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J Alzheimers Dis ; 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33216030


BACKGROUND: Plasma NfL (pNfL) levels are elevated in many neurological disorders. However, the utility of pNfL in a clinical setting has not been established. OBJECTIVE: In a cohort of diverse older participants, we examined: 1) the association of pNfL to age, sex, Hispanic ethnicity, diagnosis, and structural and amyloid imaging biomarkers; and 2) its association to baseline and longitudinal cognitive and functional performance. METHODS: 309 subjects were classified at baseline as cognitively normal (CN) or with cognitive impairment. Most subjects had structural MRI and amyloid PET scans. The most frequent etiological diagnosis was Alzheimer's disease (AD), but other neurological and neuropsychiatric disorders were also represented. We assessed the relationship of pNfL to cognitive and functional status, primary etiology, imaging biomarkers, and to cognitive and functional decline. RESULTS: pNfL increased with age, degree of hippocampal atrophy, and amyloid load, and was higher in females among CN subjects, but was not associated with Hispanic ethnicity. Compared to CN subjects, pNfL was elevated among those with AD or FTLD, but not those with neuropsychiatric or other disorders. Hippocampal atrophy, amyloid positivity and higher pNfL levels each added unique variance in predicting greater functional impairment on the CDR-SB at baseline. Higher baseline pNfL levels also predicted greater cognitive and functional decline after accounting for hippocampal atrophy and memory scores at baseline. CONCLUSION: pNfL may have a complementary and supportive role to brain imaging and cognitive testing in a memory disorder evaluation, although its diagnostic sensitivity and specificity as a stand-alone measure is modest. In the absence of expensive neuroimaging tests, pNfL could be a used for differentiating neurodegenerative disease from neuropsychiatric disorders.

Front Genet ; 10: 1139, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31803232


Introduction: Case-control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.

J Med Genet ; 56(7): 453-460, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30890586


BACKGROUND: PALB2 monoallelic loss-of-function germ-line variants confer a breast cancer risk comparable to the average BRCA2 pathogenic variant. Recommendations for risk reduction strategies in carriers are similar. Elaborating robust criteria to identify loss-of-function variants in PALB2-without incurring overprediction-is thus of paramount clinical relevance. Towards this aim, we have performed a comprehensive characterisation of alternative splicing in PALB2, analysing its relevance for the classification of truncating and splice site variants according to the 2015 American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. METHODS: Alternative splicing was characterised in RNAs extracted from blood, breast and fimbriae/ovary-related human specimens (n=112). RNAseq, RT-PCR/CE and CloneSeq experiments were performed by five contributing laboratories. Centralised revision/curation was performed to assure high-quality annotations. Additional splicing analyses were performed in PALB2 c.212-1G>A, c.1684+1G>A, c.2748+2T>G, c.3113+5G>A, c.3350+1G>A, c.3350+4A>C and c.3350+5G>A carriers. The impact of the findings on PVS1 status was evaluated for truncating and splice site variant. RESULTS: We identified 88 naturally occurring alternative splicing events (81 newly described), including 4 in-frame events predicted relevant to evaluate PVS1 status of splice site variants. We did not identify tissue-specific alternate gene transcripts in breast or ovarian-related samples, supporting the clinical relevance of blood-based splicing studies. CONCLUSIONS: PVS1 is not necessarily warranted for splice site variants targeting four PALB2 acceptor sites (exons 2, 5, 7 and 10). As a result, rare variants at these splice sites cannot be assumed pathogenic/likely pathogenic without further evidences. Our study puts a warning in up to five PALB2 genetic variants that are currently reported as pathogenic/likely pathogenic in ClinVar.

Processamento Alternativo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Alelos , Perfilação da Expressão Gênica , Estudos de Associação Genética/métodos , Mutação em Linhagem Germinativa , Humanos , Mutação , Neoplasias/diagnóstico , Neoplasias/genética , Degradação do RNAm Mediada por Códon sem Sentido , Sítios de Splice de RNA
Front Genet ; 9: 366, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30233647


In silico tools for splicing defect prediction have a key role to assess the impact of variants of uncertain significance. Our aim was to evaluate the performance of a set of commonly used splicing in silico tools comparing the predictions against RNA in vitro results. This was done for natural splice sites of clinically relevant genes in hereditary breast/ovarian cancer (HBOC) and Lynch syndrome. A study divided into two stages was used to evaluate SSF-like, MaxEntScan, NNSplice, HSF, SPANR, and dbscSNV tools. A discovery dataset of 99 variants with unequivocal results of RNA in vitro studies, located in the 10 exonic and 20 intronic nucleotides adjacent to exon-intron boundaries of BRCA1, BRCA2, MLH1, MSH2, MSH6, PMS2, ATM, BRIP1, CDH1, PALB2, PTEN, RAD51D, STK11, and TP53, was collected from four Spanish cancer genetic laboratories. The best stand-alone predictors or combinations were validated with a set of 346 variants in the same genes with clear splicing outcomes reported in the literature. Sensitivity, specificity, accuracy, negative predictive value (NPV) and Mathews Coefficient Correlation (MCC) scores were used to measure the performance. The discovery stage showed that HSF and SSF-like were the most accurate for variants at the donor and acceptor region, respectively. The further combination analysis revealed that HSF, HSF+SSF-like or HSF+SSF-like+MES achieved a high performance for predicting the disruption of donor sites, and SSF-like or a sequential combination of MES and SSF-like for predicting disruption of acceptor sites. The performance confirmation of these last results with the validation dataset, indicated that the highest sensitivity, accuracy, and NPV (99.44%, 99.44%, and 96.88, respectively) were attained with HSF+SSF-like or HSF+SSF-like+MES for donor sites and SSF-like (92.63%, 92.65%, and 84.44, respectively) for acceptor sites. We provide recommendations for combining algorithms to conduct in silico splicing analysis that achieved a high performance. The high NPV obtained allows to select the variants in which the study by in vitro RNA analysis is mandatory against those with a negligible probability of being spliceogenic. Our study also shows that the performance of each specific predictor varies depending on whether the natural splicing sites are donors or acceptors.

Hum Mutat ; 39(5): 729-741, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29460995


Although the spliceogenic nature of the BRCA2 c.68-7T > A variant has been demonstrated, its association with cancer risk remains controversial. In this study, we accurately quantified by real-time PCR and digital PCR (dPCR), the BRCA2 isoforms retaining or missing exon 3. In addition, the combined odds ratio for causality of the variant was estimated using genetic and clinical data, and its associated cancer risk was estimated by case-control analysis in 83,636 individuals. Co-occurrence in trans with pathogenic BRCA2 variants was assessed in 5,382 families. Exon 3 exclusion rate was 4.5-fold higher in variant carriers (13%) than controls (3%), indicating an exclusion rate for the c.68-7T > A allele of approximately 20%. The posterior probability of pathogenicity was 7.44 × 10-115 . There was neither evidence for increased risk of breast cancer (OR 1.03; 95% CI 0.86-1.24) nor for a deleterious effect of the variant when co-occurring with pathogenic variants. Our data provide for the first time robust evidence of the nonpathogenicity of the BRCA2 c.68-7T > A. Genetic and quantitative transcript analyses together inform the threshold for the ratio between functional and altered BRCA2 isoforms compatible with normal cell function. These findings might be exploited to assess the relevance for cancer risk of other BRCA2 spliceogenic variants.

Proteína BRCA2/genética , Variação Genética , Modelos Genéticos , Processamento de RNA/genética , Proteína BRCA2/metabolismo , Sequência de Bases , Calibragem , Linhagem Celular , Éxons/genética , Feminino , Predisposição Genética para Doença , Humanos , Mitomicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
Hum Mol Genet ; 25(11): 2256-2268, 2016 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-27008870


A recent analysis using family history weighting and co-observation classification modeling indicated that BRCA1 c.594-2A > C (IVS9-2A > C), previously described to cause exon 10 skipping (a truncating alteration), displays characteristics inconsistent with those of a high risk pathogenic BRCA1 variant. We used large-scale genetic and clinical resources from the ENIGMA, CIMBA and BCAC consortia to assess pathogenicity of c.594-2A > C. The combined odds for causality considering case-control, segregation and breast tumor pathology information was 3.23 × 10-8 Our data indicate that c.594-2A > C is always in cis with c.641A > G. The spliceogenic effect of c.[594-2A > C;641A > G] was characterized using RNA analysis of human samples and splicing minigenes. As expected, c.[594-2A > C; 641A > G] caused exon 10 skipping, albeit not due to c.594-2A > C impairing the acceptor site but rather by c.641A > G modifying exon 10 splicing regulatory element(s). Multiple blood-based RNA assays indicated that the variant allele did not produce detectable levels of full-length transcripts, with a per allele BRCA1 expression profile composed of ≈70-80% truncating transcripts, and ≈20-30% of in-frame Δ9,10 transcripts predicted to encode a BRCA1 protein with tumor suppression function.We confirm that BRCA1c.[594-2A > C;641A > G] should not be considered a high-risk pathogenic variant. Importantly, results from our detailed mRNA analysis suggest that BRCA-associated cancer risk is likely not markedly increased for individuals who carry a truncating variant in BRCA1 exons 9 or 10, or any other BRCA1 allele that permits 20-30% of tumor suppressor function. More generally, our findings highlight the importance of assessing naturally occurring alternative splicing for clinical evaluation of variants in disease-causing genes.

Proteína BRCA1/genética , Neoplasias da Mama/genética , Mutação/genética , Neoplasias Ovarianas/genética , Proteínas Supressoras de Tumor/genética , Adulto , Idoso , Processamento Alternativo/genética , Neoplasias da Mama/patologia , Análise Mutacional de DNA , Éxons/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias Ovarianas/patologia , Sítios de Splice de RNA/genética , Processamento de RNA/genética