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1.
Genes (Basel) ; 12(2)2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33671809

RESUMO

Gallbladder carcinoma (GBC) is one of the most aggressive malignancies with poor prognosis and a high fatality rate. The disease presents in advanced stages where the treatment is ineffective. Regarding GBC pathogenesis, as with other neoplasia, this tumor is a multifactorial disorder involving different causative factors such as environmental, microbial, metabolic, and molecular. Genetic alterations can be germline or somatic that involving proto-oncogenes, tumor suppressor genes, cell cycle genes, and growth factors. The ataxia telangiectasia mutated (ATM) gene, coding a serine/threonine kinase involved in the early stages of the homologous recombination (HR) mechanism, is one of the most altered genes in GBC. Here, we present the molecular characterization of a novel germline ATM large genomic rearrangement (LGR) identified by next-generation sequencing (NGS) analysis in an Italian woman diagnosed with metastatic GBC at the age of 55. The results underline the importance of expanding the NGS approach in gallbladder cancer in order to propose new molecular markers of predisposition and prognosis exploitable by novel targeted therapies that may improve the response of patients with ATM-deficient cancers.

2.
Mol Diagn Ther ; 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33710594

RESUMO

BACKGROUND AND OBJECTIVE: Congenital adrenal hyperplasia involves a series of autosomal recessive disorders where adrenal steroidogenesis is affected. We present a detailed molecular investigation of 13 newborns affected from the severe form of congenital adrenal hyperplasia related to 21-hydroxylase deficiency. METHODS: All patients were diagnosed with classical congenital adrenal hyperplasia in the neonatal period due to adrenal crisis and/or ambiguous genitalia presentation. None of the infants was identified through a congenital adrenal hyperplasia newborn screening program. A molecular analysis of the CYP21A2 gene and a familiar segregation analysis were performed. RESULTS: Adrenal crisis was the most severe manifestation in the male salt-wasting newborns while all female patients presented with atypical genitalia. Newborns were correctly genotyped and no genotype-phenotype divergences were found. Two novel severe genotypes, not previously reported, were identified. The novel CYP21A2 frameshift mutations (c.793delG and c.297dupG) were added to the other 45 variants recently reported in the literature, leading to a total count of 279 pathogenic variants affecting the gene. CONCLUSIONS: We have successfully genotyped 13 infants diagnosed with classical congenital adrenal hyperplasia after birth. Our molecular approach led to the identification of two novel frameshift CYP21A2 pathogenic variants related to the salt-wasting form of congenital adrenal hyperplasia.

3.
Mol Biol Rep ; 48(1): 983-987, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33313973

RESUMO

Recently, our lab, part of a referral center in Italy, reported its experience regarding the execution of germline BRCA1/2 (gBRCA) testing during the first months of the coronavirus disease-2019 (COVID-19) pandemic, which highlights a substantial reduction (about 60%) compared with the first 2 months of the current year. This evidence appeared to be a lockdown effect due to extraordinary restriction measures to slow down the spread of SARS-CoV-2. In this study, we aimed to evaluate the overall effects of the ongoing pandemic on gBRCA testing in our institution and to understand how COVID-19 has influenced testing after the complete lockdown (March 8-May 5, 2020). Additionally, we compared this year's trend with trends of the last 3 years to better monitor gBRCA testing progress. This detailed analysis highlights two important findings: (1) gBRCA testing did not increase significantly after the lockdown period (May-October 2020) compared with the lockdown period (March-April 2020), emphasizing that even after the lockdown period testing remained low. (2) Comparing the total tests per year (January-October 2017, 2018, 2019, with 2020), the impact of COVID-19 on gBRCA testing is apparent, with similarities of trends registered in 2017. These evidences reveal a gBRCA testing delay for cancer patients and healthy patients at this moment, and the new era of gBRCA testing in the management of ovarian, breast, pancreas and prostate cancer patients has been seriously questioned due to the COVID-19 pandemic. As consequence, we underline that measures to guarantee oncogenetic testing (e.g., gBRCA testing) along with new diagnostic/clinic strategies are mandatory. For these reasons, several proposals are presented in this study.


Assuntos
Proteína BRCA1/sangue , Neoplasias da Mama/diagnóstico , Neoplasias Ovarianas/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Pandemias , Neoplasias da Próstata/diagnóstico , Biomarcadores Tumorais/sangue , Diagnóstico Tardio/ética , Detecção Precoce de Câncer/estatística & dados numéricos , Feminino , Política de Saúde , Humanos , Itália/epidemiologia , Masculino , Quarentena/psicologia , /patogenicidade
4.
Clin Chim Acta ; 2020 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-33301768

RESUMO

BACKGROUND AND AIMS: With the introduction of Olaparib as target therapy for High Grade Serous Ovarian Cancer (HGSOC) patients with germline and somatic BRCA1/2 mutations, the genetic test performed on tumor tissue has become important like the germline test. In somatic testing the evaluation of Large Genomic Rearrangements (LGRs) represents the main challenge. We describe a droplet digital PCR (ddPCR) assay for the evaluation of target BRCA1 LGRs on blood and formalin-fixed paraffin-embedded (FFPE)/Fresh Frozen Tissue (FFT) samples. MATERIALS AND METHODS: We analyzed blood, FFPE and FFT samples in a validation setting of n=78 HGSOC patients. We applied the ddPCR to BRCA1 exons 2, 20 and 21 as some of the most common BRCA1 exons involved in LGRs in our cohort of patients. RESULTS: The ddPCR custom assays allowed the identification of LGRs in all sample types, including FFPE specimens. Moreover, we were able to clearly detect LGRs accounted as somatic event. CONCLUSION: The introduction of ddPCR in a comprehensive workflow, encompassing both germline and somatic tests, represents an improvement in BRCA1/2 testing. ddPCR can overcome challenges related to BRCA testing, especially on FFPE analysis. Finally, ddPCR represents a promising alternative strategy to the established standard methods currently used in clinical setting.

5.
Mol Biol Rep ; 47(9): 7313-7316, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32808116

RESUMO

The multiple endocrine neoplasia type 1 (MEN1) is a rare syndrome characterized by the predisposition to developing multiple endocrine and non-endocrine tumors, typically characterized by the association between parathyroid gland hyperplasia or tumors, gastroenteropancreatic tumors and pituitary adenomas. The MEN1 gene is located on the long arm of chromosome 11 (11q13) and it encodes for the protein "menin". We here reported the case of a MEN1-patient, affected by primary hyperparathyroidism, insulinoma, pituitary non-hyperfunctioning adenoma and bilateral adrenal masses, carrying a novel heterozygous pathogenic variant (c.1252_1254delGACinsAT), located in exon 9 of MEN1 gene.

6.
Mol Diagn Ther ; 24(4): 473-485, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32607951

RESUMO

BACKGROUND: Generalized glucocorticoid resistance is characterized by impaired cortisol signaling, resulting from mutations in the NR3C1 gene coding the human glucocorticoid receptor (hGR). Most of the pathogenic hGR variants are identified in the ligand-binding domain (LBD). However, we report a new case associated with a novel NR3C1 pathogenic variant in the N-terminal domain (NTD) of the hGR. METHODS: The index case was an Italian 31-year-old woman with a history of chronic fatigue, anxiety, hirsutism, irregular menstrual cycles, and infertility, but no clinical manifestations suggestive of Cushing's syndrome. Standard clinical methods were used to assess the patient's hypothalamic-pituitary-adrenal axis. Molecular analysis of the NR3C1 gene was performed by Sanger sequencing. In addition, we perform an extensive survey of all clinical pathogenic variants modifying the whole sequence of the NR3C1 gene. RESULTS: Endocrinologic evaluation revealed elevated serum cortisol, plasma adrenocorticotropic hormone, and androstenedione concentration and increased urinary free cortisol excretion. Morning serum cortisol levels remained elevated and were not suppressed during a 2-day, 2-mg dexamethasone suppression test. The identification of the novel p.(Glu123Ter) NR3C1 variant confirmed the diagnosis of glucocorticoid resistance. CONCLUSION: Our findings improve the understanding of NR3C1 mutational spectrum in patients affected by glucocorticoid resistance syndrome and contribute to precise diagnosis and genetic counseling.

7.
Mol Biol Rep ; 47(6): 4857-4860, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32388698

RESUMO

The first person-to-person transmission of the 2019-novel coronavirus in Italy on 21 February 2020 led to an infection chain that represents one of the largest known COVID-19 outbreaks outside Asia. Hospitals have been forced to reorganized their units in response to prepare for an unforeseen healthcare emergency. In this context, our laboratory (Molecular and Genomic Diagnostics Unit, Fondazione Policlinico Universitario Agostino Gemelli IRCCS) re-modulated its priorities by temporarily interrupting most of the molecular tests guaranteeing only those considered "urgent" and not postponable. In particular, this paper details changes regarding the execution of germline BRCA (gBRCA) testing in our laboratory. A substantial reduction in gBRCA testing (about 60%) compared to the first 2 months of the current year was registered, but the requests have not been reset. The requesting physicians were mainly gynaecologists and oncologists. These evidences further emphasize the new era of gBRCA testing in the management of cancer patients and confirms definitively the integration of gBRCA testing/Next Generation Sequencing (NGS) into clinical oncology. Finally, a re-organization of gBRCA testing in our Unit, mainly related to delayed and reduced arrival of tests was necessary, ensuring, however, a high-quality standard and reliability, mandatory for gBRCA testing in a clinical setting.


Assuntos
Proteína BRCA2/genética , Neoplasias da Mama/diagnóstico , Infecções por Coronavirus/epidemiologia , Detecção Precoce de Câncer/estatística & dados numéricos , Neoplasias Ovarianas/diagnóstico , Pandemias , Pneumonia Viral/epidemiologia , Betacoronavirus/genética , Betacoronavirus/patogenicidade , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Técnicas de Laboratório Clínico/estatística & dados numéricos , Infecções por Coronavirus/sangue , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/patologia , Detecção Precoce de Câncer/métodos , Diagnóstico Precoce , Feminino , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/estatística & dados numéricos , Humanos , Itália/epidemiologia , Mutação , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Pneumonia Viral/sangue , Pneumonia Viral/diagnóstico , Pneumonia Viral/patologia , Encaminhamento e Consulta/estatística & dados numéricos
8.
Mol Biol Rep ; 47(4): 3049-3052, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32185686

RESUMO

RCCX haplotypes with two copies of the CYP21A2 gene and one copy of the CYP21A1P pseudogene have been widely described in different populations. In most cases, the CYP21A2-like gene downstream of the TNXA gene showed a wild-type sequence or the c.293-13A/C > G variant while the CYP21A2 gene next to TNXB carried the p.(Gln319Ter) variant. Here is the discovery of a novel rare CYP21A2 haplotypes detected in an Italian patient with Non Classical Congenital Adrenal Hyperplasia (NC-CAH). The molecular family study was performed clarifying the previously found phenotype-genotype discrepancy.

9.
Front Biosci (Landmark Ed) ; 25: 201-228, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31585886

RESUMO

Iodothyronine deiodinases are selenoproteins that regulate thyroid hormone metabolism. Of the three types of deiodinases, type 2 is the major regulator of intracellular triiodothyronine concentration in both the hypothalamus and pituitary, and therefore the major regulator of thyrotropin secretion. A defect in iodothyronine deiodinase activity can lead to a reduced sensitivity to thyroid hormones action and the most recent literature includes these defects in the so-called "syndromes of reduced sensitivity to thyroid hormones". To date, the pathogenic variants of the selenocysteine insertion sequence-binding protein 2 (SECISBP2) gene are the first and only inherited disorder of iodothyronine metabolism described. Moreover, there is a growing interest in understanding the possible role of polymorphisms of DIO1 and DIO2 genes in some pathological conditions and in determining the requirement of levothyroxine replacement and the role of combined levothyroxine-liothyronine therapy in carrying subjects affected by hypothyroidism and who need replacement therapy. Results on this topic are still conflicting and more studies are needed to assess the efficacy of combined levothyroxine-liothyronine replacement therapy in this subset of patients.

10.
Mol Biol Rep ; 47(2): 1513-1520, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31833030

RESUMO

Correct classification of genomic variants causing potentially aberrant splicing is of utmost importance for patient management, especially in clinically actionable genes such as BRCA1/2. In this article, we report molecular evaluation of the BRCA1 c.439T>C (rs794727800, p.Leu147=) variant based on RNA of a patient suffering with high-grade serous ovarian cancer syndrome, to add new evidence to the only in silico data available for this variant. High Resolution Melting Analysis (HRMA) was used for the first time to investigate the spliceogenicity of a BRCA1 variant. HRMA with Sanger sequencing provided evidence that the c.439C allele does not cause aberrant splicing of the BRCA1 exon 7. In addition, HRMA with Sanger highlighted a different expression of the naturally occurring BRCA1 r.442_444del (c.442_444delCAG, p.Gln148del, at DNA level) isoform between blood and tumor, in this patient. HRMA is an alternative molecular approach to analyze spliceogenic properties of the c.439T>C variant and potentially for all those BRCA1/2 variants affecting splicing sites. These new evidences allowed to classify definitively the c.439T>C variant as benign. Furthermore, the different BRCA1 r.442_444del expression opens the discussion to consider a wider classification criteria for the splicing variants, including molecular evaluation at tissue level, which is an aspect currently scarcely considered in BRCA1/2 variant classification recommendations.


Assuntos
Proteína BRCA1/genética , Desnaturação de Ácido Nucleico/genética , Polimorfismo de Nucleotídeo Único/genética , Processamento de RNA/genética , Idoso , Alelos , Proteína BRCA1/sangue , Sequência de Bases , DNA/genética , Feminino , Humanos
11.
Expert Rev Mol Diagn ; 19(9): 795-802, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31429350

RESUMO

Introduction: Currently, genetic testing of BRCA1/2 genes includes screening for single-nucleotide variants, small insertions/deletions, and copy number variations (CNVs). In fact, many studies document the involvement of BRCA1/2 gene rearrangements in genetic predisposition to breast and ovarian cancer. Large genomic rearrangements (LGRs) of BRCA1 may account for up to one-third of all disease-causing alterations in various populations, while LGRs in BRCA2 are less frequently observed. Areas covered: We aimed to present an overview of current technologies employed in molecular diagnosis of BRCA1/2 LGRs. The most relevant literature papers, showing the application of new strategies, were considered. Expert opinion: Currently, the progress of next-generation sequencing (NGS) technologies allows for the validation of new pipelines able to provide rapid and effective results, ensuring the sensitivity and specificity requested for the detection of BRCA1/2 LGRs. Multiplex ligation-dependent probe amplification remains the gold standard to confirm NGS CNVs results and to perform fast screening in families where a pathogenic rearrangement has been detected in a proband.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/genética , Técnicas de Diagnóstico Molecular/métodos , Neoplasias Ovarianas/genética , Feminino , Genes BRCA1 , Genes BRCA2 , Predisposição Genética para Doença/genética , Testes Genéticos/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos
12.
Int J Mol Sci ; 20(14)2019 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-31336956

RESUMO

The aim of this report is to describe results of BRCA1 and BRCA2 Next Generation Sequencing Analysis (NGS) analysis in 132 selected Italian patients with breast/ovarian cancer. A NGS pipeline with a reliable Copy Number Variation (CNV) prediction algorithm was applied. In addition, VarSome and Priors V2.0 Software were employed for in silico analysis of novel missense variants. A total of 37 BRCA1 and BRCA2 pathogenic variants were found in 34 unrelated subjects with a frequency of positive patients of 25.7% (34/132). Twenty-four deleterious variants were detected in BRCA1 (representing the 64.9% of all identified pathogenic defects) and thirteen (35.1% of all identified pathogenic variants) in BRCA2 gene. The percentage of patients carrying a variant of unknown significance (VUS) was 7.5% (10/132). In addition, seven novel variants (five in BRCA2 and two in BRCA1 gene), never previously reported, were identified. Our approach represents a robust and easy-to-use method for full BRCA1/2 screening. However, a consistent number of our high-risk families still remained without a satisfying answer. Necessarily, further collective efforts must be directed to a definitive classification of VUSs. The future auspice is that the use of multi-gene panel and more advanced screenings, such as whole exome sequencing and/or RNA seq, in routine diagnostics increases the detection rate.


Assuntos
Neoplasias da Mama/genética , Genes BRCA1 , Genes BRCA2 , Estudos de Associação Genética , Predisposição Genética para Doença , Sequenciamento de Nucleotídeos em Larga Escala , Neoplasias Ovarianas/genética , Adulto , Idoso , Alelos , Substituição de Aminoácidos , Biomarcadores Tumorais , Neoplasias da Mama/diagnóstico , Estudos de Coortes , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética/métodos , Testes Genéticos , Genótipo , Mutação em Linhagem Germinativa , Humanos , Itália , Pessoa de Meia-Idade , Neoplasias Ovarianas/diagnóstico , Linhagem
13.
Mol Diagn Ther ; 23(5): 563-567, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31317337

RESUMO

More than 95% of congenital adrenal hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the human leukocyte antigen (HLA) class III area on the short arm of chromosome 6p21.3. In the diagnosis of 21-hydroxylase deficiency, CYP21A2 genotyping is a valuable complement to biochemical investigations. Genotyping can confirm the diagnosis (or carrier state) and, at the same time, provide accurate phenotype prediction in patients carrying severe mutations. In addition, the use of genetic testing is also helpful in prenatal diagnosis where the goal of prenatal treatment is preventing genital virilization of the female fetus. An in-depth knowledge of CYP21A2 genetics is essential to assure the correct interpretation of results obtained. To date, more than 200 small pathogenic variants of the CYP21A2 gene have been reported, showing good agreement between clinical phenotype and patient genotype. Recently, novel CYP21A2 deletions, involving one or more exons, have been reported in different populations. Since these rearrangements have never been described before in the genetic history of 21-hydroxylase deficiency, these new deletions have aroused particular interest. However, it is possible that these novel rearrangements are the result of incorrect interpretation of multiplex ligation-dependent probe amplification (MLPA).


Assuntos
Hiperplasia Suprarrenal Congênita/diagnóstico , Hiperplasia Suprarrenal Congênita/genética , Rearranjo Gênico , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Genômica , Estudos de Associação Genética/métodos , Testes Genéticos , Genômica/métodos , Humanos , Técnicas de Diagnóstico Molecular
14.
Artigo em Inglês | MEDLINE | ID: mdl-31249555

RESUMO

Introduction: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominantly inherited endocrine tumor syndrome characterized by the development of cancer in various endocrine organs, particularly in the pituitary, parathyroid and pancreas. Moreover, in some cases, also non-endocrine tumors can be diagnosed, developing atypical phenotypes. Case report: We report herein the clinical history of a patient affected by MEN-1 syndrome who developed atypical features for this disease. The patient's clinical history started in August 2015 when he was referred, at the age of 23 years, to the Emergency Department of our Hospital for the occurrence of progressive asthenia, weakness, tremors and syncope. The biochemical test documented hyper-calcemia and severe hypoglycemia. The patient was referred to our Neuroendocrine Tumor and Pituitary Unit and he was diagnosed with pancreatic insulinoma, hypercalcemic hyperparathyroidism, and a prolactin secreting pituitary adenoma. The MEN-1 syndrome was suspected and genetic tests for mutation of menin resulted positive for the pathogenic variant c1548dupG. In January 2016, the patient was diagnosed with intratubular germ cell neoplasia, consisting of a mature teratoma and yolk sac tumor and he underwent a right orchiectomy. Conclusion: This is the first case report showing the clear association of MEN-1 syndrome with yolk sac tumors and teratomas, as in our case, the c1548dupG represents a pathogenic variant rather than a SNP. This case suggests the opportunity of an accurate evaluation of the testis particularly in young MEN-1 affected patients and that a prompt screening for neoplastic disease should involve all the endocrine glands.

15.
Hum Mutat ; 40(9): 1557-1578, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31131967

RESUMO

The multifactorial likelihood analysis method has demonstrated utility for quantitative assessment of variant pathogenicity for multiple cancer syndrome genes. Independent data types currently incorporated in the model for assessing BRCA1 and BRCA2 variants include clinically calibrated prior probability of pathogenicity based on variant location and bioinformatic prediction of variant effect, co-segregation, family cancer history profile, co-occurrence with a pathogenic variant in the same gene, breast tumor pathology, and case-control information. Research and clinical data for multifactorial likelihood analysis were collated for 1,395 BRCA1/2 predominantly intronic and missense variants, enabling classification based on posterior probability of pathogenicity for 734 variants: 447 variants were classified as (likely) benign, and 94 as (likely) pathogenic; and 248 classifications were new or considerably altered relative to ClinVar submissions. Classifications were compared with information not yet included in the likelihood model, and evidence strengths aligned to those recommended for ACMG/AMP classification codes. Altered mRNA splicing or function relative to known nonpathogenic variant controls were moderately to strongly predictive of variant pathogenicity. Variant absence in population datasets provided supporting evidence for variant pathogenicity. These findings have direct relevance for BRCA1 and BRCA2 variant evaluation, and justify the need for gene-specific calibration of evidence types used for variant classification.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Biologia Computacional/métodos , Mutação de Sentido Incorreto , Neoplasias/diagnóstico , Processamento Alternativo , Detecção Precoce de Câncer , Feminino , Predisposição Genética para Doença , Humanos , Funções Verossimilhança , Masculino , Herança Multifatorial , Neoplasias/genética
16.
Mol Diagn Ther ; 23(3): 353-368, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30976996

RESUMO

Resistance to thyroid hormone beta (RTHß) is a syndrome characterized by reduced responsiveness of peripheral tissues to thyroid hormone (TH). In most cases, the disorder is associated with germline pathogenic variants in the thyroid hormone receptor beta (THRB) gene. This paper summarizes the clinical and biochemical presentation of the disease, providing a comprehensive overview on molecular genetic features. Particular care is given in reporting all identified THRB variants with an assessed or unknown clinical significance. Our aim is to offer a useful tool for clinical and genetic specialists in order to ease clinical diagnosis and genetic counseling.


Assuntos
Predisposição Genética para Doença , Mutação , Receptores beta dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/metabolismo , Síndrome da Resistência aos Hormônios Tireóideos/genética , Síndrome da Resistência aos Hormônios Tireóideos/metabolismo , Hormônios Tireóideos/metabolismo , Animais , Estudos de Associação Genética , Variação Genética , Genótipo , Humanos , Fenótipo , Síndrome da Resistência aos Hormônios Tireóideos/diagnóstico
17.
Mol Diagn Ther ; 23(1): 121-126, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30506513

RESUMO

BACKGROUND: In recent years, the number of patients being offered BRCA1/2 testing has changed dramatically. Advances in high-throughput sequencing technology have led many diagnostic laboratories to test next-generation sequencing (NGS)-based platforms as the main technology for clinical testing. As a consequence, the proportion of novel BRCA1/2 variants detected has greatly increased. Here, we describe two novel BRCA1 large deletions detected in Italian patients affected by hereditary breast and ovarian cancer syndrome (HBOC). METHODS: We applied an NGS pipeline with a reliable copy number variation (CNV) prediction algorithm. Successively, samples were investigated using the Multiplex Amplicon Quantification (MAQ) assay and array comparative genomic hybridization (CGH). In a single case, long-range polymerase chain reaction (PCR) was employed for careful detection of the breakpoint region, while the RepeatMasker program was used to identify Alu sequences at the junction point. RESULTS: A 137.8 kb deletion, involving the first six exons of BRCA1 and the full NBR2, BRCA1P1, NBR1, and TMEM106a genes, was detected in an Italian woman diagnosed with high-grade serous ovarian carcinoma. A second rearrangement, involving the deletion of BRCA1 11-14 exons, was detected in a breast cancer patient and was fully characterized and reported according to recommended Human Genome Variation Society (HGVS) nomenclature: NG_005905.2: g.125038_143266del; NM_007294.3: c.2817_4716del; NP_009225: p.Lys862Metfs? CONCLUSION: Although it was not possible to perform a familial segregation analysis and more direct evidence of the relationship between genotype and phenotype is necessary, both of the novel reported rearrangements cause the loss of crucial functional domains of the BRCA1 protein and this event supports their pathogenicity.


Assuntos
Proteína BRCA1/genética , Neoplasias da Mama/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , Neoplasias Ovarianas/genética , Idoso , Neoplasias da Mama/patologia , Feminino , Rearranjo Gênico/genética , Predisposição Genética para Doença , Genoma Humano/genética , Genômica/métodos , Síndrome Hereditária de Câncer de Mama e Ovário/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Itália , Pessoa de Meia-Idade
18.
Clin Biochem ; 63: 54-58, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30315757

RESUMO

OBJECTIVES: In silico splicing analysis, a mini-gene assay and splicing data, obtained using RNA from blood samples, have shown that the BRCA1 c.5332G > A variant induces exon 21 skipping. However, despite these evidences, up to date, this variant is unclassified. The aim of this study is to provide further molecular and clinical evidence for the BRCA1 c.5332G > A variant in a patient with high grade serous ovarian carcinoma (HGSOC) to allow a definitive classification of this variant. DESIGN AND METHOD: The effect of the BRCA1 c.5332G > A variant on RNA splicing was evaluated by amplifying regions of BRCA1 from the cDNA of the patient. Loss of heterozygosity (LOH) in tumor tissue was also investigated. RESULTS: The c.5332G > A allele causes significantly aberrant splicing of the BRCA1 exon 21. Evaluation of the c.5332A allele in tumor tissue highlights a possible loss of heterozygosity, supporting her pathogenic effect. CONCLUSIONS: Our results regarding the c.5332G > A variant confirm that it contributed to predisposition and onset of ovarian carcinoma in the patient. We propose to classify this variant as 'likely-pathogenic' (class IV).


Assuntos
Alelos , Proteína BRCA1 , Predisposição Genética para Doença , Perda de Heterozigosidade , Mutação de Sentido Incorreto , Neoplasias Ovarianas , Processamento de RNA , Substituição de Aminoácidos , Proteína BRCA1/classificação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Feminino , Humanos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia
19.
Hormones (Athens) ; 17(3): 427-435, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30083881

RESUMO

CONTEXT: Clinical phenotype variability in MEN1 syndrome exists and evidence for an established genotype-phenotype is lacking. However, a higher aggressiveness of MEN1-associated gastro-entero-pancreatic (GEP) (neuro)endocrine tumours (NETs) tumours has been reported when MEN1 gene truncating mutations are detected. We found a novel germline truncating mutation of MEN1 gene at exon 10 in a subject with an aggressive clinical behavior of GEP-NETs. Successively, other two mutant-affected familial members have been identified. OBJECTIVE: The aim of this observational study was to investigate genotype-phenotype correlation in these three members, with attention to GPE-NETs behavior over the years. DESIGN: The genetic and clinical data obtained and the follow-up screening program (2012-2016) were according to the International Guidelines in a multidisciplinary academic reference center. The familial history collected strongly suggested MEN1 GEP-NETs in at least other four members from different generations. PATIENTS: Three MEN1 patients (aged 30-69 years at MEN1 diagnosis) were clinically screened for MEN1 GEP-NETs, both functioning and nonfunctioning. METHODS: Biochemical, imaging, and nuclear medicine tests and fine-needle agobiopsy were performed, depending on found/emerging clinical symptoms/biochemical abnormalities, and made when necessary. RESULTS: Our clinical survey found strong genotype-phenotype correlation with aggressive MEN1 GEP-NETs (G1, G2-NETs, and multiple ZES/gastrinomas) over the years. The familial history strongly suggested ZES/gastrinoma in progenitors from previous generations. CONCLUSIONS: This novel MEN1 truncating mutation correlates with an aggressive evolution and behavior of MEN1 GEP-NETs in studied affected subjects, confirming the need for MEN1 individuals to be evaluated by a skilled multidisciplinary team, as also stated by International Guidelines.


Assuntos
Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/genética , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas/genética , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Adulto , Idoso , Genótipo , Mutação em Linhagem Germinativa , Humanos , Itália , Masculino , Linhagem , Fenótipo , Gêmeos Monozigóticos
20.
Case Rep Endocrinol ; 2018: 2086861, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29854486

RESUMO

Context: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease due to specific enzyme deficiencies in the adrenal steroidogenesis pathway. Case Description: A 40-year-old Chinese woman was referred to the Endocrine Unit for the work-up of a syndrome characterized by long-lasting and multidrug resistant high blood pressure, severe hypokalemia with metabolic alkalosis, and primary amenorrhea. The patient presented with sexual infantilism, lack of breast development, absence of axillary and pubic hair, tall stature, and slenderness. CT scan revealed enlarged adrenal glands bilaterally and the absence of the uterus, the ovaries, and the Fallopian tubes. Furthermore, diffuse osteopenia and osteoporosis and incomplete ossification of the growth plate cartilages were demonstrated. Chromosomal analysis showed a normal male 46,XY, karyotype, and on molecular analysis of the CYP17A1 gene she resulted homozygous for the g.4869T>A; g.4871delC (p.Y329Kfs?) mutation in exon 6. Hydrocortisone and ethinyl-estradiol supplementation therapy led to incomplete withdrawal of antihypertensive drug and breast development progression to Tanner stage B2 and slight height increase, respectively. Conclusions: We describe a late-discovered case of CAH with 46,XY disorder of sex development. Deficiency of 17α-hydroxylase/17,20-lyase due to a homozygous CYP17A1 gene mutation was the underlying cause. Laboratory, imaging, and genetic features are herein reported and discussed.

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