple café-au-lait macules (CALM) with or without freckling and Lisch nodules, but no externally visible plexiform neurofibromas or clear cutaneous neurofibromas were found. About 25% of the individuals had Noonan-like features. Pulmonic stenosis and short stature were significantly more prevalent compared with classic cohorts (P < 0.0001). Developmental delays and/or learning disabilities were reported in over 50% of patients. Melanocytes cultured from a CALM in a segmental NF1-patient showed two different somatic NF1 mutations, p.Arg1809Cys and a multi-exon deletion, providing genetic evidence that p.Arg1809Cys is a loss-of-function mutation in the melanocytes and causes a pigmentary phenotype. Constitutional missense mutations at p.Arg1809 affect 1.23% of unrelated NF1 probands in the UAB cohort, therefore this specific NF1 genotype-phenotype correlation will affect counseling and management of a significant number of patients.


Assuntos
Substituição de Aminoácidos , Códon , Mutação de Sentido Incorreto , Neurofibromina 1/genética , Síndrome de Noonan/diagnóstico , Síndrome de Noonan/genética , Fenótipo , Adolescente , Adulto , Criança , Pré-Escolar , Estudos de Coortes , Nanismo/genética , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Neurofibromina 1/química , Adulto Jovem
18.
Birth Defects Res A Clin Mol Teratol ; 103(7): 630-40, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26118977

RESUMO

BACKGROUND: Congenital microphthalmia and coloboma are severe developmental defects that are frequently associated with additional systemic anomalies and display a high level of genetic heterogeneity. METHODS: To identify the pathogenic variant in a patient with microphthalmia, coloboma, retinal dystrophy, microcephaly, and other features, whole exome sequencing analysis of the patient and parental samples was undertaken. To further explore the identified variant/gene, expression and functional studies in zebrafish were performed. RESULTS: Whole exome sequencing revealed a de novo variant, c.473_474delGA, p.(Arg158Lysfs*4), in EFTUD2 which encodes a component of the spliceosome complex. Dominant mutations in EFTUD2 cause Mandibulofacial Dysostosis, Guion-Almeida type, which does not involve microphthalmia, coloboma, or retinal dystrophy; analysis of genes known to cause these ocular phenotypes identified several variants of unknown significance but no causal alleles in the affected patient. Zebrafish eftud2 demonstrated high sequence conservation with the human gene and broad embryonic expression. TALEN-mediated disruption was employed to generate a c.378_385 del, p.(Ser127Aspfs*23) truncation mutation in eftud2. Homozygous mutants displayed a reduced head size, small eye, curved body, and early embryonic lethality. Apoptosis assays demonstrated a striking increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive cells in the developing brain, eye, spinal cord, and other tissues starting at 30 hours postfertilization. CONCLUSION: This study reports a novel mutation in EFTUD2 in a Mandibulofacial Dysostosis, Guion-Almeida type patient with unusual ocular features and the generation of a first animal model of eftud2 deficiency. The severe embryonic phenotype observed in eftud2 mutants indicates an important conserved role during development of diverse tissues in vertebrates.


Assuntos
Modelos Animais , Mutação , Fatores de Alongamento de Peptídeos/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Animais , Pré-Escolar , Exoma , Anormalidades do Olho/genética , Feminino , Humanos , Peixe-Zebra
19.
Clin J Am Soc Nephrol ; 10(6): 1011-9, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25854283

RESUMO

BACKGROUND AND OBJECTIVES: Genetic and acquired abnormalities causing dysregulation of the complement alternative pathway contribute to atypical hemolytic uremic syndrome (aHUS), a rare disorder characterized by thrombocytopenia, nonimmune microangiopathic hemolytic anemia, and acute kidney failure. However, in a substantial proportion of patients the disease-associated alterations are still unknown. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Whole-exome and whole-genome sequencing were performed in two unrelated families with infantile recessive aHUS. Sequencing of cDNA from affected individuals was used to test for the presence of aberrant mRNA species. Expression of mutant diacylglycerol kinase epsilon (DGKE) protein was evaluated with western blotting. RESULTS: Whole-exome sequencing analysis with conventional variant filtering parameters did not reveal any obvious candidate mutation in the first family. The report of aHUS-associated mutations in DGKE, encoding DGKE, led to re-examination of the noncoding DGKE variants obtained from next-generation sequencing, allowing identification of a novel intronic DGKE mutation (c.888+40A>G) that segregated with disease. Sequencing of cDNA from affected individuals revealed aberrant forms of DGKE mRNA predicted to cause profound abnormalities in the protein catalytic site. By whole-genome sequencing, the same mutation was found in compound heterozygosity with a second nonsense DGKE mutation in all affected siblings of another unrelated family. Homozygous and compound heterozygous patients presented similar clinical features, including aHUS presentation in the first year of life, multiple relapsing episodes, and proteinuria, which are prototypical of DGKE-associated aHUS. CONCLUSIONS: This is the first report of a mutation located beyond the exon-intron boundaries in aHUS. Intronic mutations such as these are underreported because conventional filtering parameters used to process next-generation sequencing data routinely exclude these regions from downstream analyses in both research and clinical settings. The results suggest that analysis of noncoding regions of aHUS-associated genes coupled with mRNA sequencing might provide a tool to explain genetically unsolved aHUS cases.


Assuntos
Síndrome Hemolítico-Urêmica Atípica/genética , Diacilglicerol Quinase/genética , Íntrons , Mutação , Adolescente , Síndrome Hemolítico-Urêmica Atípica/diagnóstico , Síndrome Hemolítico-Urêmica Atípica/enzimologia , Sequência de Bases , Western Blotting , Criança , Análise Mutacional de DNA , Diacilglicerol Quinase/metabolismo , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Lactente , Masculino , Dados de Sequência Molecular , Fenótipo , Valor Preditivo dos Testes , Fatores de Risco
20.
Genet Med ; 17(5): 405-24, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25741868

RESUMO

The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.(1) In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next-generation sequencing. By adopting and leveraging next-generation sequencing, clinical laboratories are now performing an ever-increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes, and epigenetic assays for genetic disorders. By virtue of increased complexity, this shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP, and College of American Pathologists stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. This report recommends the use of specific standard terminology-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"-to describe variants identified in genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments-approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.


Assuntos
Testes Genéticos/normas , Variação Genética , Análise de Sequência de DNA/normas , Estudos de Casos e Controles , Frequência do Gene , Humanos , Mutação , Razão de Chances
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