Simona Agata

Characterization of BRCA1 and BRCA2 splicing variants: A collaborative report by ENIGMA consortium members

Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.

FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor

Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer.

DNA copy number alterations correlate with survival of esophageal adenocarcinoma patients

Despite recent advances in surgical and multidisciplinary treatment, prognosis for patients with esophageal adenocarcinoma remains poor, and the low prognostic significance of pTNM staging suggests that additional parameters are needed. To identify genomic abnormalities characteristic of esophageal adenocarcinoma, a panel of 33 samples obtained at surgery from previously untreated patients were analyzed by muliplex ligation-dependent probe amplification technique. We detected frequent gains of 6p, 8q, 13q, 17q, 20q, and losses of 4q, 5q, 15q, and 18q. When DNA copy number changes were correlated to clinicopathological features of patients no association was found between the number of chromosomal aberrations and gender, age, tumor grade or pTNM staging. However, interestingly, a significant correlation between patient survival and total number of chromosomal aberrations was found when esophageal adenocarcinoma cases were stratified according to the median of survival (20 months) (P=0.002) or the median of aberrations (12 aberrations) (P=0.014). Evaluation of the distribution of gains and losses at the level of single chromosomes indicated that gains on chromosomes 5, 6, 8, 11, 20 and losses on chromosomes 1, 3, 5, 11, and 18 were significantly different in the two survival groups. Furthermore, when single gene imbalances were analyzed in further details, we found that besides alterations that involve genes shared by both survival groups, a few genes (KIAA0170, EMS1, ABCC4, F3, and MIF) were altered only in samples from patients with poor survival. Thus, we established a good correlation between the total number of chromosomal alterations and survival, suggesting that the estimation of total imbalances might represent an additional indicator of disease outcome. In addition, the finding of alterations specific for the more aggressive esophageal adenocarcinoma subset might represent promising biomarkers to increase the accuracy of clinical outcome prediction.

CDKN2A Unclassified Variants in Familial Malignant Melanoma: Combining Functional and Computational Approaches for Their Assessment

CDKN2A codes for two oncosuppressors by alternative splicing of two first exons: p16INK4a and p14ARF. Germline mutations are found in about 40% of melanoma‐prone families, and most of them are missense mutations mainly affecting p16INK4a. A growing number of p16INK4a variants of uncertain significance (VUS) are being identified but, unless their pathogenic role can be demonstrated, they cannot be used for identification of carriers at risk. Predicting the effect of these VUS by either a “standard” in silico approach, or functional tests alone, is rather difficult. Here, we report a protocol for the assessment of any p16INK4a VUS, which combines experimental and computational tools in an integrated approach. We analyzed p16INK4a VUS from melanoma patients as well as variants derived through permutation of conserved p16INK4a amino acids. Variants were expressed in a p16INK4a‐null cell line (U2‐OS) and tested for their ability to block proliferation. In parallel, these VUS underwent in silico prediction analysis and molecular dynamics simulations. Evaluation of in silico and functional data disclosed a high agreement for 15/16 missense mutations, suggesting that this approach could represent a pilot study for the definition of a protocol applicable to VUS in general, involved in other diseases, as well.

Functional impairment of p16INK4A due to CDKN2A p.Gly23Asp missense mutation

The CDKN2A locus encodes for two distinct tumor suppressor proteins, p16(INK4A) and p14(ARF), involved in cell cycle regulation. CDKN2A germline mutations have been associated with familial predisposition to melanoma and other tumor types. Besides bona-fide pathogenic mutations, many sequence variants have been identified, but their effect is not well known. We detected the p.Gly23Asp missense mutation in one of the two tested melanoma patients of a family with three melanoma cases. Even though the mutated amino acid is located in a conserved domain that specifically binds to and blocks the function of CDK4/6, its lack of segregation with disease suggested a series of functional assays to discriminate between a pathogenic variant and a neutral polymorphism. The effect of this mutation has been investigated exploiting four p16(INK4A) properties: its ability (i) to bind CDK4, (ii) to inhibit pRb phosphorylation, (iii) to evenly localize in the cell, and (iv) to cause cell cycle arrest. The mutant protein properties were evaluated transfecting three different cell lines (U2-OS and NM-39, both p16-null, and SaOS 2, p53 and pRb-null) with plasmids expressing either p16(wt), p16(23Asp), or the p16(32Pro) pathogenic variant. We found that p16(23Asp) was less efficient than p16(wt) in CDK4 binding, in inhibiting pRb phosphorylation, in inducing G1 cell cycle arrest; moreover, its pattern of distribution throughout the cell was suggestive of protein aggregation, thus assessing a pathogenic role for p16(23Asp) in familial melanoma.

Contribution of susceptibility gene variants to melanoma risk in families from the Veneto region of Italy

to take out a personal subscription, please click here More information about Pigment Cell & Melanoma Research at www.pigment.org Contribution of susceptibility gene variants to melanoma risk in families from the Veneto region of italy Chiara menin, antonella Vecchiato, maria Chiara scaini, lisa Elefanti, gloria Funari, gian luca de salvo, monica Quaggio, silvia tognazzo, simona agata, silvia dalla santa, marco montagna, mauro alaibac, Vanna Chiarion-sileni6 and Emma d’andrea

Whole-exome sequencing and targeted gene sequencing provide insights into the role of PALB2 as a male breast cancer susceptibility gene

Male breast cancer (MBC) is a rare disease whose etiology appears to be largely associated with genetic factors. BRCA1 and BRCA2 mutations account for about 10% of all MBC cases. Thus, a fraction of MBC cases are expected to be due to genetic factors not yet identified. To further explain the genetic susceptibility for MBC, whole‐exome sequencing (WES) and targeted gene sequencing were applied to high‐risk, BRCA1/2 mutation–negative MBC cases.