A Novel Pathogenic Variant in the N-Terminal Domain of the Glucocorticoid Receptor, Causing Glucocorticoid Resistance.

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.

BRCA1 and BRCA2 Testing through Next Generation Sequencing in a Small Cohort of Italian Breast/Ovarian Cancer Patients: Novel Pathogenic and Unknown Clinical Significance Variants.

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.

Mutational Landscape of Resistance to Thyroid Hormone Beta (RTHß).

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.

High-resolution melting analysis coupled with next-generation sequencing as a simple tool for the identification of a novel somatic BRCA2 variant: a case report.

In a 72-year-old woman with no associated personal or family history of breast and/or ovarian cancers, we identified a novel somatic pathogenic BRCA2 variant (c.18_28delAGAGAGGCCAA, p.Lys6Asnfs*4) using next-generation sequencing (NGS). The variant allele frequency (VAF) was 16%, and Sanger sequencing was unable to identify this variant. Adopting a high-resolution melting analysis strategy coupled with NGS, we successfully highlighted the presence of the c.18_28delAGAGAGGCCAA allele.

Preliminary molecular evidence associating a novel BRCA1 synonymous variant with hereditary ovarian cancer syndrome.

Extensive molecular screening of the BRCA1/2 (BRCA) genes by massively parallel sequencing (MPS) identified variants of uncertain (or unknown) significance (VUS) and novel variants. We performed a molecular characterization of a novel BRCA1 synonymous variant discovered in a family with hereditary ovarian cancer (HOC) syndrome. We showed that the BRCA1 c.5073 A > T variant might play a pathogenic role in HOC syndrome in this family.

Congenital Adrenal Hyperplasia (CAH) due to 21-Hydroxylase Deficiency: A Comprehensive Focus on 233 Pathogenic Variants of CYP21A2 Gene.

Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by complete or partial defects in one of the several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 95-99% of all cases of CAH are caused by deficiency of steroid 21-hydroxylase, an enzyme encoded by the CYP21A2 gene. Currently, CYP21A2 genotyping is considered a valuable complement to biochemical investigations in the diagnosis of 21-hydroxylase deficiency. More than 200 mutations have been described in literature reports, and much energy is still focused on the clinical classification of new variants. In this review, we focus on molecular genetic features of 21-hydroxylase deficiency, performing an extensive survey of all clinical pathogenic variants modifying the whole sequence of the CYP21A2 gene. Our aim is to offer a very useful tool for clinical and genetic specialists in order to ease clinical diagnosis and genetic counseling.

Identification and Characterization of a New BRCA2 Rearrangement in an Italian Family with Hereditary Breast and Ovarian Cancer Syndrome.

INTRODUCTION: Many studies document the involvement of BRCA1/2 gene rearrangements in genetic predisposition to breast and ovarian cancer. Large genomic rearrangements (LGRs) of BRCA1 account for 0-27% of all disease-causing mutations in various populations, while LGRs in BRCA2 are rarer. Here, we describe a novel BRCA2 LGR, involving the duplication of exons 4-26, in an Italian family with hereditary breast and ovarian cancer (HBOC) syndrome. OBJECTIVE: Our purpose was to provide an effective characterization of this variant using a combination of different methods able to establish the exact breakpoints of the duplication. METHODS: A multiplex amplicon quantification (MAQ) assay was used as the primary screening method in the detection of LGRs. Array comparative genomic hybridization (CGH), reverse transcriptase polymerase chain reaction (RT-PCR) and long-range PCR were used for the careful characterization of the rearrangement and breakpoint regions. The Repeat Masker program was employed to identify Alu sequences at breakpoint junctions. RESULTS: Array CGH and long-range PCR strategies revealed that the BRCA2 exons 4-26 duplication (g.12016_87170dup) involved exactly 75,154 bp nucleotides between intron 3 and intron 26 of the gene. Given that no Alu repeats were found at the junction sites, we support the hypothesis that the new duplication could be the result of a microhomology-mediated event (MH) involving very short homologous sequences at an upstream breakpoint. DISCUSSION: LGR investigation is mandatory in BRCA1/2 routine testing in order to provide a complete result for a targeted therapeutic decision. Nevertheless, the characterization and classification of novel BRCA1/2 variants represents a crucial step in the support of genetic counselling. Our results, including a comprehensive co-segregation analysis, indicate that the novel duplication identifed has a pathogenic role and would be considered a causing-disease variant in genetic and oncologic counselling.

CYP21A2 intronic variants causing 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase gene (CYP21A2). Most of CYP21A2 mutations result from intergenic recombinations between CYP21A2 and closely linked CYP21A1P pseudogene. Rare mutations not generated by gene conversion account for 5-10% of 21-hydroxylase deficiency alleles. Intronic variants represent only a little part of these but their effect on the protein is generally deleterious. The aim of this paper is to provide a comprehensive literary review regarding all intronic CYP21A2 pathological variants reported to date. In addition, we describe three novel causing disease variants in our patients affected by the classic form of CAH: IVS4-1G>A, IVS5-8T>A, IVS8-2A>G. In silico analysis revealed that all these substitutions affect the splicing process leading to a non-functional protein. Based on these results, we are able to classify them as pathological variants according to the patient's phenotype.

Multiple endocrine neoplasia type 1 (MEN1): An update of 208 new germline variants reported in the last nine years.

This review will focus on the germline MEN1 mutations that have been reported in patients with MEN1 and other hereditary endocrine disorders from 2007 to September 2015. A comprehensive review regarding the analysis of 1336 MEN1 mutations reported in the first decade following the gene's identification was performed by Lemos and Thakker in 2008. No other similar papers are available in literature apart from these data. We also checked for the list of Locus-Specific DataBases (LSDBs) and we found five MEN1 free-online mutational databases. 151 articles from the NCBI PubMed literature database were read and evaluated and a total of 75 MEN1 variants were found. On the contrary, 67, 22 and 44 novel MEN1 variants were obtained from ClinVar, MEN1 at Café Variome and HGMD (The Human Gene Mutation Database) databases respectively. A final careful analysis of MEN1 mutations affecting the coding region was performed.

Clinical impact on ovarian cancer patients of massive parallel sequencing for BRCA mutation detection: the experience at Gemelli hospital and a literature review.

OBJECTIVE: Massive parallel sequencing (MPS) is the new frontier for molecular diagnostics. Twenty-four papers regarding BRCA analysis were considered for reviewing all pipelines evaluated in this field. METHODS: Proposed here is an integrated MPS workflow able to successfully identify BRCA1/2 mutational status on 212 Italian ovarian cancer patients. The review of literature data is reported. RESULT: The pipeline can be routinely used as robust molecular diagnostic strategy, being highly sensitive and specific. CONCLUSION: Literature data report that efforts are being made in order to fully translate MPS-based BRCA1/2 gene assay into routine clinical diagnostics. However, this study highlights the need of an integrated MPS BRCA1/2 molecular workflow fulfilling the standardized requirements needed in the routine clinical laboratory practice.

A preliminary Quality Control (QC) for next generation sequencing (NGS) library evaluation turns out to be a very useful tool for a rapid detection of BRCA1/2 deleterious mutations.

BACKGROUND: Recent advances in next generation sequencing (NGS) technology have enabled comprehensive and accurate screening of the entire genomic region of BRCA1/2 genes and, to date, many studies report the effectiveness of these technologies. Here we show that Gene Scan (GS) labeling Quality Control (QC), performed before massive parallel pyrosequencing, coupled with Multiple Amplicon Quantification software (MAQ-S) analysis is a rapid and powerful tool in the detection of deleterious BRCA mutations carried by different patients. METHODS: GS labeling QC assay was performed according to the manufacturers' instructions and MAQ-S software was employed for analysis results. RESULTS: GS labeling QC was able to detect 14 different BRCA frameshift mutations in our patients. In addition, two novel BRCA mutations (c.1893_1894insTTAAGCCCACAAAT in BRCA1 gene and c.9413_9414insT in BRCA2 gene) were identified. CONCLUSION: We prove that a simple QC step may represent a valid and useful tool for a rapid detection of frameshift mutations in BRCA genes. For this reason, we recommend using this approach before massive parallel sequencing.