The BRCA1 c.4096+1G>A Is a Founder Variant Which Originated in Ancient Times.

Approximately 30-50% of hereditary breast and ovarian cancer (HBOC) is due to the presence of germline pathogenic variants in the BRCA1 (OMIM 113705) and BRCA2 (OMIM 600185) onco-suppressor genes, which are involved in DNA damage response. Women who carry pathogenic BRCA1 variants are particularly likely to develop breast cancer (BC) and ovarian cancer (OC), with a 45-79 percent and 39-48 percent chance, respectively. The BRCA1 c.4096+1G>A variant has been frequently ascertained in Tuscany, Italy, and it has also been detected in other Italian regions and other countries. Its pathogenetic status has been repeatedly changed from a variant of uncertain significance, to pathogenic, to likely pathogenic. In our study, 48 subjects (38 of whom are carriers) from 27 families were genotyped with the Illumina OncoArray Infinium platform (533,531 SNPs); a 20 Mb region (24.6 cM) around BRCA1, including 4130 SNPs (21 inside BRCA1) was selected for haplotype analysis. We used a phylogenetic method to estimate the time to the most recent common ancestor (MRCA) of BRCA1 c.4096+1G>A founder pathogenic variant. This analysis suggests that the MRCA lived about 155 generations ago-around 3000 years ago.

Sclerosing Paragangliomas: Correlations of Histological Features with Patients' Genotype and Vesicular Monoamine Transporter Expression.

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score ≥ 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas.

Validation and Data-Integration of Yeast-Based Assays for Functional Classification of BRCA1 Missense Variants.

Germline mutations in the BRCA1 gene have been reported to increase the lifetime risk of developing breast and/or ovarian cancer (BOC). By new sequencing technologies, numerous variants of uncertain significance (VUS) are identified. It is mandatory to develop new tools to evaluate their functional impact and pathogenicity. As the expression of pathogenic BRCA1 variants in Saccharomyces cerevisiae increases the frequency of intra- and inter-chromosomal homologous recombination (HR), and gene reversion (GR), we validated the two HR and the GR assays by testing 23 benign and 23 pathogenic variants and compared the results with those that were obtained in the small colony phenotype (SCP) assay, an additional yeast-based assay, that was validated previously. We demonstrated that they scored high accuracy, sensitivity, and sensibility. By using a classifier that was based on majority of voting, we have integrated data from HR, GR, and SCP assays and developed a reliable method, named yBRCA1, with high sensitivity to obtain an accurate VUS functional classification (benign or pathogenic). The classification of BRCA1 variants, important for assessing the risk of developing BOC, is often difficult to establish with genetic methods because they occur rarely in the population. This study provides a new tool to get insights on the functional impact of the BRCA1 variants.

Detection of Germline Variants in 450 Breast/Ovarian Cancer Families with a Multi-Gene Panel Including Coding and Regulatory Regions.

With the progress of sequencing technologies, an ever-increasing number of variants of unknown functional and clinical significance (VUS) have been identified in both coding and non-coding regions of the main Breast Cancer (BC) predisposition genes. The aim of this study is to identify a mutational profile of coding and intron-exon junction regions of 12 moderate penetrance genes (ATM, BRIP1, CDH1, CHEK2, NBN, PALB2, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53) in a cohort of 450 Italian patients with Hereditary Breast/Ovarian Cancer Syndrome, wild type for germline mutation in BRCA1/2 genes. The analysis was extended to 5'UTR and 3'UTR of all the genes listed above and to the BRCA1 and BRCA2 known regulatory regions in a subset of 120 patients. The screening was performed through NGS target resequencing on the Illumina platform MiSeq. 8.7% of the patients analyzed is carriers of class 5/4 coding variants in the ATM (3.6%), BRIP1 (1.6%), CHEK2 (1.8%), PALB2 (0.7%), RAD51C (0.4%), RAD51D (0.4%), and TP53 (0.2%) genes, while variants of uncertain pathological significance (VUSs)/class 3 were identified in 9.1% of the samples. In intron-exon junctions and in regulatory regions, variants were detected respectively in 5.1% and in 32.5% of the cases analyzed. The average age of disease onset of 44.4 in non-coding variant carriers is absolutely similar to the average age of disease onset in coding variant carriers for each proband's group with the same cancer type. Furthermore, there is not a statistically significant difference in the proportion of cases with a tumor onset under age of 40 between the two groups, but the presence of multiple non-coding variants in the same patient may affect the aggressiveness of the tumor and it is worth underlining that 25% of patients with an aggressive tumor are carriers of a PTEN 3'UTR-variant. This data provides initial information on how important it might be to extend mutational screening to the regulatory regions in clinical practice.

Functional Interaction Between BRCA1 and DNA Repair in Yeast May Uncover a Role of RAD50, RAD51, MRE11A, and MSH6 Somatic Variants in Cancer Development.

In this study, we determined if BRCA1 partners involved in DNA double-strand break (DSB) and mismatch repair (MMR) may contribute to breast and ovarian cancer development. Taking advantage the functional conservation of DNA repair pathways between yeast and human, we expressed several BRCA1 missense variants in DNA repair yeast mutants to identify functional interaction between BRCA1 and DNA repair in BRCA1-induced genome instability. The pathogenic p.C61G, pA1708E, p.M775R, and p.I1766S, and the neutral pS1512I BRCA1 variants increased intra-chromosomal recombination in the DNA-repair proficient strain RSY6. In the mre11, rad50, rad51, and msh6 deletion strains, the BRCA1 variants p.C61G, pA1708E, p.M775R, p.I1766S, and pS1215I did not increase intra-chromosomal recombination suggesting that a functional DNA repair pathway is necessary for BRCA1 variants to determine genome instability. The pathogenic p.C61G and p.I1766S and the neutral p.N132K, p.Y179C, and p.N550H variants induced a significant increase of reversion in the msh2Δ strain; the neutral p.Y179C and the pathogenic p.I1766S variant induced gene reversion also, in the msh6Δ strain. These results imply a functional interaction between MMR and BRCA1 in modulating genome instability. We also performed a somatic mutational screening of MSH6, RAD50, MRE11A, and RAD51 genes in tumor samples from 34 patients and identified eight pathogenic or predicted pathogenic rare missense variants: four in MSH6, one in RAD50, one in MRE11A, and two in RAD51. Although we found no correlation between BRCA1 status and these somatic DNA repair variants, this study suggests that somatic missense variants in DNA repair genes may contribute to breast and ovarian tumor development.

Whole-exome analysis of a Li-Fraumeni family trio with a novel TP53 PRD mutation and anticipation profile.

Li-Fraumeni syndrome is a clinically heterogeneous familial cancer predisposition syndrome with autosomal-dominant inheritance caused by heterozygous germline mutations in the TP53 gene. We here analyze the genetic background of a family with a 4-year-proband presented with a Li-Fraumeni tumor. The mother developed breast cancer at age 37 and the proband died at age 8. We performed Sanger sequencing and whole-exome sequencing on peripheral blood DNA from proband and relatives. Data analysis selected only high-quality score and depth reads, rare variants and protein impact involving missense, non-sense, frameshift and splice disrupt mutations. Disease implicated variants and predicted deleterious alterations were also chosen. TP53 genetic testing revealed a never reported TP53 deletion arose as de novo mutation in the mother and inherited by the proband. We then performed whole-exome analysis of the trio to uncover inherited variants from the father that potentially worsen the already altered genetic background in the proband. No pathogenic variants were inherited in autosomal recessive, de novo dominant or X-linked recessive manner. Comparing proband and father exome we detected 25 predicted deleterious variants including a nonsense mutation in ERCC3. Those inherited mutations are possible candidate modifiers linked to TP53, explaining the proband accelerated tumor onset compared to the mother and providing a possible explanation of the genetic anticipation event in this Li-Fraumeni family.

Characterization of an Italian founder mutation in the RING-finger domain of BRCA1.

The identification of founder mutations in cancer predisposing genes is important to improve risk assessment in geographically defined populations, since it may provide specific targets resulting in cost-effective genetic testing. Here, we report the characterization of the BRCA1 c.190T>C (p.Cys64Arg) mutation, mapped to the RING-finger domain coding region, that we detected in 43 hereditary breast/ovarian cancer (HBOC) families, for the large part originating from the province of Bergamo (Northern Italy). Haplotype analysis was performed in 21 families, and led to the identification of a shared haplotype extending over three BRCA1-associated marker loci (0.4 cM). Using the DMLE+2.2 software program and regional population demographic data, we were able to estimate the age of the mutation to vary between 3,100 and 3,350 years old. Functional characterization of the mutation was carried out at both transcript and protein level. Reverse transcriptase-PCR analysis on lymphoblastoid cells revealed expression of full length mRNA from the mutant allele. A green fluorescent protein (GFP)-fragment reassembly assay showed that the p.Cys64Arg substitution prevents the binding of the BRCA1 protein to the interacting protein BARD1, in a similar way as proven deleterious mutations in the RING-domain. Overall, 55 of 83 (66%) female mutation carriers had a diagnosis of breast and/or ovarian cancer. Our observations indicate that the BRCA1 c.190T>C is a pathogenic founder mutation present in the Italian population. Further analyses will evaluate whether screening for this mutation can be suggested as an effective strategy for the rapid identification of at-risk individuals in the Bergamo area.

Role of 18F-DOPA PET/CT in diagnosis and follow-up of adrenal and extra-adrenal paragangliomas.

PURPOSE: The objective of this study was to establish the clinical value of F-DOPA PET/CT in patients with adrenal and extra-adrenal paragangliomas (PGLs). METHODS: Twenty-six consecutive patients with suspected or recurrent PGL underwent MR (and/or CT) and F-DOPA PET/CT. Histopathology confirmation was obtained in 20 cases. Genetic analysis on known susceptibility genes for PGL (VHL, RET, SDHx, TMEM127) was available in 13 patients. RESULTS: Fourteen patients were affected by PGL (8 with head/neck location, 6 with abdominal/thoracic location), whereas 12 showed masses of other origin. Three patients proved to be SDHD, 1 SDHB, 2 SDHC, and 1 TMEM127 mutation carriers. F-DOPA PET/CT showed pathological uptake in 13 of 26 patients. The procedure identified all PGLs except one with bone metastases (previous malignant adrenal PGL). No uptake was found in patients without proven PGL. Thus, in the whole group, F-DOPA PET/CT sensitivity was 92.8%, and specificity was 100% with positive and negative predictive values of 100% and 92.3%, respectively. Total diagnostic accuracy was 96.2%. In the head/neck subgroup, sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy were 100%. In the abdominal location, sensitivity was 80% and specificity was 100%, and positive and negative predictive values were 100% and 91.7%, respectively. Abdominal diagnostic accuracy was 93.7%. Radiotracer uptake was superimposable in head/neck PGLs versus abdominal PGLs and in mutated versus wild-type patients. CONCLUSIONS: The high diagnostic performance of F-DOPA PET/CT showed this technique to be a useful tool in detecting PGLs, above all those located at the head/neck site, regardless of the genetic pattern.

Sporadic or familial head neck paragangliomas enrolled in a single center: clinical presentation and genotype/phenotype correlations.

BACKGROUND: The purpose of this study was to investigate clinical features and prevalence of germline mutations of patients with head/neck paragangliomas. METHODS: Genetic analysis on known susceptibility genes for paragangliomas (VHL, RET, SDHB, SDHC, SDHD, and SDHAF2) was performed in 17 consecutive patients with head/neck paraganglioma (age range, 14-82 years) and 17 relatives. RESULTS: Head/neck paragangliomas were usually symptomatic with "mass effect" (88.2%), without family history (82.3%), often multifocal (41.2%), never functioning, and malignant. Germline mutations were detected in 7 of 17 patients (41%; 6 SDHD and 1 SDHB). Patients with mutations were younger, with head/neck paragangliomas usually multifocal and with higher biologic aggressiveness than wild-type subjects. To date, 4 families have been studied and the prevalence of carriers was elevated (58.8%). These mutated relatives (age range, 17-71 years) were disease-free, except 4 patients in whom multiple head/neck paragangliomas were detected. CONCLUSION: Adequate morpho-functional screening and follow-up and, if possible, genetic testing is advisable in patients with head/neck paraganglioma.

Acro-cardio-facial syndrome: a microdeletion syndrome?

Acro-cardio-facial syndrome (ACFS) is an infrequently reported, variable condition characterized by split-hand and split-foot malformation and congenital heart defect (CHD), along with cleft lip and palate, genital anomalies, unusual face and intellectual disability. An autosomal recessive pattern of inheritance has been suggested because of affected sibs born to unaffected parents and parental consanguinity; the cause is unknown. We describe a newborn with the clinical manifestations of ACFS in whom a deletion of the region 6q21-q22.3 was detected by array CGH. We compare the clinical features of the present patient with earlier reported patients with similar 6q deletions and patients diagnosed with ACFS. The similarities between these patient groups suggest that ACFS may be a microdeletion syndrome caused by loss of the 6q21-22.3 region. The recurrence in families may be explained by prenatal germline mosaicism. Alternatively, ACFS may be a genetically heterogeneous disorder which can also be caused by biallelic mutations of an autosomal recessive gene.

A PALB2 germline mutation associated with hereditary breast cancer in Italy.

Recently, it has been demonstrated that monoallelic PALB2 mutations predispose to familial breast cancer. We investigated the contribution of PALB2 mutations in a set of 132 Italian BRCA1/BRCA2-negative breast cancer families; one truncating PALB2 mutation, c.2257C>T, resulting in p.Arg753X, was identified in a woman and her daughter, with breast cancer diagnosed at 60 and 31 years old, respectively. This study supports the recent observation that PALB2 mutation are present, although infrequently, in familial BRCA1/BRCA2-negative breast cancer cases; moreover, it sustains latest evidences that some PALB2 mutations are associated with a substantially increased risk of breast cancer.

Germline mutations in MEN1 and BRCA1 genes in a woman with familial multiple endocrine neoplasia type 1 and inherited breast-ovarian cancer syndromes: a case report.

The simultaneous occurrence of mutations in two different tumor suppressor genes in the same individual is a very rare event. Here we report the case of a woman in whom germline mutations in both MEN1 and BRCA1 were identified. The severity of MEN1-related biochemical and clinical findings did not significantly differ from that for other affected family members lacking the BRCA1 mutation, except for the development of an extremely large visceral lipoma; the proband has not developed any BRCA1-related malignancies. We explore genetic and molecular rationales for an association between these neoplastic processes.

Founder mutations account for the majority of BRCA1-attributable hereditary breast/ovarian cancer cases in a population from Tuscany, Central Italy.

BACKGROUND: Germline mutations in the BRCA1 and BRCA2 tumour-suppressor genes predispose to early-onset breast and ovarian cancer. Although both genes display a highly heterogeneous mutation spectrum, a number of alterations recur in some populations. Only a limited number of founder mutations have been identified in the Italian population so far. OBJECTIVE: To investigate the spectrum of BRCA1/BRCA2 mutations in a set of families originary from the Central-Eastern part of Tuscany and to ascertain the presence of founder effects. We also wanted to approximate the age of the most frequent BRCA1 founder mutation. RESULTS: Overall, four distinct BRCA1 mutations accounted for a large fraction (72.7%) of BRCA1-attributable hereditary breast/ovarian cancer in families originary from this area. We identified common haplotypes for two newly recognised recurrent BRCA1 mutations, c.3228_3229delAG and c.3285delA. The c.3228_3229delAG mutation was estimated to have originated about 129 generations ago. Interestingly, male breast cancer cases were present in 3 out of 11 families with the c.3228_3229delAG mutation. CONCLUSIONS: The observation that a high proportion of families with BRCA1 alterations from Central-Eastern Tuscany harbours a limited number of founder mutations can have significant impact on clinical management of at risk subjects from this area. In addition, the identification of a large set of families carrying an identical mutation that predisposes to breast and ovarian cancer provides unique opportunities to study the effect of other genetic and environmental factors on penetrance and disease phenotype.

Interleukin-10 promoter polymorphisms influence susceptibility to ulcerative colitis in a gender-specific manner.

OBJECTIVE: Pathological evidence supports a potential role of the pro- and anti-inflammatory cytokine network in the pathogenesis of inflammatory bowel disease (IBD). Moreover, associated studies suggest a possible involvement of cytokine-related genes in IBD susceptibility. In this study, we evaluated the effect of the anti-inflammatory interleukin-10 (IL10) gene on ulcerative colitis (UC). MATERIAL AND METHODS: Two functional single nucleotide polymorphisms (-1082 G/A, -819 T/C) in the IL10 promoter in 203 Italian sporadic UC patients and 391 controls were determined using high-resolution melting analysis. RESULTS: The frequency of the -1082A allele was significantly higher in the UC patients than in controls (p=0.00003); -1082 genotype frequencies were also significantly different between UC patients and controls (p=0.0001). Allele and genotype frequencies of -819 T/C were not significantly associated with UC. Furthermore, the frequencies of haplotypes -1082A/-819C and -1082A/-819T, which have been reported to have a lower promoter activity, were significantly higher in UC patients than in controls (p=0.0004). After gender stratification, we found a significant difference in the -1082A allele (p=0.00004) and genotype (p=0.0002) frequencies only between female UC patients and controls; the same result was obtained for the -1082A/-819C and -1082A/-819T haplotypes (p=0.0006). CONCLUSIONS: A gender effect is observed, with women of AG/AA IL10 genotypes and AC/AT haplotypes having a higher risk of developing UC at a younger age. This finding could be related to the previously documented lower IL10 production associated with the -1082A allele and to the IL10 down-regulating effect of estrogens.