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1.
Crit Rev Oncol Hematol ; 140: 67-72, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176273

RESUMO

The current availability of new Poly(ADP-ribose) Polymerase (PARP)-inhibitors for the treatment of ovarian cancer patients independently of the presence of a BRCA pathogenic variant, together with the validation of somatic test for the analysis of BRCA1/2 genes, involves the need to optimise the guidelines for BRCA testing. The AIOM-SIGU-SIBIOC-SIAPEC-IAP Italian Scientific Societies, in this position paper, recommend the implementation of BRCA testing with 2 main objectives: the first is the identification of ovarian cancer patients with higher probability of benefit from specific anticancer treatments (test for response to therapy); the second goal, through BRCA testing in the family members of ovarian cancer patients, is the identification of carriers of pathogenic variant, who have inheredited predisposition to cancer development (test for cancer risk). These individuals with increased risk of cancer, should be encouraged to participate in dedicated high-risk surveillance clinics and specific risk-reducing measures (primary and/or secondary prevention programs).


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Testes Genéticos/normas , Mutação em Linhagem Germinativa , Neoplasias Ovarianas/genética , Sociedades Médicas , Bioquímica , Feminino , Genética , Humanos , Itália , Oncologia , Neoplasias Ovarianas/diagnóstico
2.
Eur J Med Genet ; 62(5): 397-404, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31085343

RESUMO

Personal genome screening (PGenS) is increasingly being offered as a screen for future health management, and to identify carrier status pertinent to reproductive decision-making. The aim of this study was therefore to explore the experience of individuals who undertook PGenS through the 2014 Sydney "Understand Your Genome (UYG)" event and a 2015 offer of PGenS by Australian biotechnology company Life Letters (LL). Eligible individuals were invited to participate by their clinical geneticist (UYG), or email from Director of LL. Semi-structured telephone interviews with 17 individuals were audio-recorded, transcribed, de-identified and analyzed by two coders using thematic analysis with an inductive approach. Nine participants had genetic/genomics expertise and eight were well-informed health and business professionals. Individual participant PGenS results included: an autosomal dominant condition not previously clinically identified (n = 1); carrier status for recessive condition(s) (n = 8); a number of disease-causing variants associated with an increased susceptibility to an inherited disorder (n = 7); variants of uncertain significance (n = 5); and a few pharmacogenomically-relevant variants (n = 4). The majority of participants described the importance of pre-test genetic counseling, information and/or consent (n = 12). Some barriers to uptake were identified, including scepticism by GPs (n = 6), colleagues (n = 3), and family members (n = 2), as well as privacy concerns (n = 4). Those without genetic/genomics expertise were mostly motivated to have testing by curiosity or interest in personal health (6/8), one seeking a diagnosis for an inherited medical condition and another for future health management. For many with genetic/genomics experience, the motivation was professional interest (8/9) and/or curiosity (5/9), without concern for personal health risk (4/9). On reflection, despite this initial motivation by the latter, the test result had unanticipated personal impact for some of this group, which changed over time (4/5). Several later recognized this, as health problems developed or family history was interrogated more closely. For all participants, disclosure of results to extended family members was limited. Most participants felt personal and family implications and communication (5/17) and/or expectations (3/17) should be addressed at the pre-test session, including more emphasis on residual risk and changes in interpretation with developing phenotypes. Those without genetics/genomics expertise highlighted the need for easy to understand pre-test information and/or an example report to be provided (7/8). These results are consistent with a need to develop more accessible resources, and more personalized counseling approaches to address expectations, dissemination of results, and preparedness for unexpected findings.


Assuntos
Triagem e Testes Direto ao Consumidor/métodos , Testes Genéticos/métodos , Competência em Informação , Adulto , Atitude , Triagem e Testes Direto ao Consumidor/psicologia , Triagem e Testes Direto ao Consumidor/normas , Feminino , Testes Genéticos/normas , Humanos , Masculino , Pessoa de Meia-Idade
3.
Ann Hematol ; 98(8): 1835-1844, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30953085

RESUMO

The manual verification of gene tests is time-consuming and error prone. In this study, we try to explore a high-efficiency, clinically useful auto-verification system for gene detection of thalassemia. A series of verification elements were rooted in the auto-verification system. Consistency check was applied initially as one of the essential elements in our study. One hundred twenty-four archived cases were used to choose the consistency-check rules' indices from routine blood examination and hemoglobin electrophoresis by the receiver operating characteristic curves. Rule 1 and rule 2 established by the chosen indices were compared by their passing rate, consistency with manual validation, and error rate. Finally, 748 cases were used for verifying the system's feasibility by evaluating the passing rate, turn-around time (TAT), and error rate. The rule 2 had a higher passing rate (67.7% vs. 50.8%) and consistency (0.623 vs. 0.364) than the rule 1 with an error rate of zero. In a "live" valuation, the auto-verification system can reduce the TAT and error rate of verification by 51.5% and 0.13%, respectively, with a high passing rate of 82.8%. The auto-verification system for gene detection of thalassemia in this study can shorten the validation time, reduce errors, and enhance efficiency.


Assuntos
Testes Genéticos/normas , Talassemia/diagnóstico , Talassemia/genética , alfa-Globinas/genética , Globinas beta/genética , Algoritmos , Feminino , Deleção de Genes , Expressão Gênica , Genótipo , Humanos , Lactente , Recém-Nascido , Masculino , Controle de Qualidade , Curva ROC , Talassemia/classificação , Talassemia/patologia , alfa-Globinas/deficiência , Globinas beta/deficiência
4.
J Genet ; 982019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30945666

RESUMO

The aim of the present study was to evaluate the diagnostic yield of prenatal cytogenetic microarray (CMA) in structurally normal and abnormal foetuses and record the acceptance rate of CMA for prenatal diagnosis over a course of five year. In 128 structurally normal and abnormal foetuses, CMA was performed along with foetal karyotype, after exclusion of aneuploidy by quantitative fluorescence polymerase chain reaction. The microarray was able to detect the pathogenic variants in 5.5% cases; the diagnostic yield in structurally abnormal foetuses was 8.8% and 4.7% in foetuses with a high aneuploidy risk. Balanced and unbalanced translocations, and low level mosaicism were detected. Reanalysis of variants of uncertain significance identified pathogenic variant. The study shows higher diagnostic yield in structurally abnormal cases, the importance of foetal karyotype and reanalysis in microarray. The acceptance rate of prenatal CMA increased five-fold over a period of five year.


Assuntos
Aberrações Cromossômicas , Transtornos Cromossômicos/genética , Análise Citogenética/métodos , Doenças Fetais/genética , Testes Genéticos/normas , Análise em Microsséries/métodos , Diagnóstico Pré-Natal/métodos , Cariótipo Anormal , Adulto , Transtornos Cromossômicos/diagnóstico , Feminino , Doenças Fetais/diagnóstico , Humanos , Gravidez , Prognóstico
5.
Mol Genet Genomic Med ; 7(5): e630, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30900393

RESUMO

BACKGROUND: Advances in sequencing technology have led to expanded use of multi-gene panel tests (MGPTs) for clinical diagnostics. Well-designed MGPTs must balance increased detection of clinically significant findings while mitigating the increase in variants of uncertain significance (VUS). To maximize clinical utililty, design of such panels should include comprehensive gene vetting using a standardized clinical validity (CV) scoring system. METHODS: To assess the impact of CV-based gene vetting on MGPT results, data from MGPTs for cardiovascular indications were retrospectively analyzed. Using our CV scoring system, genes were categorized as having definitive, strong, moderate, or limited evidence. The rates of reported pathogenic or likely pathogenic variants and VUS were then determined for each CV category. RESULTS: Of 106 total genes, 42% had definitive, 17% had strong, 29% had moderate, and 12% had limited CV. The detection rate of variants classified as pathogenic or likely pathogenic was higher for genes with greater CV, while the VUS rate showed an inverse relationship with CV score. No pathogenic or likely pathogenic findings were observed in genes with a limited CV. CONCLUSION: These results demonstrate the importance of a standardized, evidence-based vetting process to establish CV for genes on MGPTs. Using our proposed system may help to increase the detection rate while mitigating higher VUS rates.


Assuntos
Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/normas , Humanos , Herança Multifatorial
6.
Genome Res ; 29(3): 428-438, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30787035

RESUMO

In the last decade, noninvasive prenatal diagnosis (NIPD) has emerged as an effective procedure for early detection of inherited diseases during pregnancy. This technique is based on using cell-free DNA (cfDNA) and fetal cfDNA (cffDNA) in maternal blood, and hence, has minimal risk for the mother and fetus compared with invasive techniques. NIPD is currently used for identifying chromosomal abnormalities (in some instances) and for single-gene disorders (SGDs) of paternal origin. However, for SGDs of maternal origin, sensitivity poses a challenge that limits the testing to one genetic disorder at a time. Here, we present a Bayesian method for the NIPD of monogenic diseases that is independent of the mode of inheritance and parental origin. Furthermore, we show that accounting for differences in the length distribution of fetal- and maternal-derived cfDNA fragments results in increased accuracy. Our model is the first to predict inherited insertions-deletions (indels). The method described can serve as a general framework for the NIPD of SGDs; this will facilitate easy integration of further improvements. One such improvement that is presented in the current study is a machine learning model that corrects errors based on patterns found in previously processed data. Overall, we show that next-generation sequencing (NGS) can be used for the NIPD of a wide range of monogenic diseases, simultaneously. We believe that our study will lead to the achievement of a comprehensive NIPD for monogenic diseases.


Assuntos
Doenças Genéticas Inatas/genética , Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Teorema de Bayes , Ácidos Nucleicos Livres/genética , Doenças Genéticas Inatas/diagnóstico , Testes Genéticos/normas , Humanos , Mutação INDEL , Aprendizado de Máquina , Diagnóstico Pré-Natal/normas
7.
Mol Genet Genomic Med ; 7(3): e545, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30706702

RESUMO

BACKGROUND: We evaluated the performance of a cell-free DNA (cfDNA) prenatal screening assay for trisomies 21, 18, and 13, and sex chromosome aneuploidies (SCAs) among a population of pregnant women that included both those at average and high risk. METHODS: Specimen collection, cfDNA extraction, massively parallel sequencing, and bioinformatics analysis were conducted per laboratory protocol. Assay results, concordance with pregnancy outcomes, and performance characteristics were evaluated. RESULTS: A total 75,658 specimens from 72,176 individual pregnant women were received. Technical reasons accounted for 288 (0.4% of all received samples) tests not performed. In the final analysis cohort (N = 69,794), 13% of pregnancies were considered at average risk and 87% at high risk. Mean gestational age at specimen collection was 15.1 weeks. Of the 69,794 unique pregnancies, 1,359 (1.9%) had positive test results. Among the results with confirmed outcomes, PPV for trisomies 21, 18, and 13 was 98.1%, 88.2%, and 59.3%, respectively; the PPV was 69.0% for SCAs and 75.0% for microdeletions. Overall, PPV was 87.2%, sensitivity was 97.9%, and specificity was 99.9%. CONCLUSION: This cfDNA prenatal screening assay provides highly accurate discrimination between affected and unaffected pregnancies among a population of pregnant women at average and high risk for fetal genetic abnormalities.


Assuntos
Aneuploidia , Ácidos Nucleicos Livres/genética , Transtornos Cromossômicos/diagnóstico , Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Adulto , Ácidos Nucleicos Livres/química , Transtornos Cromossômicos/genética , Feminino , Testes Genéticos/normas , Humanos , Gravidez , Diagnóstico Pré-Natal/normas , Reprodutibilidade dos Testes , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
8.
Mol Genet Genomic Med ; 7(4): e00597, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30767419

RESUMO

BACKGROUND: The noninvasive prenatal testing (NIPT) has been successfully used in the clinical screening of fetal trisomy 13, 18, and 21 in the last few years and researches on detecting sub-chromosomal copy number variations (CNVs) and monogenic diseases are also in progress. To date, multiple tests are needed in order to complete a full set of fetus disorder screening, which is costly and time consuming. Therefore, an integrated 3-in-1 NIPT approach will be in great demand by routine clinical practice in the near future. METHODS: We designed a target capture sequencing panel with an associate bioinformatics pipeline to create a novel multi-functional NIPT method and we evaluated its performance by testing 22 clinical samples containing aneuploidy, CNV, and single-gene disorder. Chromosomal aneuploidy and CNV were detected based on the Z-value approach, whereas single-gene disorder was identified by using the "pseudo-tetraploid" model to estimate the best-suited genotype for each locus. RESULTS: The performance of this newly constructed 3-in-1 system was promising. We achieved a 100% detection rate for chromosomal aneuploidies (7/7), a 100% diagnosis rate for fetus CNVs larger than 20 Mb (3/3), and an 86.4% accuracy for single-gene disorder screening (19/22). CONCLUSION: For the first time, we showed that it is possible to use just a single NIPT test to detect three distinct types of fetus disorder and laid a foundation for developing a cheaper, faster, and multi-functional NIPT method in the future.


Assuntos
Aneuploidia , Variações do Número de Cópias de DNA , Testes Genéticos/métodos , Mutação , Diagnóstico Pré-Natal/métodos , Transtornos Cromossômicos/diagnóstico , Transtornos Cromossômicos/genética , Feminino , Doenças Genéticas Inatas/diagnóstico , Doenças Genéticas Inatas/genética , Testes Genéticos/normas , Humanos , Projetos Piloto , Gravidez , Diagnóstico Pré-Natal/normas , Sensibilidade e Especificidade , Análise de Sequência de DNA/métodos , Análise de Sequência de DNA/normas
9.
Mol Genet Genomic Med ; 7(4): e00575, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30793526

RESUMO

BACKGROUND: Detailed analysis of imprinting center (IC) defects in individuals with Prader-Willi syndrome (PWS) is not readily available beyond chromosomal microarray (MA) analysis, and such testing is important for a more accurate diagnosis and recurrence risks. This is the first feasibility study of newly developed droplet digital polymerase chain reaction (ddPCR) examining DNA copy number differences in the PWS IC region of those with IC defects. METHODS: The study cohort included 17 individuals without 15q11-q13 deletions or maternal disomy but with IC defects as determined by genotype analysis showing biparental inheritance. Seven sets of parents and two healthy, unrelated controls were also analyzed. RESULTS: Copy number differences were distinguished by comparing the number of positive droplets detected by IC probes to those from a chromosome 15 reference probe, GABRß3. The ddPCR findings were compared to results from other methods including MA, and whole-exome sequencing (WES) with 100% concordance. The study also estimated the frequency of IC microdeletions and identified gene variants by WES that may impact phenotypes including CPT2 and NTRK1 genes. CONCLUSION: Droplet digital polymerase chain reaction is a cost-effective method that can be used to confirm the presence of microdeletions in PWS with impact on genetic counseling and recurrence risks for families.


Assuntos
Testes Genéticos/métodos , Impressão Genômica , Síndrome de Prader-Willi/genética , Sequenciamento Completo do Exoma/métodos , Adolescente , Adulto , Criança , Pré-Escolar , Cromossomos Humanos Par 15/genética , Feminino , Deleção de Genes , Testes Genéticos/economia , Testes Genéticos/normas , Humanos , Masculino , Síndrome de Prader-Willi/diagnóstico , Sequenciamento Completo do Exoma/economia , Sequenciamento Completo do Exoma/normas
10.
Diabetes Care ; 42(3): 406-415, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30659077

RESUMO

OBJECTIVE: Genetic risk scores (GRS) have been developed that differentiate individuals with type 1 diabetes from those with other forms of diabetes and are starting to be used for population screening; however, most studies were conducted in European-ancestry populations. This study identifies novel genetic variants associated with type 1 diabetes risk in African-ancestry participants and develops an African-specific GRS. RESEARCH DESIGN AND METHODS: We generated single nucleotide polymorphism (SNP) data with the ImmunoChip on 1,021 African-ancestry participants with type 1 diabetes and 2,928 control participants. HLA class I and class II alleles were imputed using SNP2HLA. Logistic regression models were used to identify genome-wide significant (P < 5.0 × 10-8) SNPs associated with type 1 diabetes in the African-ancestry samples and validate SNPs associated with risk in known European-ancestry loci (P < 2.79 × 10-5). RESULTS: African-specific (HLA-DQA1*03:01-HLA-DQB1*02:01) and known European-ancestry HLA haplotypes (HLA-DRB1*03:01-HLA-DQA1*05:01-HLA-DQB1*02:01, HLA-DRB1*04:01-HLA-DQA1*03:01-HLA-DQB1*03:02) were significantly associated with type 1 diabetes risk. Among European-ancestry defined non-HLA risk loci, six risk loci were significantly associated with type 1 diabetes in subjects of African ancestry. An African-specific GRS provided strong prediction of type 1 diabetes risk (area under the curve 0.871), performing significantly better than a European-based GRS and two polygenic risk scores in independent discovery and validation cohorts. CONCLUSIONS: Genetic risk of type 1 diabetes includes ancestry-specific, disease-associated variants. The GRS developed here provides improved prediction of type 1 diabetes in African-ancestry subjects and a means to identify groups of individuals who would benefit from immune monitoring for early detection of islet autoimmunity.


Assuntos
Grupo com Ancestrais do Continente Africano/genética , Diabetes Mellitus Tipo 1/etnologia , Diabetes Mellitus Tipo 1/genética , Testes Genéticos , Antígenos HLA-D/genética , Grupo com Ancestrais do Continente Africano/estatística & dados numéricos , Alelos , Estudos de Casos e Controles , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/normas , Estudo de Associação Genômica Ampla , Cadeias alfa de HLA-DQ/genética , Cadeias beta de HLA-DQ/genética , Cadeias HLA-DRB1/genética , Haplótipos , Humanos , Masculino , Polimorfismo de Nucleotídeo Único , Valor Preditivo dos Testes , Projetos de Pesquisa , Fatores de Risco
11.
Mol Genet Genomic Med ; 7(3): e564, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30637984

RESUMO

BACKGROUND: Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA using long reads. METHODS: We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing. RESULTS: We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55-base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed. CONCLUSION: The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants.


Assuntos
Doença de Gaucher/genética , Testes Genéticos/métodos , Glucosilceramidase/genética , Mutação de Sentido Incorreto , Análise de Sequência de DNA/métodos , Doença de Gaucher/diagnóstico , Testes Genéticos/normas , Humanos , Kit de Reagentes para Diagnóstico/normas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas
12.
Mol Genet Genomic Med ; 7(3): e493, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30652428

RESUMO

BACKGROUND: Ductal carcinoma in situ (DCIS) is considered a component of the clinical spectrum of breast cancer even in those with BRCA1/2 mutation. The aim of this study was to report the feature of DCIS raised in Japanese women with BRCA1/2 mutations. METHODS: A total of 325 Japanese women with breast cancer (BC) (with or without invasive cancer) were referred for genetic counseling and underwent genetic testing for mutations in the BRCA1 and BRCA2 genes in Showa University Hospital between December 2011 and August 2016. And 49 of them who were pathologically diagnosed as DCIS were included in this study. Logistic regression models were fit to determine the associations between potential predictive factors and BRCA status. A Cox proportional hazards model is used to predictive value of parameters for Ipsilateral breast tumor recurrence (IBTR) and contralateral breast tumor recurrence (CBTR). RESULTS: (a) Of 325 patients (with or without invasive cancer), 19.1% (62/325) tested positive for BRCA1/BRCA2 mutations. And 18.4% (9/49) was positive for BRCA1/BRCA2 mutations in DCIS, compared with 19.2% (53/276) in IDC (p = 1.000). Among BRCA mutations, 14.5% (9/62) had DCIS compared with nonmutations (15.2%, 40/263). Incidence of DCIS was 3.0% (1/33) of BRCA1 mutations and 27.5% (8/29) of BRCA2 mutation (p = 0.009). (b) Median age of diagnosis in BRCA mutation carriers was 39 years, compared with 46 years in noncarriers. Age, Family history (FH) of BC, FH of first or second BC and total number of relatives with BC diagnosis (DX) has significant difference between BRCA mutation carriers and noncarriers in univariate analysis. In a multivariate logistic model, total relatives with BC DX ≥ 2 (odds ratio [OR], 5.128; 95% confidence interval [CI], 1.266-20.763; p = 0.022), age at diagnosis ≤35 years (OR 0.149, 95% CI 0.023-0.954, p = 0.045) and ER+/HER2+ status (OR 5.034, 95% CI 1.092-23.210, p = 0.038) remained as independent significant predictors for BRCA mutation. Ki67 index (cut off by 14% or 30%) did not differ between BRCA mutation carriers and noncarriers (p = 0.459 and p = 0.651). (c) There was a significant difference in ER-positive tumors among BRCA2 carriers and noncarriers (p = 0.042). Subgroup analysis showed BRCA2 carriers tend to be of higher grade (Grade 2 and 3), more frequently ER+/PR+ (p = 0.041) and lower proliferation (Ki67 index) than noncarriers, whereas differences in nuclear grade and ki67 index were not found significantly in our study. (d) BRCA mutation was not associated with an increased risk of IBTR and CBTR. CONCLUSION: DCIS is equally as prevalent in patients who were BRCA mutation carriers as in high familial-risk women who were noncarriers, but occurs at earlier age. BRCA2 carriers have higher incidence in DCIS than that of BRCA1 carriers, and tend to be higher grade and more frequently ER positive and lower proliferation. Total relatives with BC DX ≥2, age at diagnosis ≤35 years and ER+/HER2+ might be independent predictors for BRCA mutation in Japanese women with DCIS and patients of these risk factors should be recommended to receive genetic counseling and BRCA testing.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Mutação , Adulto , Neoplasias da Mama/epidemiologia , Carcinoma Ductal de Mama/epidemiologia , Feminino , Testes Genéticos/normas , Humanos , Incidência , Japão
13.
Mol Genet Genomic Med ; 7(3): e565, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30666819

RESUMO

INTRODUCTION: Facioscapulohumeral muscular dystrophy 1 (FSHD1) is a relatively common autosomal dominant adult muscular dystrophy with variable disease penetrance. The disease is caused by shortening of a D4Z4 repeat array located near the telomere of chromosome 4 at 4q35. This causes activation of a dormant gene DUX4, permitting aberrant DUX4 expression which is toxic to muscles. Molecular diagnosis of FSHD1 by Southern blot hybridization or FISH combing is difficult and time consuming, requiring specialist laboratories. As an alternative, we apply a novel approach for the diagnosis of FSHD1 utilizing single-molecule optical mapping (SMOM). METHODS: Long DNA molecules with BssS1 enzyme marking were subjected to SMOM on the Bionano Genomics platform to determine the number of D4Z4 repeats. Southern blot and molecular combing were used to confirm the FSHD1 haplotypes. RESULTS: In a study of a five-generation FSHD1 pedigree, SMOM correctly diagnosed the disease and normal haplotypes, identifying the founder 4qA disease allele as having 4 D4Z4 repeat units. Southern blot and molecular combing analysis confirmed the SMOM results for the 4qA disease and 4qB nondisease alleles. CONCLUSION: Based on our findings, we propose that SMOM is a reliable and accurate technique suitable for the molecular diagnosis of FSHD1.


Assuntos
Testes Genéticos/métodos , Distrofia Muscular Facioescapuloumeral/genética , Mutação , Mapeamento por Restrição Óptica/métodos , Testes Genéticos/normas , Humanos , Distrofia Muscular Facioescapuloumeral/diagnóstico , Mapeamento por Restrição Óptica/normas , Linhagem
14.
Fam Cancer ; 18(2): 273-280, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30607672

RESUMO

Early-onset breast cancer may be due to Li-Fraumeni Syndrome (LFS). Current national and international guidelines recommend that TP53 genetic testing should be considered for women with breast cancer diagnosed before the age of 31 years. However, large studies investigating TP53 mutation prevalence in this population are scarce. We collected nationwide laboratory records for all young breast cancer patients tested for TP53 mutations in the Netherlands. Between 2005 and 2016, 370 women diagnosed with breast cancer younger than 30 years of age were tested for TP53 germline mutations, and eight (2.2%) were found to carry a (likely) pathogenic TP53 sequence variant. Among BRCA1/BRCA2 mutation negative women without a family history suggestive of LFS or a personal history of multiple LFS-related tumours, the TP53 mutation frequency was < 1% (2/233). Taking into consideration that TP53 mutation prevalence was comparable or even higher in some studies selecting patients with breast cancer onset at older ages or HER2-positive breast cancers, raises the question of whether a very early age of onset is an appropriate single TP53 genetic testing criterion.


Assuntos
Neoplasias da Mama/genética , Aconselhamento Genético/normas , Testes Genéticos/normas , Síndrome de Li-Fraumeni/genética , Proteína Supressora de Tumor p53/genética , Adolescente , Adulto , Fatores Etários , Idade de Início , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Neoplasias da Mama/epidemiologia , Análise Mutacional de DNA , Feminino , Aconselhamento Genético/estatística & dados numéricos , Predisposição Genética para Doença , Testes Genéticos/estatística & dados numéricos , Mutação em Linhagem Germinativa , Humanos , Síndrome de Li-Fraumeni/diagnóstico , Síndrome de Li-Fraumeni/epidemiologia , Anamnese , Países Baixos/epidemiologia , Guias de Prática Clínica como Assunto , Estudos Retrospectivos , Adulto Jovem
15.
Prenat Diagn ; 39(2): 70-80, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30625249

RESUMO

Cell-free DNA (cfDNA) testing for fetal aneuploidy is one of the most important technical advances in prenatal care. Additional chromosome targets beyond common aneuploidies, including the 22q11.2 microdeletion, are now available because of this clinical testing technology. While there are numerous potential benefits, 22q11.2 microdeletion screening using cfDNA testing also presents significant limitations and pitfalls. Practitioners who are offering this test should provide comprehensive pretest and posttest prenatal counselling. The discussion should include the possibility of an absence of a result, as well as the risk of possible discordance between cfDNA screening results and the actual fetal genetic chromosomal constitution. The goal of this review is to provide an overview of the cfDNA testing technologies for 22q11.2 microdeletions screening, describe the current state of test validation and clinical experience, review "no results" and discordant findings based on differing technologies, and discuss management options.


Assuntos
Síndrome da Deleção 22q11/diagnóstico , Ácidos Nucleicos Livres/análise , Testes Genéticos/métodos , Síndrome da Deleção 22q11/sangue , Síndrome da Deleção 22q11/genética , Aneuploidia , Ácidos Nucleicos Livres/sangue , Deleção Cromossômica , Síndrome de DiGeorge/diagnóstico , Síndrome de Down/diagnóstico , Feminino , Testes Genéticos/normas , Humanos , Gravidez , Diagnóstico Pré-Natal/métodos , Diagnóstico Pré-Natal/normas
16.
Diabetes Care ; 42(2): 200-207, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30655379

RESUMO

OBJECTIVE: Previously generated genetic risk scores (GRSs) for type 1 diabetes (T1D) have not captured all known information at non-HLA loci or, particularly, at HLA risk loci. We aimed to more completely incorporate HLA alleles, their interactions, and recently discovered non-HLA loci into an improved T1D GRS (termed the "T1D GRS2") to better discriminate diabetes subtypes and to predict T1D in newborn screening studies. RESEARCH DESIGN AND METHODS: In 6,481 case and 9,247 control subjects from the Type 1 Diabetes Genetics Consortium, we analyzed variants associated with T1D both in the HLA region and across the genome. We modeled interactions between variants marking strongly associated HLA haplotypes and generated odds ratios to create the improved GRS, the T1D GRS2. We validated our findings in UK Biobank. We assessed the impact of the T1D GRS2 in newborn screening and diabetes classification and sought to provide a framework for comparison with previous scores. RESULTS: The T1D GRS2 used 67 single nucleotide polymorphisms (SNPs) and accounted for interactions between 18 HLA DR-DQ haplotype combinations. The T1D GRS2 was highly discriminative for all T1D (area under the curve [AUC] 0.92; P < 0.0001 vs. older scores) and even more discriminative for early-onset T1D (AUC 0.96). In simulated newborn screening, the T1D GRS2 was nearly twice as efficient as HLA genotyping alone and 50% better than current genetic scores in general population T1D prediction. CONCLUSIONS: An improved T1D GRS, the T1D GRS2, is highly useful for classifying adult incident diabetes type and improving newborn screening. Given the cost-effectiveness of SNP genotyping, this approach has great clinical and research potential in T1D.


Assuntos
Diabetes Mellitus Tipo 1/diagnóstico , Diabetes Mellitus Tipo 1/genética , Testes Genéticos , Triagem Neonatal/métodos , Triagem Neonatal/normas , Alelos , Estudos de Casos e Controles , Diabetes Mellitus Tipo 1/epidemiologia , Feminino , Predisposição Genética para Doença , Testes Genéticos/métodos , Testes Genéticos/normas , Antígenos HLA/genética , Haplótipos , Humanos , Incidência , Recém-Nascido , Masculino , Polimorfismo de Nucleotídeo Único , Melhoria de Qualidade , Padrões de Referência , Projetos de Pesquisa/normas , Fatores de Risco , Reino Unido
17.
J Obstet Gynaecol Res ; 45(4): 830-840, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30632238

RESUMO

AIM: Chromosome analysis of prenatal samples and products of conception (POC) has conventionally been done by karyotyping (KT). Shortcomings of KT like high turnaround time and culture failure led to technology innovations, such as the bacterial artificial chromosomes (BAC)s-on-Beads (BoBs)-based tests, Prenatal BoBs (prenatal samples) and KaryoLite BoBs (POC samples). In the present study, we validated and evaluated the utility of each test on prenatal, POC and blood samples. METHODS: Study A (n = 305; 259 prenatal + 46 blood/POC) and Study B (n = 176; 146 POC/chorionic vill + 30 blood/amniotic fluid) samples were analyzed using Prenatal and KaryoLite BoBs kits, respectively. KT, array-based Comparative Genomic Hybridization (arrayCGH) and fluorescence in situ hybridization (FISH) were used for comparison of results. Ability of KaryoLite BoBs to identify ring chromosomes was tested. RESULTS: Prenatal BoBs had zero test failure rate and results of all samples were concordant with KT results. Totally four microdeletions were identified by Prenatal BoBs but not by KT. In Study B, all but two POC samples (one triploid and one tetraploid) were concordant with KT and arrayCGH. Partial chromosomal imbalance detection rate was ~64% and KaryoLite BoBs indicated the presence of a ring chromosome in all four cases. The failure rate of KaryoLite BoBs was 3%. CONCLUSION: We conclude that Prenatal BoBs (common aneuploidies and nine microdeletions) together with KT constitutes more comprehensive prenatal testing compared to FISH and KT. KaryoLite BoBs for aneuploidies of all chromosomes is highly successful in POC analysis and the ability to indicate presence of ring chromosomes improves its clinical sensitivity. Both tests are robust and could also be used for different specimens.


Assuntos
Bioensaio/normas , Aberrações Cromossômicas , Transtornos Cromossômicos/diagnóstico , Cromossomos Artificiais Bacterianos , Testes Genéticos/normas , Diagnóstico Pré-Natal/normas , Adulto , Feminino , Humanos , Cariotipagem , Gravidez
18.
Genome Med ; 11(1): 5, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696458

RESUMO

BACKGROUND: International guidelines for variant interpretation in Mendelian disease set stringent criteria to report a variant as (likely) pathogenic, prioritising control of false-positive rate over test sensitivity and diagnostic yield. Genetic testing is also more likely informative in individuals with well-characterised variants from extensively studied European-ancestry populations. Inherited cardiomyopathies are relatively common Mendelian diseases that allow empirical calibration and assessment of this framework. METHODS: We compared rare variants in large hypertrophic cardiomyopathy (HCM) cohorts (up to 6179 cases) to reference populations to identify variant classes with high prior likelihoods of pathogenicity, as defined by etiological fraction (EF). We analysed the distribution of variants using a bespoke unsupervised clustering algorithm to identify gene regions in which variants are significantly clustered in cases. RESULTS: Analysis of variant distribution identified regions in which variants are significantly enriched in cases and variant location was a better discriminator of pathogenicity than generic computational functional prediction algorithms. Non-truncating variant classes with an EF ≥ 0.95 were identified in five established HCM genes. Applying this approach leads to an estimated 14-20% increase in cases with actionable HCM variants, i.e. variants classified as pathogenic/likely pathogenic that might be used for predictive testing in probands' relatives. CONCLUSIONS: When found in a patient confirmed to have disease, novel variants in some genes and regions are empirically shown to have a sufficiently high probability of pathogenicity to support a "likely pathogenic" classification, even without additional segregation or functional data. This could increase the yield of high confidence actionable variants, consistent with the framework and recommendations of current guidelines. The techniques outlined offer a consistent and unbiased approach to variant interpretation for Mendelian disease genetic testing. We propose adaptations to ACMG/AMP guidelines to incorporate such evidence in a quantitative and transparent manner.


Assuntos
Cardiomiopatia Hipertrófica/genética , Testes Genéticos/normas , Mutação , Cardiomiopatia Hipertrófica/patologia , Humanos , Guias de Prática Clínica como Assunto
19.
Mol Genet Genomic Med ; 7(1): e00513, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30565893

RESUMO

BACKGROUND: Targeted gene capture and next-generation sequencing (NGS) has been widely utilized as a robust and cost-effective approach for detecting small variants among a group of disease genes. Copy number variations (CNV) can also be inferred from the read-depth information but the accuracy of CNVs called from panel-based NGS data has not been well evaluated. METHODS: Sequencing data were acquired from patients underwent routine clinical targeted panel sequencing testing. Pathogenic CNVs detected from targeted panel sequencing data were evaluated using CNVs generated by two clinical accepted platforms, namely chromosome microarray analysis (CMA) and multiple ligation-dependent probe amplification (MLPA) as benchmarks. CNVkit was used in our study to call CNVs from sequencing data using read-depth information. CMA and MLPA tests were used to confirm and further assess the size and breakpoints of CNVs. RESULTS: The size of CNVs detected using panel-based NGS data are over 300 kb. The sizes of CNVs detected are slightly larger (102.3% on average) using the NGS platform than using the CMA platform, and the size accuracy improved as the size of variants increases. The breakpoints of CNVs detected using NGS data are quite close (within 2.3% of margin) to the breakpoints detected by CMA. CNVs on sex chromosomes, however, are less concordant between NGS and CMA platforms. CONCLUSION: Copy number variations covering adequate exons on autosomes can be accurately detected using targeted panel sequencing data as using CMA. CNVs detected from sex chromosomes need further evaluation and validation. Except for exon-level deletion/duplication and CNV on sex chromosome, our data support the use of panel-based NGS data for routine clinical detection of pathogenic CNVs.


Assuntos
Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Variações do Número de Cópias de DNA , Testes Genéticos/normas , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Distrofia Muscular de Duchenne/genética , Sensibilidade e Especificidade , Análise de Sequência de DNA/normas
20.
Mol Genet Genomic Med ; 7(2): e00527, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30548430

RESUMO

BACKGROUND: The diagnostic workup in patients with a clinical suspicion of lysosomal storage diseases (LSD) is often difficult due to the variability in the clinical phenotype. The gold standard for diagnosis of LSDs consists of enzymatic testing. However, due to the sequential nature of this methodology and inconsistent genotype-phenotype correlations of certain LSDs, finding a diagnosis can be challenging. METHOD: We developed and clinically implemented a gene panel covering 50 genes known to cause LSDs when mutated. Over a period of 18 months, we analyzed 150 patients who were referred for LSD testing and compared these results with the data of patients who were previously enrolled in a scheme of classical biochemical testing. RESULTS: Our panel was able to determine the molecular cause of the disease in 22 cases (15%), representing an increase in diagnostic yield compared to biochemical tests developed for 21 LSDs (4.6%). We were furthermore able to redirect the diagnosis of a mucolipidosis patient who was initially suspected to be affected with galactosialidosis. Several patients were identified as being affected with neuronal ceroid lipofuscinosis, which cannot readily be detected by enzyme testing. Finally, several carriers of pathogenic mutations in LSD genes related to the disease phenotype were identified as well, thus potentially increasing the diagnostic yield of the panel as heterozygous deletions cannot be detected. CONCLUSION: We show that the implementation of a gene panel for LSD diagnostics results in an increased yield in comparison to classical biochemical testing. As the panel is able to cover a wider range of diseases, we propose to implement this methodology as a first-tier test in cases of an aspecific LSD presentation, while enzymatic testing remains the first choice in patients with a more distinctive clinical presentation. Positive panel results should however still be enzymatically confirmed whenever possible.


Assuntos
Testes Genéticos/métodos , Doenças por Armazenamento dos Lisossomos/genética , Análise de Sequência de DNA/métodos , Células Cultivadas , Fibroblastos/metabolismo , Testes Genéticos/normas , Humanos , Imunoensaio/métodos , Doenças por Armazenamento dos Lisossomos/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de DNA/normas
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