Ovarian cancer pathology characteristics as predictors of variant pathogenicity in BRCA1 and BRCA2.

BACKGROUND: The distribution of ovarian tumour characteristics differs between germline BRCA1 and BRCA2 pathogenic variant carriers and non-carriers. In this study, we assessed the utility of ovarian tumour characteristics as predictors of BRCA1 and BRCA2 variant pathogenicity, for application using the American College of Medical Genetics and the Association for Molecular Pathology (ACMG/AMP) variant classification system. METHODS: Data for 10,373 ovarian cancer cases, including carriers and non-carriers of BRCA1 or BRCA2 pathogenic variants, were collected from unpublished international cohorts and consortia and published studies. Likelihood ratios (LR) were calculated for the association of ovarian cancer histology and other characteristics, with BRCA1 and BRCA2 variant pathogenicity. Estimates were aligned to ACMG/AMP code strengths (supporting, moderate, strong). RESULTS: No histological subtype provided informative ACMG/AMP evidence in favour of BRCA1 and BRCA2 variant pathogenicity. Evidence against variant pathogenicity was estimated for the mucinous and clear cell histologies (supporting) and borderline cases (moderate). Refined associations are provided according to tumour grade, invasion and age at diagnosis. CONCLUSIONS: We provide detailed estimates for predicting BRCA1 and BRCA2 variant pathogenicity based on ovarian tumour characteristics. This evidence can be combined with other variant information under the ACMG/AMP classification system, to improve classification and carrier clinical management.

Relative validity and reproducibility of the CyFFQ semiquantitative food frequency questionnaire for assessing dietary intake in Cypriot adults.

BACKGROUND: Assessment of dietary intake is fundamental for evaluating the interrelationships between diet and disease. The present study aimed to develop and validate the semiquantitative Cypriot food frequency questionnaire (CyFFQ). METHODS: A 171-item paper-and-pencil semiquantitative interview-administered FFQ was developed, including local foods and culturally specific meals commonly consumed among Cypriot adults. FFQ reproducibility was assessed by comparing the energy-adjusted daily macro- and micronutrients intake at baseline (FFQ1) and 1 year later (FFQ2) using a Wilcoxon matched pairs signed rank test and intraclass correlation coefficients (ICCs) in a random sample of Cypriot adults. FFQ relative validity was evaluated by comparing the intake as estimated by FFQ2 with that obtained from the average of three 24-h recalls taken over the year between FFQ1 and FFQ2. Associations between nutrient intakes estimated using FFQ2 and the 24-h recalls were assessed using Spearman rank correlation and Bland-Altman plots were used to assess agreement between the FFQ and the 24-h recalls. RESULTS: Among eligible participants, 68 (78%) completed the study (44.1% males, aged 30.5-47.5 years). The energy-adjusted intakes of macro- and micronutrients did not significantly differ between the two FFQs, excluding magnesium. The FFQ2 and the averaged 24-h recalls were significantly correlated for most macro- and micronutrients. The median (interquartile) ICC for all macro- and micronutrients was 0.46 (0.38-0.52) (p < 0.05). Agreement was satisfactory (>30%) for most micro- and macronutrients. Bland-Altman plots also confirmed good agreement between the two methods. CONCLUSIONS: The CyFFQ is a valid and reliable tool for assessing dietary consumption in Cypriot adults.

Genetic Study of Early Onset Parkinson's Disease in Cyprus.

Parkinson's Disease (PD) is a multifactorial neurodegenerative disease characterized by motor and non-motor symptoms. The etiology of PD remains unclear. However, several studies have demonstrated the interplay of genetic, epigenetic, and environmental factors in PD. Early-onset PD (EOPD) is a subgroup of PD diagnosed between the ages of 21 and 50. Population genetic studies have demonstrated great genetic variability amongst EOPD patients. Hence, this study aimed to obtain a genetic landscape of EOPD in the Cypriot population. Greek-Cypriot EOPD patients (n = 48) were screened for variants in the six most common EOPD-associated genes (PINK1, PRKN, FBXO7, SNCA, PLA2G6, and DJ-1). This included DNA sequencing and Multiplex ligation-dependent probe amplification (MLPA). One previously described frameshift variant in PINK1 (NM_032409.3:c.889del) was detected in five patients (10.4%)-the largest number to be detected to date. Copy number variations in the PRKN gene were identified in one homozygous and 3 compound heterozygous patients (8.3%). To date, the pathogenic variants identified in this study have explained the PD phenotype for 18.8% of the EOPD cases. The results of this study may contribute to the genetic screening of EOPD in Cyprus.

Performance evaluation of pipelines for mapping, variant calling and interval padding, for the analysis of NGS germline panels.

BACKGROUND: Next-generation sequencing (NGS) represents a significant advancement in clinical genetics. However, its use creates several technical, data interpretation and management challenges. It is essential to follow a consistent data analysis pipeline to achieve the highest possible accuracy and avoid false variant calls. Herein, we aimed to compare the performance of twenty-eight combinations of NGS data analysis pipeline compartments, including short-read mapping (BWA-MEM, Bowtie2, Stampy), variant calling (GATK-HaplotypeCaller, GATK-UnifiedGenotyper, SAMtools) and interval padding (null, 50 bp, 100 bp) methods, along with a commercially available pipeline (BWA Enrichment, Illumina®). Fourteen germline DNA samples from breast cancer patients were sequenced using a targeted NGS panel approach and subjected to data analysis. RESULTS: We highlight that interval padding is required for the accurate detection of intronic variants including spliceogenic pathogenic variants (PVs). In addition, using nearly default parameters, the BWA Enrichment algorithm, failed to detect these spliceogenic PVs and a missense PV in the TP53 gene. We also recommend the BWA-MEM algorithm for sequence alignment, whereas variant calling should be performed using a combination of variant calling algorithms; GATK-HaplotypeCaller and SAMtools for the accurate detection of insertions/deletions and GATK-UnifiedGenotyper for the efficient detection of single nucleotide variant calls. CONCLUSIONS: These findings have important implications towards the identification of clinically actionable variants through panel testing in a clinical laboratory setting, when dedicated bioinformatics personnel might not always be available. The results also reveal the necessity of improving the existing tools and/or at the same time developing new pipelines to generate more reliable and more consistent data.

Implementation of multigene panel NGS diagnosis in the national primary ciliary dyskinesia cohort of Cyprus: An island with a high disease prevalence.

We aimed to determine a genetic diagnosis in the national primary ciliary dyskinesia (PCD) cohort of Cyprus, an island with a high disease prevalence. We used targeted next-generation sequencing (NGS) of 39 PCD genes in 48 patients of Greek-Cypriot and other ancestries. We achieved a molecular diagnosis in 74% of the unrelated families tested. We identified 24 different mutations in 11 genes, 12 of which are novel. Homozygosity was more common in Greek-Cypriot than non-Greek-Cypriot patients (88% vs. 46.2%, p = .016). Four mutations (DNAH11:c.5095-2A>G, CFAP300:c.95_103delGCCGGCTCC, TTC25:c.716G>A, RSPH9:c.670+2T>C) were found in 74% of the diagnosed Greek-Cypriot families. Patients with RSPH9 mutations demonstrated higher nasal nitric oxide (57 vs. 15 nl/min, p <.001), higher forced expiratory volume in 1 s (-0.89 vs. -2.37, p = .018) and forced vital capacity (-1.00 vs. -2.16, p = .029) z scores than the rest of the cohort. Targeted multigene-panel NGS is an efficient tool for early diagnosis of PCD, providing insight into genetic disease epidemiology and improved patient stratification.

Alport syndrome: Proteomic analysis identifies early molecular pathway alterations in Col4a3 knock out mice.

AIM: Alport syndrome (AS) is the second most common hereditary kidney disease caused by mutations in collagen IV genes. Patients present with microhaematuria that progressively leads to proteinuria and end stage renal disease. Currently, no specific treatment exists for AS. Using mass spectrometry based proteomics, we aimed to detect early alterations in molecular pathways implicated in AS before the stage of overt proteinuria, which could be amenable to therapeutic intervention. METHODS: Kidneys were harvested from male Col4a3-/- knock out and sex and age-matched Col4a3+/+ wild-type mice at 4 weeks of age. Purified peptides were separated by liquid chromatography and analysed by high resolution mass spectrometry. The Cytoscape bioinformatics tool was used for function enrichment and pathway analysis. PPARα expression levels were evaluated by immunofluorescence and immunoblotting. RESULTS: Proteomic analysis identified 415 significantly differentially expressed proteins, which were mainly involved in metabolic and cellular processes, the extracellular matrix, binding and catalytic activity. Pathway enrichment analysis revealed among others, downregulation of the proteasome and PPAR pathways. PPARα protein expression levels were observed to be downregulated in Alport mice, supporting further the results of the discovery proteomics. CONCLUSION: This study provides additional evidence that alterations in proteins which participate in cellular metabolism and mitochondrial homeostasis in kidney cells are early events in the development of chronic kidney disease in AS. Of note is the dysregulation of the PPAR pathway, which is amenable to therapeutic intervention and provides a new potential target for therapy in AS.

The spectrum of BRCA1 and BRCA2 pathogenic sequence variants in Middle Eastern, North African, and South European countries.

BRCA1 BRCA2 mutational spectrum in the Middle East, North Africa, and Southern Europe is not well characterized. The unique history and cultural practices characterizing these regions, often involving consanguinity and inbreeding, plausibly led to the accumulation of population-specific founder pathogenic sequence variants (PSVs). To determine recurring BRCA PSVs in these locales, a search in PUBMED, EMBASE, BIC, and CIMBA was carried out combined with outreach to researchers from the relevant countries for unpublished data. We identified 232 PSVs in BRCA1 and 239 in BRCA2 in 25 of 33 countries surveyed. Common PSVs that were detected in four or more countries were c.5266dup (p.Gln1756Profs), c.181T>G (p.Cys61Gly), c.68_69del (p.Glu23Valfs), c.5030_5033del (p.Thr1677Ilefs), c.4327C>T (p.Arg1443Ter), c.5251C>T (p.Arg1751Ter), c.1016dup (p.Val340Glyfs), c.3700_3704del (p.Val1234Glnfs), c.4065_4068del (p.Asn1355Lysfs), c.1504_1508del (p.Leu502Alafs), c.843_846del (p.Ser282Tyrfs), c.798_799del (p.Ser267Lysfs), and c.3607C>T (p.Arg1203Ter) in BRCA1 and c.2808_2811del (p.Ala938Profs), c.5722_5723del (p.Leu1908Argfs), c.9097dup (p.Thr3033Asnfs), c.1310_1313del (p. p.Lys437Ilefs), and c.5946del (p.Ser1982Argfs) for BRCA2. Notably, some mutations (e.g., p.Asn257Lysfs (c.771_775del)) were observed in unrelated populations. Thus, seemingly genotyping recurring BRCA PSVs in specific populations may provide first pass BRCA genotyping platform.

Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac 19F MRS/MRI and temporal tracking of progenitor cells.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and 19F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENEHD-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine 19F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold 19F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac 19F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with 19F MRI.

The effect of l-Arginine on Ciliary Beat Frequency in PCD patients, non-PCD respiratory patients and healthy controls.

OBJECTIVES: Few studies have examined the potentially therapeutic effect of increasing the production of endogenous nitric oxide (NO) in Primary Ciliary Dyskinesia (PCD) and other chronic respiratory conditions. Nasal NO is low in PCD and has been found to correlate with compromised Ciliary Beat Frequency (CBF). In this study we assessed the effect of increasing l-Arginine, as the substrate of NO synthases, on CBF in biopsies of human respiratory ciliated epithelium. METHODOLOGY: A total of 28 suspect cases with chronic respiratory manifestations referred for PCD diagnostic testing and 8 healthy controls underwent nasal brushing. Obtained epithelial cells were divided between three culture medium 199 solutions, containing different levels of l-Arginine (0.33 mM as baseline, 1 mM and 10 Mm as increased levels). CBF measurements were obtained at 37 °C and 25 °C at 1, 3 and 24 h after sample acquisition. RESULTS: Among a total of 36 recruited subjects, 8 had PCD confirmed (PCD n = 8), 20 had PCD excluded (non-PCD n = 20) and 8 were healthy controls (Healthy Controls = 8). Among PCD subjects, ciliary motility was characterized by rotational (n = 5) or dyskinetic (n = 3) beating. At 37 °C, compared to baseline, higher levels of l-Arginine resulted in up to 9% CBF increase at 1 h (p = 0.007), up to 9% CBF increase at 3 h (p < 0.001) and up to 12% CBF increase at 24 h (p = 0.002). Similar although smaller scale increases were recorded at 25 °C. The effect of l-Arginine was time dependent (interaction p = 0.002) and was similar in PCD patients, non-PCD chronic respiratory patients and healthy controls (interaction p = 0.800). CONCLUSIONS: l-Arginine increases CBF and merits to be evaluated as a potential stimulator of mucociliary clearance in chronic respiratory conditions and congenital ciliary disorders with residual motility. Larger human studies are needed to confirm these findings.

Biomarkers of systemic lupus erythematosus identified using mass spectrometry-based proteomics: a systematic review.

Advances in mass spectrometry technologies have created new opportunities for discovering novel protein biomarkers in systemic lupus erythematosus (SLE). We performed a systematic review of published reports on proteomic biomarkers identified in SLE patients using mass spectrometry-based proteomics and highlight their potential disease association and clinical utility. Two electronic databases, MEDLINE and EMBASE, were systematically searched up to July 2015. The methodological quality of studies included in the review was performed according to Preferred Reporting Items for Systematic Reviews and Meta-analyses guidelines. Twenty-five studies were included in the review, identifying 241 SLE candidate proteomic biomarkers related to various aspects of the disease including disease diagnosis and activity or pinpointing specific organ involvement. Furthermore, 13 of the 25 studies validated their results for a selected number of biomarkers in an independent cohort, resulting in the validation of 28 candidate biomarkers. It is noteworthy that 11 candidate biomarkers were identified in more than one study. A significant number of potential proteomic biomarkers that are related to a number of aspects of SLE have been identified using mass spectrometry proteomic approaches. However, further studies are required to assess the utility of these biomarkers in routine clinical practice.

The synergistic effect between the Mediterranean diet and GSTP1 or NAT2 SNPs decreases breast cancer risk in Greek-Cypriot women.

PURPOSE: Xenobiotic metabolism is related to the interplay between diet and breast cancer (BC) risk. This involves detoxification enzymes, which are polymorphic and metabolise various dietary metabolites. An important characteristic of this pathway is that chemoprotective micronutrients can act not only as substrates but also as inducers for these enzymes. We investigated whether functional GSTP1 (p.Ile105Val-rs1695), NAT2 (590G>A-rs1799930) SNPs and GSTM1 and GSTT1 deletion polymorphisms could modulate the effect of the Mediterranean diet (MD) on BC risk, in Greek-Cypriot women. METHODS: Genotyping was performed on women from the MASTOS case-control study of BC in Cyprus. A 32-item food-frequency questionnaire was used to obtain dietary intake information. A dietary pattern, which closely resembles the MD (high loadings of vegetables, fruit, legumes and fish), was previously derived with principal component analysis and was used as our dietary variable. RESULTS: GSTT1 null genotype increased BC risk compared with the homozygous non-null GSTT1 genotype (OR 1.21, 95 % CI 1.01-1.45). Increasing adherence to the MD reduced BC risk in women with at least one GSTP1 Ile allele (OR for Ile/Ile = 0.84, 95 % CI 0.74-0.95, for Ile/Val = 0.73, 95 % CI 0.62-0.85) or one NAT2 590G allele (OR for 590 GG = 0.73, 95 % CI 0.63-0.83, for 590 GA = 0.81, 95 % CI 0.70-0.94). p interaction values were not, however, statistically significant. CONCLUSION: The homozygous null GSTT1 genotype could be a risk allele for BC among Greek-Cypriot women. The anticarcinogenic effects of the high adherence to MD against BC risk could also be further enhanced when combined with the wild-type alleles of the detoxification GSTP1 or NAT2 SNPs.

Association between Echinococcus granulosus infection and cancer risk - a pilot study in Cyprus.

BACKGROUND: Infections from microorganisms and parasites have been connected with either increased or decreased cancer risk. The objective of this study was to investigate whether infection by Echinococcus granulosus is associated with cancer risk. METHODS: We assembled a pilot retrospective cohort of patients who were diagnosed as being infected by E. granulosus in Cyprus between 1930 and 2011. Age/gender-matched non-infected family members and neighbors were selected as references. Medical history was ascertained from each study subject through in-person interview. Cox proportional hazards regression analysis was performed to assess the association of being infected by E. granulosus with cancer risk. RESULTS: Individuals with prior infection by E. granulosus (n=249) were more likely to have cancer compared to those without infection (n=753), 11.65% vs. 8.37% (p=0.0492). Survival analysis also showed that subjects with prior infection had a higher risk for developing cancer. The hazards ratio (HR) was 1.595, [95% confidence interval (CI) between 1.008 and 2.525]. The risk ratio did not change significantly (HR=1.536; 95% CI: 0.965-2.445) after adjusting for gender, year of birth, smoking status, alcohol consumption, and family history of cancer. CONCLUSIONS: Our study suggests that infection by E. granulosus may increase cancer risk. If this observation can be confirmed independently, further investigation of the mechanisms underlying the association is warranted.

MnSOD and CAT polymorphisms modulate the effect of the Mediterranean diet on breast cancer risk among Greek-Cypriot women.

PURPOSE: Oxidative stress arises due to a cellular imbalance in oxidants and antioxidants and/or due to an altered activity of antioxidant enzymes, caused by SNPs. Oxidative stress increases susceptibility to breast cancer (BC) risk, and we previously showed that the Mediterranean diet (MD), which is rich in antioxidants, reduces BC risk in Greek-Cypriot women. Here, we investigated the effect of MnSOD (p.Val16Ala, rs4880) and CAT (-262C>T, rs1001179) SNPs on the association between the MD and BC risk in the case-control study of BC MASTOS in Cyprus. METHODS: Dietary intake data were obtained using a 32-item food frequency questionnaire, from which a dietary pattern was previously derived, using principal component analysis. This pattern included high loadings of vegetables, fruit, legumes and fish, a combination that closely resembles the MD and was used as our dietary variable. RESULTS: High vegetable intake lowered BC risk in women with at least one MnSOD Val allele (ORHigh vs. Low for Val/Val = 0.56, 95 % CI 0.35-0.88, for Val/Ala = 0.57, 95 % CI 0.39-0.82), or one CAT -262C allele (ORHigh vs. Low for -262CC = 0.66, 95 % CI 0.47-0.92, for -262CT = 0.53, 95 % CI 0.35-0.81). High fish intake conferred a decreased BC risk of CAT -262CC women (ORQ4 vs. Q1 0.66, 95 % CI 0.47-0.92) compared with the CAT -262TT women and low fish intake (ORQ2 vs. Q1 2.79, 95 % CI 1.08-7.17). Additionally, high fish intake reduced BC risk in MnSOD Val/Val women (ORQ4 vs. Q1 0.63, 95 % CI 0.40-0.98). p interaction values were, however, not statistically significant. CONCLUSION: Our results demonstrate that the antioxidative effects of the MD against BC risk may be enhanced by the wild-type alleles of the MnSOD or CAT SNPs among Greek-Cypriot women.

The role of ubiquitin-binding domains in human pathophysiology.

Ubiquitination, a fundamental post-translational modification (PTM) resulting in the covalent attachment of ubiquitin (Ub) to a target protein, is currently implicated in several key cellular processes. Although ubiquitination was initially associated with protein degradation, it is becoming increasingly evident that proteins labeled with polyUb chains of specific topology and length are activated in an ever-expanding repertoire of specific cellular processes. In addition to their involvement in the classical protein degradation pathways they are involved in DNA repair, kinase regulation and nuclear factor-κB (NF-κB) signaling. The sorting and processing of distinct Ub signals is mediated by small protein motifs, known as Ub-binding domains (UBDs), which are found in proteins that execute disparate biological functions. The involvement of UBDs in several biological pathways has been revealed by several studies which have highlighted the vital role of UBDs in cellular homeostasis. Importantly, functional impairment of UBDs in key regulatory pathways has been related to the development of pathophysiological conditions, including immune disorders and cancer. In this review, we present an up-to-date account of the crucial role of UBDs and their functions, with a special emphasis on their functional impairment in key biological pathways and the pathogenesis of several human diseases. The still under-investigated topic of Ub-UBD interactions as a target for developing novel therapeutic strategies against many diseases is also discussed.

The bifacial role of helminths in cancer: involvement of immune and non-immune mechanisms.

Infectious agents have been associated with cancer due to activation of pro-carcinogenic inflammatory processes within their host. Several reports, however, indicate that specific pathogens may be able to elicit anti-tumor immune responses that can lead to protection from tumorigenesis or cancer regression. Amongst these "beneficial" pathogens are some helminthic parasites that have already been connected with prevention of autoimmune diseases and allergies, immune conditions increasingly associated with cancer. Even though helminths have co-existed with humans and their ancestors for millions of years, investigations of their impact on human (patho)physiology are relatively new and the functions of components that can explain the helminth bi-directional influence on carcinogenesis are not well understood. This review aims to discuss evidence for the helminth-induced immune, genetic, epigenetic, proteomic, hormonal and metabolic changes that may ultimately mediate the potential pro- or anti-carcinogenic role of helminths. This overview may serve future investigations in clarifying the tumorigenic role of the most common helminthic parasites. It may also inspire the development of anti-cancer regimens and vaccines, in parallel to ongoing efforts of using helminth-based components for the prevention and/or treatment of autoimmune diseases and allergies.

Identification of stage-specific breast markers using quantitative proteomics.

Matched healthy and diseased tissues from breast cancer patients were analyzed by quantitative proteomics. By comparing proteomic profiles of fibroadenoma (benign tumors, three patients), DCIS (noninvasive cancer, three patients), and invasive ductal carcinoma (four patients), we identified protein alterations that correlated with breast cancer progression. Three 8-plex iTRAQ experiments generated an average of 826 protein identifications, of which 402 were common. After excluding those originating from blood, 59 proteins were significantly changed in tumor compared with normal tissues, with the majority associated with invasive carcinomas. Bioinformatics analysis identified relationships between proteins in this subset including roles in redox regulation, lipid transport, protein folding, and proteasomal degradation, with a substantial number increased in expression due to Myc oncogene activation. Three target proteins, cofilin-1 and p23 (increased in invasive carcinoma) and membrane copper amine oxidase 3 (decreased in invasive carcinoma), were subjected to further validation. All three were observed in phenotype-specific breast cancer cell lines, normal (nontransformed) breast cell lines, and primary breast epithelial cells by Western blotting, but only cofilin-1 and p23 were detected by multiple reaction monitoring mass spectrometry analysis. All three proteins were detected by both analytical approaches in matched tissue biopsies emulating the response observed with proteomics analysis. Tissue microarray analysis (361 patients) indicated cofilin-1 staining positively correlating with tumor grade and p23 staining with ER positive status; both therefore merit further investigation as potential biomarkers.

Multi-gene panel testing and association analysis in Cypriot breast cancer cases and controls.

Introduction: It is estimated that around 5% of breast cancer cases carry pathogenic variants in established breast cancer susceptibility genes. However, the underlying prevalence and gene-specific population risk estimates in Cyprus are currently unknown. Methods: We performed sequencing on a population-based case-control study of 990 breast cancer cases and 1094 controls from Cyprus using the BRIDGES sequencing panel. Analyses were conducted separately for protein-truncating and rare missense variants. Results: Protein-truncating variants in established breast cancer susceptibility genes were detected in 3.54% of cases and 0.37% of controls. Protein-truncating variants in BRCA2 and ATM were associated with a high risk of breast cancer, whereas PTVs in BRCA1 and PALB2 were associated with a high risk of estrogen receptor (ER)-negative disease. Among participants with a family history of breast cancer, PTVs in ATM, BRCA2, BRCA1, PALB2 and RAD50 were associated with an increased risk of breast cancer. Furthermore, an additional 19.70% of cases and 17.18% of controls had at least one rare missense variant in established breast cancer susceptibility genes. For BRCA1 and PALB2, rare missense variants were associated with an increased risk of overall and triple-negative breast cancer, respectively. Rare missense variants in BRCA1, ATM, CHEK2 and PALB2 domains, were associated with increased risk of disease subtypes. Conclusion: This study provides population-based prevalence and gene-specific risk estimates for protein-truncating and rare missense variants. These results may have important clinical implications for women who undergo genetic testing and be pivotal for a substantial proportion of breast cancer patients in Cyprus.

Exome sequencing identifies breast cancer susceptibility genes and defines the contribution of coding variants to breast cancer risk.

Linkage and candidate gene studies have identified several breast cancer susceptibility genes, but the overall contribution of coding variation to breast cancer is unclear. To evaluate the role of rare coding variants more comprehensively, we performed a meta-analysis across three large whole-exome sequencing datasets, containing 26,368 female cases and 217,673 female controls. Burden tests were performed for protein-truncating and rare missense variants in 15,616 and 18,601 genes, respectively. Associations between protein-truncating variants and breast cancer were identified for the following six genes at exome-wide significance (P < 2.5 × 10-6): the five known susceptibility genes ATM, BRCA1, BRCA2, CHEK2 and PALB2, together with MAP3K1. Associations were also observed for LZTR1, ATR and BARD1 with P < 1 × 10-4. Associations between predicted deleterious rare missense or protein-truncating variants and breast cancer were additionally identified for CDKN2A at exome-wide significance. The overall contribution of coding variants in genes beyond the previously known genes is estimated to be small.

FANCM missense variants and breast cancer risk: a case-control association study of 75,156 European women.

Evidence from literature, including the BRIDGES study, indicates that germline protein truncating variants (PTVs) in FANCM confer moderately increased risk of ER-negative and triple-negative breast cancer (TNBC), especially for women with a family history of the disease. Association between FANCM missense variants (MVs) and breast cancer risk has been postulated. In this study, we further used the BRIDGES study to test 689 FANCM MVs for association with breast cancer risk, overall and in ER-negative and TNBC subtypes, in 39,885 cases (7566 selected for family history) and 35,271 controls of European ancestry. Sixteen common MVs were tested individually; the remaining rare 673 MVs were tested by burden analyses considering their position and pathogenicity score. We also conducted a meta-analysis of our results and those from published studies. We did not find evidence for association for any of the 16 variants individually tested. The rare MVs were significantly associated with increased risk of ER-negative breast cancer by burden analysis comparing familial cases to controls (OR = 1.48; 95% CI 1.07-2.04; P = 0.017). Higher ORs were found for the subgroup of MVs located in functional domains or predicted to be pathogenic. The meta-analysis indicated that FANCM MVs overall are associated with breast cancer risk (OR = 1.22; 95% CI 1.08-1.38; P = 0.002). Our results support the definition from previous analyses of FANCM as a moderate-risk breast cancer gene and provide evidence that FANCM MVs could be low/moderate risk factors for ER-negative and TNBC subtypes. Further genetic and functional analyses are necessary to clarify better the increased risks due to FANCM MVs.