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.

Protein kinase CK2 potentiates translation efficiency by phosphorylating eIF3j at Ser127.

In eukaryotic protein synthesis the translation initiation factor 3 (eIF3) is a key player in the recruitment and assembly of the translation initiation machinery. Mammalian eIF3 consists of 13 subunits, including the loosely associated eIF3j subunit that plays a stabilizing role in the eIF3 complex formation and interaction with the 40S ribosomal subunit. By means of both co-immunoprecipitation and mass spectrometry analyses we demonstrate that the protein kinase CK2 interacts with and phosphorylates eIF3j at Ser127. Inhibition of CK2 activity by CX-4945 or down-regulation of the expression of CK2 catalytic subunit by siRNA cause the dissociation of j-subunit from the eIF3 complex as judged from glycerol gradient sedimentation. This finding proves that CK2-phosphorylation of eIF3j is a prerequisite for its association with the eIF3 complex. Expression of Ser127Ala-eIF3j mutant impairs both the interaction of mutated j-subunit with the other eIF3 subunits and the overall protein synthesis. Taken together our data demonstrate that CK2-phosphorylation of eIF3j at Ser127 promotes the assembly of the eIF3 complex, a crucial step in the activation of the translation initiation machinery.

Genetic instability of the tumor suppressor gene FHIT in normal human cells.

Common fragile sites are hotspots for chromosome instability and co-localize to cancer genomic rearrangements. Whether these loci may be considered stable in human subjects under physiological conditions remains an open question. Here we show by molecular combing that a small but significant percentage of normal human cells carry an abnormal sequence pattern within the tumor suppressor gene FHIT (3p14.2) at FRA3B. Each sequence variation represents a unique pattern within a normal cell population, and therefore it would remain undetected or not interpreted by genome-wide analyses. Remarkably, the region is the same as in FHIT rearrangements described in tumors. By analyses on several normal cell lines (proliferating and resting primary lymphocytes, primary fibroblasts, lymphoblastoid cells including clonal cell cultures) we verified that: (a) each cell type displays altered sequence patterns at FHIT; (b) the presence of abnormal sequence patterns is specific for the FHIT locus; and (c) FHIT instability occurs de novo during cell proliferation, and heterogeneous sequence variants progressively accumulate in the cell populations. FHIT has been widely investigated in cancer cells, but to our knowledge this is the first direct evidence of spontaneous and recurrent occurrence of genomic instability at this gene in human subjects, at the same region involved in cancer rearrangements. Our results suggest that common fragile site activity is not restricted to in vitro cell culture and that genomic instability may pre-exist in normal cells in the absence of exogenous replication stress.

Atypical cancer risk profile in carriers of Italian founder BRCA1 variant p.His1673del: Implications for classification and clinical management.

BACKGROUND: BRCA1:c.5017_5019del (p.His1673del) is a founder variant relatively frequent in Northern Italy. Despite previous suggestion of pathogenicity, variant classification in public databases is still conflicting, needing additional evidence. METHODS: Maximum likelihood penetrance of breast/ovarian and other cancer types was estimated using full pedigree data from 53 informative Italian families. The effect of the variant on BRCA1-ABRAXAS1 interaction was assessed using a GFP-fragment reassembly-based PPI assay. Results were combined with additional data from multiple sources to classify the variant according to ACMG/AMP classification rules specified for BRCA1/2. RESULTS: Variant-carriers displayed increased risk for ovarian cancer (HR = 33.0, 95% CI = 7.0-155.0; cumulative risk at age 70 = 27.6%, 95% CI = 12.6-40.0%) but not for breast cancer (HR = 0.7, 95% CI = 0.2-2.2). An increased risk of uterine cancer (HR = 8.0, 95% CI = 1.03-61.6) emerged, warranting further evaluation. Likelihood-ratio in favor of pathogenicity was 98898642.82 under assumption of standard BRCA1 breast and ovarian penetrance, and 104240832.84 after excluding breast cancer diagnoses (based on penetrance results). Functional analysis demonstrated that the variant abrogates the BRCA1-ABRAXAS1 binding, supporting the PS3 code assignment within the ACMG/AMP rule-based model. Collectively, these findings allowed to classify the variant as pathogenic. CONCLUSION: Pathogenicity of BRCA1:c.5017_5019del(p.His1673del) has been confirmed; however, breast cancer risk in Italian families is not increased, unlike in families from other countries and in carriers of most BRCA1 pathogenic variants. The knowledge of atypical risk profiles for this and other variants will pave the way for personalized management based on specific genotype.

Replication dynamics at common fragile site FRA6E.

The replication dynamics at common fragile site FRA6E has been evaluated by molecular combing and interphase fluorescent in situ hybridisation (FISH) in primary human lymphocytes cultured under normal or aphidicolin-induced stress conditions. FRA6E is one of the most frequently expressed common fragile sites of the human genome. It harbours several genes, PARK2 being regarded as the most relevant one. According to the results obtained from interphase FISH analysis, FRA6E can be considered a mid-late-replicating sequence characterised by heterogeneous replication timing. Molecular combing did not reveal specific replication parameters at the fragile site: fork rates were highly comparable to those detected at an early replicating locus (LMNB2) used as control and in very good agreement with the whole-genome data obtained in parallel. The same indication applied to the density of initiation zones, the inter-origin distances from adjacent ongoing forks, the frequencies of unidirectional forks, fork arrest events and asynchronous forks. Interestingly, PARK2 appeared embedded in an early/late replication transition zone, corresponding to intron 8 (162 kb) and to the fragility core of FRA6E. In cells exposed to aphidicolin, few forks progressing at a rather slow rate were observed, the majority of them being unidirectional, but again a specific response of the fragile site was not observed. In summary, at FRA6E the replication process is not impaired per se, but chromosome breakages occur preferentially at an early/late replication transition zone. Aphidicolin might increase the occurrence of breakage events at FRA6E by prolonging the time interval separating the replication of early and late replication domains. These results may be of general significance to address the problem of fragile site instability.

Segregation analysis of the BRCA2 c.9227G>T variant in multiple families suggests a pathogenic role in breast and ovarian cancer predisposition.

Classification of variants in the BRCA1 and BRCA2 genes has a major impact on the clinical management of subjects at high risk for breast and ovarian cancer. The identification of a pathogenic variant allows for early detection/prevention strategies in healthy carriers as well as targeted treatments in patients affected by BRCA-associated tumors. The BRCA2 c.9227G>T p.(Gly3076Val) variant recurs in families from Northeast Italy and is rarely reported in international databases. This variant substitutes the evolutionary invariant glycine 3076 with a valine in the DNA binding domain of the BRCA2 protein, thus suggesting a high probability of pathogenicity. We analysed clinical and genealogic data of carriers from 15 breast/ovarian cancer families in whom no other pathogenic variants were detected. The variant was shown to co-segregate with breast and ovarian cancer in the most informative families. Combined segregation data led to a likelihood ratio of 81,527:1 of pathogenicity vs. neutrality. We conclude that c.9227G>T is a BRCA2 pathogenic variant that recurs in Northeast Italy. It can now be safely used for the predictive testing of healthy family members to guide preventive surgery and/or early tumor detection strategies, as well as for PARP inhibitors treatments in patients with BRCA2-associated tumors.

Candidate genetic modifiers for breast and ovarian cancer risk in BRCA1 and BRCA2 mutation carriers.

BACKGROUND: BRCA1 and BRCA2 mutation carriers are at substantially increased risk for developing breast and ovarian cancer. The incomplete penetrance coupled with the variable age at diagnosis in carriers of the same mutation suggests the existence of genetic and nongenetic modifying factors. In this study, we evaluated the putative role of variants in many candidate modifier genes. METHODS: Genotyping data from 15,252 BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n = 3,248) located within or around 445 candidate genes, were available through the iCOGS custom-designed array. Breast and ovarian cancer association analysis was performed within a retrospective cohort approach. RESULTS: The observed P values of association ranged between 0.005 and 1.000. None of the variants was significantly associated with breast or ovarian cancer risk in either BRCA1 or BRCA2 mutation carriers, after multiple testing adjustments. CONCLUSION: There is little evidence that any of the evaluated candidate variants act as modifiers of breast and/or ovarian cancer risk in BRCA1 or BRCA2 mutation carriers. IMPACT: Genome-wide association studies have been more successful at identifying genetic modifiers of BRCA1/2 penetrance than candidate gene studies.