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1.
NPJ Genom Med ; 6(1): 92, 2021 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-34750377

RESUMO

TET3 encodes an essential dioxygenase involved in epigenetic regulation through DNA demethylation. TET3 deficiency, or Beck-Fahrner syndrome (BEFAHRS; MIM: 618798), is a recently described neurodevelopmental disorder of the DNA demethylation machinery with a nonspecific phenotype resembling other chromatin-modifying disorders, but inconsistent variant types and inheritance patterns pose diagnostic challenges. Given TET3's direct role in regulating 5-methylcytosine and recent identification of syndrome-specific DNA methylation profiles, we analyzed genome-wide DNA methylation in whole blood of TET3-deficient individuals and identified an episignature that distinguishes affected and unaffected individuals and those with mono-allelic and bi-allelic pathogenic variants. Validation and testing of the episignature correctly categorized known TET3 variants and determined pathogenicity of variants of uncertain significance. Clinical utility was demonstrated when the episignature alone identified an affected individual from over 1000 undiagnosed cases and was confirmed upon distinguishing TET3-deficient individuals from those with 46 other disorders. The TET3-deficient signature - and the signature resulting from activating mutations in DNMT1 which normally opposes TET3 - are characterized by hypermethylation, which for BEFAHRS involves CpG sites that may be biologically relevant. This work expands the role of epi-phenotyping in molecular diagnosis and reveals genome-wide DNA methylation profiling as a quantitative, functional readout for characterization of this new biochemical category of disease.

2.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445317

RESUMO

The 22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder in humans and is the result of a recurrent 1.5 to 2.5 Mb deletion, encompassing approximately 20-40 genes, respectively. The clinical presentation of the typical deletion includes: Velocardiofacial, Di George, Opitz G/BBB and Conotruncalanomaly face syndromes. Atypical deletions (proximal, distal or nested) are rare and characterized mainly by normal phenotype or mild intellectual disability and variable clinical features. The pathogenetic mechanisms underlying this disorder are not completely understood. Because the 22q11.2 region harbours genes coding for transcriptional factors and chromatin remodelers, in this study, we performed analysis of genome-wide DNA methylation of peripheral blood from 49 patients with 22q11.2DS using the Illumina Infinium Methylation EPIC bead chip arrays. This cohort comprises 43 typical, 2 proximal and 4 distal deletions. We demonstrated the evidence of a unique and highly specific episignature in all typical and proximal 22q11.2DS. The sensitivity and specificity of this signature was further confirmed by comparing it to over 1500 patients with other neurodevelopmental disorders with known episignatures. Mapping the 22q11.2DS DNA methylation episignature provides both novel insights into the molecular pathogenesis of this disorder and an effective tool in the molecular diagnosis of 22q11.2DS.


Assuntos
Metilação de DNA , Síndrome de DiGeorge/genética , Epigenoma , Feminino , Humanos , Lactente , Masculino
3.
Front Genet ; 12: 698595, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34326862

RESUMO

Background: Hereditary cancer predisposition syndromes account for approximately 10% of cancer cases. Next generation sequencing (NGS) based multi-gene targeted panels is now a frontline approach to identify pathogenic mutations in cancer predisposition genes in high-risk families. Recent evolvement of NGS technologies have allowed simultaneous detection of sequence and copy number variants (CNVs) using a single platform. In this study, we have analyzed frequency and nature of sequence variants and CNVs, in a Canadian cohort of patients, suspected with hereditary cancer syndrome, referred for genetic testing following specific genetic testing guidelines based on patient's personal and/or family history of cancer. Methods: A 2870 patients were subjected to a single NGS based multi-gene targeted hereditary cancer panel testing algorithm to identify sequence variants and CNVs in cancer predisposition genes at our reference laboratory in Southwestern Ontario. CNVs identified by NGS were confirmed by alternative techniques like Multiplex ligation-dependent probe amplification (MLPA). Results: A 15% (431/2870) patients had a pathogenic variant and 36% (1032/2870) had a variant of unknown significance (VUS), in a cancer susceptibility gene. A total of 287 unique pathogenic variant were identified, out of which 23 (8%) were novel. CNVs identified by NGS based approach accounted for 9.5% (27/287) of pathogenic variants, confirmed by alternate techniques with high accuracy. Conclusion: This study emphasizes the utility of NGS based targeted testing approach to identify both sequence and CNVs in patients suspected with hereditary cancer syndromes in clinical setting and expands the mutational spectrum of high and moderate penetrance cancer predisposition genes.

5.
Genet Med ; 23(6): 1065-1074, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33547396

RESUMO

PURPOSE: We describe the clinical implementation of genome-wide DNA methylation analysis in rare disorders across the EpiSign diagnostic laboratory network and the assessment of results and clinical impact in the first subjects tested. METHODS: We outline the logistics and data flow between an integrated network of clinical diagnostics laboratories in Europe, the United States, and Canada. We describe the clinical validation of EpiSign using 211 specimens and assess the test performance and diagnostic yield in the first 207 subjects tested involving two patient subgroups: the targeted cohort (subjects with previous ambiguous/inconclusive genetic findings including genetic variants of unknown clinical significance) and the screening cohort (subjects with clinical findings consistent with hereditary neurodevelopmental syndromes and no previous conclusive genetic findings). RESULTS: Among the 207 subjects tested, 57 (27.6%) were positive for a diagnostic episignature including 48/136 (35.3%) in the targeted cohort and 8/71 (11.3%) in the screening cohort, with 4/207 (1.9%) remaining inconclusive after EpiSign analysis. CONCLUSION: This study describes the implementation of diagnostic clinical genomic DNA methylation testing in patients with rare disorders. It provides strong evidence of clinical utility of EpiSign analysis, including the ability to provide conclusive findings in the majority of subjects tested.


Assuntos
Metilação de DNA , Epigenômica , Canadá , Europa (Continente) , Humanos , Síndrome
7.
Int J Mol Sci ; 22(3)2021 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-33498634

RESUMO

A growing number of genetic neurodevelopmental disorders are known to be associated with unique genomic DNA methylation patterns, called episignatures, which are detectable in peripheral blood. The intellectual developmental disorder, X-linked, syndromic, Armfield type (MRXSA) is caused by missense variants in FAM50A. Functional studies revealed the pathogenesis to be a spliceosomopathy that is characterized by atypical mRNA processing during development. In this study, we assessed the peripheral blood specimens in a cohort of individuals with MRXSA and detected a unique and highly specific DNA methylation episignature associated with this disorder. We used this episignature to construct a support vector machine model capable of sensitive and specific identification of individuals with pathogenic variants in FAM50A. This study contributes to the expanding number of genetic neurodevelopmental disorders with defined DNA methylation episignatures, provides an additional understanding of the associated molecular mechanisms, and further enhances our ability to diagnose patients with rare disorders.


Assuntos
Metilação de DNA , Retardo Mental Ligado ao Cromossomo X/genética , Adulto , Estudos de Casos e Controles , Criança , Proteínas de Ligação a DNA/genética , Epigenoma , Humanos , Masculino , Retardo Mental Ligado ao Cromossomo X/etiologia , Pessoa de Meia-Idade , Modelos Genéticos , Transtornos do Neurodesenvolvimento/genética , Proteínas de Ligação a RNA/genética
8.
J Hum Genet ; 66(5): 451-464, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33093641

RESUMO

The adaptation of a broad genomic sequencing approach in the clinical setting has been accompanied by considerations regarding the clinical utility, technical performance, and diagnostic yield compared to targeted genetic approaches. We have developed MedExome, an integrated framework for sequencing, variant calling (SNVs, Indels, and CNVs), and clinical assessment of ~4600 medically relevant genes. We compared the technical performance of MedExome with the whole-exome and targeted gene-panel sequencing, assessed the reasons for discordance, and evaluated the added clinical yield of MedExome in a cohort of unresolved subjects suspected of genetic disease. Our analysis showed that despite a higher average read depth in panels (3058 vs. 855), MedExome yielded full coverage of the enriched regions (>20X) and 99% variant concordance rate with panels. The discordance rate was associated with low-complexity regions, high-GC content, and low allele fractions, observed in both platforms. MedExome yielded full sensitivity in detecting clinically actionable variants, and the assessment of 138 patients with suspected genetic conditions resulted in 76 clinical reports (31 full [22.1%], 3 partial, and 42 uncertain/possible molecular diagnoses). MedExome sequencing has comparable performance in variant detection to gene panels. Added diagnostic yield justifies expanded implementation of broad genomic approaches in unresolved patients; however, cost-benefit and health systems impact warrants assessment.


Assuntos
Doenças Genéticas Inatas/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Diagnóstico Molecular/métodos , Sequenciamento Completo do Exoma/métodos , Alelos , Composição de Bases , Consanguinidade , Variações do Número de Cópias de DNA , Exoma , Biblioteca Gênica , Variação Genética , Homozigoto , Humanos , Mutação INDEL , Ontário , Mutação Puntual , Alinhamento de Sequência , Fluxo de Trabalho
9.
Br J Haematol ; 192(4): 729-736, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32588428

RESUMO

Next-generation sequencing (NGS) increasingly influences diagnosis, prognosis and management of myelodysplastic syndrome (MDS). In addition to marrow morphology and flow cytometry, our institution performs cytogenetics (CG) and NGS-based testing routinely in patients with suspected MDS. We evaluated the relative value of NGS in the assessment of patients with suspected MDS. We initially compared the diagnostic and prognostic information derived from CG and NGS in 134 patients. NGS enhanced the diagnostic yield compared to CG for clonal myeloid disorders (sensitivity 77% vs. 42·2%; specificity 90·2% vs. 78%; positive predictive value 92·8% vs. 76%; and negative predictive value 70·8% vs. 45·5%). The identification of poor prognosis mutations by NGS altered risk category in 27/39 (69·2%) patients with MDS with good/intermediate risk CG. Subsequently, we prospectively evaluated 70 patients with suspected MDS using an 'NGS-first approach' with CG restricted to samples with morphological abnormalities. We rarely identified mutations or CG abnormalities in patients without dysplastic features. NGS has a superior diagnostic performance compared to CG in patients with suspected MDS. We estimate that by using an 'NGS-first approach' we could reduce karyotyping by approximately 30%.


Assuntos
Análise Citogenética , Sequenciamento de Nucleotídeos em Larga Escala , Síndromes Mielodisplásicas/genética , Aberrações Cromossômicas , Humanos , Mutação , Síndromes Mielodisplásicas/diagnóstico , Prognóstico , Estudos Retrospectivos
10.
J Med Genet ; 58(4): 284-288, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-32376792

RESUMO

Charcot-Marie-Tooth disease (CMT) is one of the most common Mendelian disorders characterised by genetic heterogeneity, progressive distal muscle weakness and atrophy, foot deformities and distal sensory loss. In this report, we describe genetic testing data including comprehensive sequencing and copy number analysis of 34 CMT-related genes in a Canadian cohort of patients with suspected CMT. We have demonstrated a notable gender testing bias, with an overall diagnostic yield of 15% in males and 21% in females. We have identified a large number of novel pathogenic variants as well as variants of unknown clinical significance in CMT-related genes. In this largest to date analysis of gene CNVs in CMT, in addition to the common PMP22 deletion/duplication, we have described a significant contribution of pathogenic CNVs in several CMT-related genes. This study significantly expand the mutational spectrum of CMT genes, while demonstrating the clinical utility of a comprehensive sequence and copy number next-generation sequencing-based clinical genetic testing in patients with suspected diagnosis of CMT.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Variações do Número de Cópias de DNA/genética , Miopatias Distais/genética , Testes Genéticos , Adulto , Idoso , Canadá/epidemiologia , Doença de Charcot-Marie-Tooth/diagnóstico , Doença de Charcot-Marie-Tooth/epidemiologia , Doença de Charcot-Marie-Tooth/patologia , Estudos de Coortes , Miopatias Distais/diagnóstico , Miopatias Distais/epidemiologia , Miopatias Distais/patologia , Feminino , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Fenótipo
11.
Am J Med Genet A ; 185(2): 486-499, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33300680

RESUMO

Diagnosing mitochondrial disorders is a challenge due to the heterogeneous clinical presentation and large number of associated genes. A custom next generation sequencing (NGS) panel was developed incorporating the full mitochondrial genome (mtDNA) plus 19 nuclear genes involved in structural mitochondrial defects and mtDNA maintenance. This assay is capable of simultaneously detecting small gene sequence variations and larger copy number variants (CNVs) in both the nuclear and mitochondrial components along with heteroplasmy detection down to 5%. We describe technical validations of this panel and its implementation for clinical testing in a Canadian reference laboratory, and report its clinical performance in the initial 950 patients tested. Using this assay, we demonstrate a diagnostic yield of 18.1% of patients with known pathogenic variants. In addition to the common 5 kb mtDNA deletion, we describe significant contribution of pathogenic CNVs in both the mitochondrial genome and nuclear genes in this patient population.


Assuntos
Variações do Número de Cópias de DNA/genética , Genética Populacional , Sequenciamento de Nucleotídeos em Larga Escala , Doenças Mitocondriais/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Canadá/epidemiologia , Núcleo Celular/genética , Criança , Pré-Escolar , DNA Mitocondrial/genética , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Mitocôndrias/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/epidemiologia , Doenças Mitocondriais/patologia , Adulto Jovem
12.
Genet Med ; 22(11): 1838-1850, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32694869

RESUMO

PURPOSE: Nontruncating variants in SMARCA2, encoding a catalytic subunit of SWI/SNF chromatin remodeling complex, cause Nicolaides-Baraitser syndrome (NCBRS), a condition with intellectual disability and multiple congenital anomalies. Other disorders due to SMARCA2 are unknown. METHODS: By next-generation sequencing, we identified candidate variants in SMARCA2 in 20 individuals from 18 families with a syndromic neurodevelopmental disorder not consistent with NCBRS. To stratify variant interpretation, we functionally analyzed SMARCA2 variants in yeasts and performed transcriptomic and genome methylation analyses on blood leukocytes. RESULTS: Of 20 individuals, 14 showed a recognizable phenotype with recurrent features including epicanthal folds, blepharophimosis, and downturned nasal tip along with variable degree of intellectual disability (or blepharophimosis intellectual disability syndrome [BIS]). In contrast to most NCBRS variants, all SMARCA2 variants associated with BIS are localized outside the helicase domains. Yeast phenotype assays differentiated NCBRS from non-NCBRS SMARCA2 variants. Transcriptomic and DNA methylation signatures differentiated NCBRS from BIS and those with nonspecific phenotype. In the remaining six individuals with nonspecific dysmorphic features, clinical and molecular data did not permit variant reclassification. CONCLUSION: We identified a novel recognizable syndrome named BIS associated with clustered de novo SMARCA2 variants outside the helicase domains, phenotypically and molecularly distinct from NCBRS.


Assuntos
Blefarofimose , Hipotricose , Deficiência Intelectual , Facies , Deformidades Congênitas do Pé , Humanos , Deficiência Intelectual/genética , Fenótipo , Fatores de Transcrição/genética
13.
J Hum Genet ; 65(10): 865-873, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32483276

RESUMO

Poly-ADP-ribose-polymerase inhibitor (PARPi) treatment is indicated for advanced-stage ovarian tumors with BRCA1/2 deficiency. The "BRCAness" status is thought to be attributed to a tumor phenotype associated with a specific epigenomic DNA methylation profile. Here, we examined the diagnostic impact of combined BRCA1/2 sequence, copy number, and promoter DNA methylation analysis, and evaluated whether genomic DNA methylation patterns can predict the BRCAness in ovarian tumors. DNA sequencing of 172 human tissue samples of advanced-stage ovarian adenocarcinoma identified 36 samples with a clinically significant tier 1/2 sequence variants (point mutations and in/dels) and 9 samples with a CNV causing a loss of function in BRCA1/2. DNA methylation analysis of the promoter of BRCA1/2 identified promoter hypermethylation of BRCA1 in two mutation-negative samples. Computational modeling of genome-wide methylation markers, measured using Infinium EPIC arrays, resulted in a total accuracy of 0.75, sensitivity: 0.83, specificity: 0.64, positive predictive value: 0.76, negative predictive value: 0.74, and area under the receiver's operating curve (AUC): 0.77, in classifying tumors harboring a BRCA1/2 defect from the rest. These findings indicate that the assessment of CNV and promoter DNA methylation in BRCA1/2 increases the cumulative diagnostic yield by 10%, compared with the 20% yield achieved by sequence variant analysis alone. Genomic DNA methylation data can partially predict BRCAness in ovarian tumors; however, further investigation in expanded BRCA1/2 cohorts is needed, and the effect of other double strand DNA repair gene defects in these tumors warrants further investigations.


Assuntos
Adenocarcinoma/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Metilação de DNA , Genes BRCA1 , Genes BRCA2 , Técnicas de Diagnóstico Molecular , Síndromes Neoplásicas Hereditárias/genética , Neoplasias Ovarianas/genética , Adenocarcinoma/diagnóstico , Adenocarcinoma/tratamento farmacológico , Adulto , Idoso , Antineoplásicos/uso terapêutico , Área Sob a Curva , Variações do Número de Cópias de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Feminino , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Pessoa de Meia-Idade , Síndromes Neoplásicas Hereditárias/diagnóstico , Síndromes Neoplásicas Hereditárias/tratamento farmacológico , Neoplasias Ovarianas/diagnóstico , Neoplasias Ovarianas/tratamento farmacológico , Mutação Puntual , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Valor Preditivo dos Testes , Regiões Promotoras Genéticas/genética , Curva ROC , Sensibilidade e Especificidade
14.
FASEB J ; 34(7): 9297-9306, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32441840

RESUMO

Studies have shown that mitochondrial DNA (mtDNA) can be exchanged between tissues; however, the mechanism(s) behind this phenomenon remain unclear. Exosomes and other extracellular vesicles (EVs) including microvesicles (MV) have been shown to contain mtDNA. EVs can be derived from a number of tissues; however, the source and relative proportion of EVs containing mtDNA remains unknown. We sampled whole blood and the EV fractions (exosome-enriched, MV-enriched, and apoptotic body-enriched) as well as several tissues (epithelial-cheek and urine sediment), connective (fibroblasts), and skeletal muscle in two subjects who received allogenic bone marrow transplants. Next generation sequencing of the mtDNA confirmed that all EV fractions contained mtDNA and most was derived from the donor, confirming that most of the EV fractions in the serum are bone marrow/blood cell-derived. Even after exposure to the donor mtDNA in EV fractions (and potentially free in the plasma) for years, there was little to no transfer of the donor mtDNA to the host mtDNA fraction in epithelial, connective, or skeletal muscle tissues. These data call into question the potential therapeutic use of bone marrow transplant or EV-based delivery systems for mtDNA-based disorders and establish bone marrow as the primary source of most of the mtDNA enriched EVs in serum.


Assuntos
Transtornos da Insuficiência da Medula Óssea/terapia , Transplante de Medula Óssea/métodos , Medula Óssea/metabolismo , DNA Mitocondrial/genética , Vesículas Extracelulares/patologia , Mitocôndrias/patologia , Mutação , Adulto , Medula Óssea/patologia , Vesículas Extracelulares/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Masculino , Mitocôndrias/metabolismo , Doadores de Tecidos , Adulto Jovem
15.
Am J Hum Genet ; 106(3): 356-370, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32109418

RESUMO

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders.


Assuntos
Metilação de DNA , Transtornos do Neurodesenvolvimento/genética , Fenótipo , Estudos de Coortes , Heterogeneidade Genética , Humanos , Síndrome
16.
Clin Epigenetics ; 12(1): 7, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31910894

RESUMO

BACKGROUND: We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern. RESULTS: Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This "episignature" was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject. CONCLUSIONS: We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis.


Assuntos
Metilação de DNA , Deficiências do Desenvolvimento/genética , Mutação da Fase de Leitura , Histonas/genética , Deficiência Intelectual/genética , Encéfalo/metabolismo , Deficiências do Desenvolvimento/metabolismo , Epigênese Genética , Humanos , Deficiência Intelectual/metabolismo , Neurônios/metabolismo , Transdução de Sinais/genética , Síndrome
17.
Hum Mutat ; 40(10): 1684-1689, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31268616

RESUMO

Nontruncating sequence variants represent a major challenge in variant interpretation and classification. Here, we report a patient with features of Kabuki syndrome who carries two rare heterozygous variants in KMT2D: c.12935C>T, p.(Ser4312Phe) and c.15785-10T>G. The clinical significance of these variants were discordantly interpreted by different diagnostic laboratories. Parental testing showed that the missense variant was inherited from the father with a mild Kabuki phenotype and the intronic variant from the mother with mosaic status. Through genome-wide DNA methylation analysis of peripheral blood, we confirmed that the proband exhibited a previously described episignature of Kabuki syndrome. Parental samples had normal DNA methylation profiles, thus ruling out the involvement of the paternally inherited missense variant. RNA analysis revealed that the intronic change resulted in exon 49 skipping and frameshift, thereby providing a molecular diagnosis of Kabuki syndrome. This study demonstrates the utility of epigenomic and RNA analyses in resolving ambiguous clinical cases.


Assuntos
Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Metilação de DNA , Face/anormalidades , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Doenças Hematológicas/diagnóstico , Doenças Hematológicas/genética , Fenótipo , RNA/genética , Doenças Vestibulares/diagnóstico , Doenças Vestibulares/genética , Alelos , Criança , Epigênese Genética , Éxons , Feminino , Humanos , Análise de Sequência de DNA
18.
Eur J Haematol ; 103(3): 178-189, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31177553

RESUMO

OBJECTIVES: The diagnosis of hematologic malignancies integrates multiple diagnostic and clinical disciplines. Historically, targeted (single-analyte) genetic testing has been used as reflex to initial prescreening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. Given the wide range of mutations associated with hematologic malignancies a DNA/RNA-based NGS panel can provide a more effective and economical approach to comprehensive testing of patients as an initial, tier-1 screen. METHODS: Using a cohort of 380 patients, we performed clinical validation of a gene panel designed to assess 40 genes (DNA), and 29 fusion driver genes with over 600 gene fusion partners (RNA), including sample exchange data across three clinical laboratories, and correlation with cytogenetic testing results. RESULTS: The clinical validation of this technology demonstrated that its accuracy, sensitivity, and specificity are comparable to the majority of targeted single-gene approaches, while assessment of the initial patient cohort data demonstrated a high diagnostic yield of 50.5%. CONCLUSIONS: Implementation of a tier-1 NGS-based protocol for gene panel screening provides a comprehensive alternative to targeted molecular testing in patients with suspected hematologic malignancies, with increased diagnostic yield, scalability, reproducibility, and cost effectiveness, making it ideally suited for implementation in clinical laboratories.


Assuntos
Biomarcadores Tumorais , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Fusão Oncogênica/genética , Biologia Computacional/métodos , Predisposição Genética para Doença , Testes Genéticos , Variação Genética , Genômica/métodos , Neoplasias Hematológicas/epidemiologia , Humanos , Mutação , Estudos Retrospectivos
19.
Clin Epigenetics ; 11(1): 64, 2019 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-31029150

RESUMO

BACKGROUND: ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome. RESULTS: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The "epi-ADNP-1" episignature included ~ 6000 mostly hypomethylated CpGs, and the "epi-ADNP-2" episignature included ~ 1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N- and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model. CONCLUSIONS: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome.


Assuntos
Metilação de DNA , Proteínas de Homeodomínio/genética , Mutação , Proteínas do Tecido Nervoso/genética , Transtornos do Neurodesenvolvimento/diagnóstico , Transtornos do Neurodesenvolvimento/genética , Transtorno do Espectro Autista/genética , Criança , Pré-Escolar , Biologia Computacional/métodos , Ilhas de CpG , Diagnóstico Precoce , Epigênese Genética , Feminino , Humanos , Deficiência Intelectual/genética , Masculino , Modelos Genéticos
20.
Am J Hum Genet ; 104(4): 685-700, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929737

RESUMO

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs.


Assuntos
Anormalidades Congênitas/genética , Metilação de DNA , Doenças Genéticas Inatas/diagnóstico , Estudo de Associação Genômica Ampla , Estudos de Coortes , Simulação por Computador , Anormalidades Congênitas/diagnóstico , Variações do Número de Cópias de DNA , Epigenômica , Dosagem de Genes , Doenças Genéticas Inatas/genética , Variação Genética , Impressão Genômica , Humanos , Fenótipo , Análise de Sequência de DNA , Síndrome , Expansão das Repetições de Trinucleotídeos
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