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ObjectiveOlaparib plus bevacizumab maintenance therapy improves survival outcomes in women with newly diagnosed, advanced, high-grade ovarian cancer with a deficiency in homologous recombination. We report data from the first year of routine homologous recombination deficiency testing in the National Health Service (NHS) in England, Wales, and Northern Ireland between April 2021 and April 2022.MethodsThe Myriad myChoice companion diagnostic was used to test DNA extracted from formalin-fixed, paraffin-embedded tumor tissue in women with newly diagnosed International Federation of Gynecology and Obstetrics (FIGO) stage III/IV high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer. Tumors with homologous recombination deficiency were those with aBRCA1/2mutation and/or a Genomic Instability Score (GIS) ≥42. Testing was coordinated by the NHS Genomic Laboratory Hub network.ResultsThe myChoice assay was performed on 2829 tumors. Of these, 2474 (87%) and 2178 (77%) successfully underwentBRCA1/2and GIS testing, respectively. All complete and partial assay failures occurred due to low tumor cellularity and/or low tumor DNA yield. 385 tumors (16%) contained aBRCA1/2mutation and 814 (37%) had a GIS ≥42. Tumors with a GIS ≥42 were more likely to beBRCA1/2wild-type (n=510) thanBRCA1/2 mutant (n=304). The distribution of GIS was bimodal, withBRCA1/2mutant tumors having a higher mean score thanBRCA1/2wild-type tumors (61 vs 33, respectively, χ2test pConclusionThis is the largest real-world evaluation of homologous recombination deficiency testing in newly diagnosed FIGO stage III/IV high-grade epithelial ovarian, fallopian tube, or primary peritoneal cancer. It is important to select tumor tissue with adequate tumor content and quality to reduce the risk of assay failure. The rapid uptake of testing across England, Wales, and Northern Ireland demonstrates the power of centralized NHS funding, center specialization, and the NHS Genomic Laboratory Hub network.

The British Gynecological Cancer Society and the British Association of Gynecological Pathologists established a multidisciplinary consensus group comprising experts in surgical gynecological oncology, medical oncology, genetics, and laboratory science, and clinical nurse specialists to identify the optimal pathways to BRCA germline and tumor testing in patients with ovarian cancer in routine clinical practice. In particular, the group explored models of consent, quality standards identified at pathology laboratories, and experience and data from pioneering cancer centers. The group liaised with representatives from ovarian cancer charities to also identify patient perspectives that would be important to implementation. Recommendations from these consensus group deliberations are presented in this manuscript.

Ashkenazi-Jewish (AJ) population-based BRCA testing is acceptable, cost-effective and amplifies primary prevention for breastovarian cancer. However, data describing lifestyle impact are lacking. We report long-term results of population-based BRCA testing on lifestyle behaviour and cancer risk perception.Two-arm randomised controlled trials (ISRCTN73338115, GCaPPS): (a) population-screening (PS); (b) family history (FH)/clinical criteria testing.North London AJ-population.AJ women/men18 years.prior BRCA testing or first-degree relatives of BRCA-carriers.Participants were recruited through self-referral. All participants received informed pre-test genetic counselling. The intervention included genetic testing for three AJ BRCA-mutations: 185delAG(c.68_69delAG), 5382insC(c.5266dupC) and 6174delT(c.5946delT). This was undertaken for all participants in the PS arm and participants fulfilling FH/clinical criteria in the FH arm. Patients filled out customised/validated questionnaires at baseline/1-year/2-year/3-year follow-ups. Generalised linear-mixed models adjusted for covariates and appropriate contrast tests were used for between-group/within-group analysis of lifestyle and behavioural outcomes along with evaluating factors associated with these outcomes. Outcomes are adjusted for multiple testing (Bonferroni method), with P 0.0039 considered significant.Lifestyle/behavioural outcomes at baseline/1-year/2-year/3-year follow-ups.1034 participants were randomised to PS (n = 530) or FH (n = 504) arms. No significant difference was identified between PS- and FH-based BRCA testing approaches in terms of dietary fruit/vegetable/meat consumption, vitamin intake, alcohol quantity/ frequency, smoking behaviour (frequency/cessation), physical activity/exercise or routine breast mammogram screening behaviour, with outcomes not affected by BRCA test result. Cancer risk perception decreased with time following BRCA testing, with no difference between FH/PS approaches, and the perception of risk was lowest in BRCA-negative participants. Men consumed fewer fruits/vegetables/vitamins and more meat/alcohol than women (P 0.001).Population-based and FH-based AJ BRCA testing have similar long-term lifestyle impacts on smoking, alcohol, dietary fruit/vegetable/meat/vitamin, exercise, breast screening participation and reduced cancer risk perception.

Background/objective: Epidemiological and phenotypic differences exist in breast and ovarian cancers among patients of different ethnicity. Reasons for this are unclear and likely multifactorial. In Asian countries breast and ovarian cancers occur a decade earlier (1). In India, 15% of breast and ovarian cancer is diagnosed in patients over 65 years compared to approximately 50% in the UK, despite an increased UK incidence(1). Triple negative breast cancer is also more prevalent in India(1). A recent systematic review by our group examined the prevalence and spectra of BRCA1/2 mutations in the Indian population and concluded that there is a lack of high-quality genetic and histopathology data (2). This study examines BRCA mutation prevalence, spectra, and clinico-pathological features of breast and ovarian cancers in patients of Asian origin with familial risk. Methods: 89 patients of Asian origin were identified out of a total of 3359 patients referred to the regional genetics service for BRCA1/2 testing between 2006-2016. Genetics data were correlated with recorded histopathological including the type of surgical excision, tumour type, size, grade and receptor status (breast cancers). Outcome data, where available, was also recorded. The BRCA1/2 genetic variants were searched in the ClinVar (3) and BIC (Breast cancer Information Core)databases (4) to confirm their pathogenicity. Results: 51 patients were of Indian (57.30%), 21 of Pakistani (23.59%) and 3 of Bangladeshi (3.37%) origin with the remaining 14 of other unspecified or mixed Asian ethnicities (15.73%). 25 BRCA variants across 21 patients were identified. 16.85% (n=15) had a pathogenic variant (BRCA1 n= 11, BRCA2 n= 4) and 11.23% (n=10) had a variant of unknown significance (VUS) (BRCA1 n=1, BRCA2 n=9). One BRCA1 variant (c.3770_3771delAG, p.Glu1257GlyfsX9) occurred in 2 different patients, both of Indian origin. The Genome Aggregation Database (gnomAD) allele frequency of this variant in the South Asian population is reported as 3.27e-5. Pathology data for one or more parameters was available for 78 patients (87.64%). The majority were breast cancers, with a median age of 39 years. 90.90% of the BRCA1 and BRCA2 pathogenic variant breast cancers (PVBC) were of no special type (n =10, total 11). 87.50% of BRCA1 PVBCs (n=7, total 8) were HER2/oestrogen receptor (ER) negative and 85.71% were grade 3 (n=6, total 7). 100% (n=4, total 4) of BRCA2 PVBCs were grade 3 and ER positive. Two patients with a BRCA2 PVBC were male. Conclusion: This study shows, for the first time, the real-time genetic and pathological correlation of BRCA status in breast and ovarian cancer patients of Asian origin with familial risk. The higher number of pathogenic BRCA1 alterations compared to BRCA2 as well as the higher number of BRCA2 VUS compared to BRCA1 VUS is consistent with our previous work of the Indian population(2). The BRCA1/2 histological and molecular features are consistent with profiles reported in the literature. The genetic makeup may explain, at least partly, the phenotypic and epidemiological breast and ovarian cancer differences observed within different ethnicities. References: 1. Sundar S et al. Harnessing genomics to improve outcomes for women with cancer in India: key priorities for research. Lancet Oncol. 2018 Feb;19(2):e102-e112 2. Sharma-Oates A et al. Heterogeneity of germline variants in high risk breast and ovarian cancer susceptibility genes in India. Precis. Clin. Med. 2018 Sept; 1(2):75-87. 3. Landrum MJ et al. ClinVar: improving access to variant interpretations and supporting evidence. Nucleic Acids Res. 2018 Jan 4;46(D1):D1062-D1067. 4. Breast Cancer Information Core. An Open Access On-Line Breast Cancer Mutation Database. https://research.nhgri.nih.gov/bic/ Citation Format: Aisling Longworth, Jonathan Hoffman, Archana Sharma-Oates, Kai-Ren Ong, Rachel Hart, Yvonne Wallis, Anna Considine, Pooja Dasani, Raji Ganesan, Ian Tomlinson, Sudha Sundar, Abeer Shaaban. Genetics and pathological correlation of BRCA status in patients of Asian origin diagnosed with breast or ovarian cancer; a UK Reference Genetics Laboratory Study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-10-13.

Introduction/Background* Population-based BRCA-testing in the Ashkenazi Jewish (AJ) population is feasible, acceptable, reduces anxiety, identifies more BRCA-carriers and is cost-effective. The Jewish population is the first population for whom unselected population testing is now being implemented with Israel recently adopted this into national policy. Guideline change is being advocated in the UK and by others too. It is unknown whether BRCA-testing differs across Jewish denominational affiliations and religious or cultural outlook. We evaluate the association of Jewish cultural and religious identity and denominational affiliation with interest-in, intention-to undertake and uptake-of population-based BRCA-testing Methodology Design Cohort-study set within recruitment to GCaPPS-trial (ISRCTN73338115). AJ men and women, >18years, from London, self-referred, and attended recruitment clinics(clusters) for pre-test counselling. Subsequently consenting individuals underwent BRCA-testing. Mina oucome measures were: Interest, intention, uptake-of BRCA-testing. Participants self-identified to one Jewish denomination: Conservative/Liberal/Reform/Traditional/Orthodox/Unaffiliated. Validated scales measured Jewish Cultural-Identity (JI) and Jewish Religious-identity (JR). 4-item Likert-scales analysed initial ‘interest’ and ‘intention-to-test’ pre-counselling. Item-Response-Theory and graded-response-models, modelled responses to JI and JR scales. Ordered/multinomial logistic regression modelling evaluated association of JI-scale, JR-scale and Jewish Denominational affiliation on interest, intention and uptake-of BRCA-testing. Result(s)* 935 AJ women/men of mean-age=53.8 (S.D=15.02) years, received pre-test education and counselling through 256 recruitment clinic clusters (mean cluster size=3.64). Denominational affiliations included Conservative/Masorti=91(10.2%); Liberal=82(9.2%), Reform=135(15.1%), Traditional=212(23.7%), Orthodox=239(26.7%); and Unaffiliated/Non-practising=135(15.1%). Overall BRCA-testing uptake was 88%. Pre-counselling 96% expressed interest and 60% intention-to test. JI and JR scores were highest for Orthodox, followed by Conservative/Masorti, Traditional, Reform, Liberal and Unaffiliated Jewish denominations. Regression modelling showed no significant association between overall Jewish Cultural or Religious Identity with either interest, intention or uptake-of BRCA-testing. Interest, intention and uptake of BRCA-testing was not significantly associated with denominational affiliation. Conclusion* Jewish religious/cultural identity and denominational affiliation do not appear to influence interest, intention or uptake of population-based BRCA-testing. BRCA-testing was robust across all Jewish denominations.

OBJECTIVE To evaluate the association of Jewish cultural and religious identity and denominational affiliation with interest in, intention to undertake and uptake of population-based BRCA (Breast Cancer Gene)-testing. DESIGN Cohort-study set within recruitment to GCaPPS-trial (ISRCTN73338115). SETTING London Ashkenazi-Jewish (AJ) population. POPULATION OR SAMPLE AJ men and women, >18 years. METHODS Participants were self-referred, and attended recruitment clinics (clusters) for pre-test counselling. Subsequently consenting individuals underwent BRCA testing. Participants self-identified to one Jewish denomination: Conservative/Liberal/Reform/Traditional/Orthodox/Unaffiliated. Validated scales measured Jewish Cultural-Identity (JI) and Jewish Religious-identity (JR). Four-item Likert-scales analysed initial 'interest' and 'intention to test' pre-counselling. Item-Response-Theory and graded-response models, modelled responses to JI and JR scales. Ordered/multinomial logistic regression modelling evaluated association of JI-scale, JR-scale and Jewish Denominational affiliation on interest, intention and uptake of BRCA testing. MAIN OUTCOME MEASURES Interest, intention, uptake of BRCA testing. RESULTS In all, 935 AJ women/men of mean age = 53.8 (S.D = 15.02) years, received pre-test education and counselling through 256 recruitment clinic clusters (median cluster size = 3). Denominational affiliations included Conservative/Masorti = 91 (10.2%); Liberal = 82 (9.2%), Reform = 135 (15.1%), Traditional = 212 (23.7%), Orthodox = 239 (26.7%); and Unaffiliated/Non-practising = 135 (15.1%). Overall BRCA testing uptake was 88%. Pre-counselling, 96% expressed interest and 60% intention to test. JI and JR scores were highest for Orthodox, followed by Conservative/Masorti, Traditional, Reform, Liberal and Unaffiliated Jewish denominations. Regression modelling showed no significant association between overall Jewish Cultural or Religious Identity with either interest, intention or uptake of BRCA testing. Interest, intention and uptake of BRCA testing was not significantly associated with denominational affiliation. CONCLUSIONS Jewish religious/cultural identity and denominational affiliation do not appear to influence interest, intention or uptake of population-based BRCA testing. BRCA testing was robust across all Jewish denominations. TWEETABLE ABSTRACT Jewish cultural/religious factors do not affect BRCA testing, with robust uptake seen across all denominational affiliations.

The British Gynecological Cancer Society and the British Association of Gynecological Pathologists established a multidisciplinary consensus group comprising experts in surgical gynecological oncology, medical oncology, genetics, and laboratory science, and clinical nurse specialists to identify the optimal pathways to

Unselected population-based personalised ovarian cancer (OC) risk assessment combining genetic/epidemiology/hormonal data has not previously been undertaken. We aimed to perform a feasibility study of OC risk stratification of general population women using a personalised OC risk tool followed by risk management. Volunteers were recruited through London primary care networks. Inclusion criteria: women &ge, 18 years. Exclusion criteria: prior ovarian/tubal/peritoneal cancer, previous genetic testing for OC genes. Participants accessed an online/web-based decision aid along with optional telephone helpline use. Consenting individuals completed risk assessment and underwent genetic testing (BRCA1/BRCA2/RAD51C/RAD51D/BRIP1, OC susceptibility single-nucleotide polymorphisms). A validated OC risk prediction algorithm provided a personalised OC risk estimate using genetic/lifestyle/hormonal OC risk factors. Population genetic testing (PGT)/OC risk stratification uptake/acceptability, satisfaction, decision aid/telephone helpline use, psychological health and quality of life were assessed using validated/customised questionnaires over six months. Linear-mixed models/contrast tests analysed impact on study outcomes. Main outcomes: feasibility/acceptability, uptake, decision aid/telephone helpline use, satisfaction/regret, and impact on psychological health/quality of life. In total, 123 volunteers (mean age = 48.5 (SD = 15.4) years) used the decision aid, 105 (85%) consented. None fulfilled NHS genetic testing clinical criteria. OC risk stratification revealed 1/103 at &ge, 10% (high), 0/103 at &ge, 5%&ndash, &lt, 10% (intermediate), and 100/103 at &lt, 5% (low) lifetime OC risk. Decision aid satisfaction was 92.2%. The telephone helpline use rate was 13% and the questionnaire response rate at six months was 75%. Contrast tests indicated that overall depression (p = 0.30), anxiety (p = 0.10), quality-of-life (p = 0.99), and distress (p = 0.25) levels did not jointly change, while OC worry (p = 0.021) and general cancer risk perception (p = 0.015) decreased over six months. In total, 85.5&ndash, 98.7% were satisfied with their decision. Findings suggest population-based personalised OC risk stratification is feasible and acceptable, has high satisfaction, reduces cancer worry/risk perception, and does not negatively impact psychological health/quality of life.

Introduction/Background Unselected population-based BRCA-testing enables application of genomics on a population-scale to maximise primary-prevention for breast-&-ovarian cancer. We present factors affecting uptake and long-term follow-up results of the GCaPPS-trial comparing population-based and Family-History (FH)/Clinical-criteria based BRCA-testing. Methodology Design: Randomised-Controlled-Trial (ISRCTN73338115) GCaPPS, with two-arms: (a)Population-Screening (PS); (b)FH/Clinical-criteria based testing. Setting: North-London Ashkenazi-Jewish(AJ) population. Population-based RCT (1:1). Participants recruited through self-referral, from North-London AJ-population. Inclusion-criteria: AJ women/men >18years. Exclusion-criteria: prior BRCA-testing or first-degree-relatives of BRCA-carriers. Interventions: Pre-test counselling for BRCA-testing through recruitment-clinics (clusters). Genetic-testing for Jewish BRCA founder-mutations: 185delAG(c.68_69delAG), 5382insC(c.5266dupC) and 6174delT(c.5946delT), for (a)all participants in PS-arm; (b)those fulfilling FH/clinical-criteria in FH-arm. Validated questionnaires (HADS/MICRA/HAI/SF12) analysed psychological well-being/quality-of-life outcomes at baseline, 1-year, 2-years and 3-years follow-up. Linear mixed-models and appropriate contrast-tests analysed the impact of BRCA-testing on psychological and quality-of-life outcomes over 3-years. Socio-demographic/family-history/knowledge/psychological well-being data along-with benefits/risks/cultural-influences (18-item-questionnaire measuring ‘attitude’) were collected. Logistic-regression models evaluated factors affecting uptake/interest/intention-to undergo BRCA-testing. Results 1034 (women=691/men=343) participants randomized to PS (n=530) or FH (n=504) arms. There was a statistically significant decrease in anxiety(p=0.046) and total anxiety-&-depression scores(p=0.0.012) in the PS-arm compared to FH-arm over 3years. No significant difference was observed between FH/PS arms for depression, health-anxiety, distress, uncertainty, quality-of-life or experience scores associated with BRCA-testing. 18/30(60%) BRCA-carriers identified did not fulfil BRCA-testing clinical-criteria. The total BRCA-prevalence= 2.9%(CI:1.97%,4.12%) (BRCA1=1.55%(CI:0.89%,2.5%); BRCA2=1.35%(CI:0.74%,2.26%)). The BRCA-testing uptake was 88%. Being married/cohabiting had four-fold higher-odds for BRCA-testing uptake(p=0.009). Reduced uncertainty/reassurance were the most important factors affecting decision-making. Increased importance/concern towards risks/limitations (confidentiality/insurance/emotional-impact/inability to prevent cancer/marriage-ability/ethnic-focus/stigmatization) were significantly associated with lower-odds of undergoing BRCA-testing, discriminating between acceptors and decliners. Conclusion Population-based AJ BRCA-testing has high acceptability, doesn’t adversely affect long-term psychological well-being or quality-of-life, decreases anxiety and could identify up-to 150% additional BRCA-carriers. Pre-test counselling increases awareness of disadvantages/limitations of BRCA-testing, influencing final cost-benefit perception and decision-making on undergoing testing. Disclosure IJ and UM have a financial interest in Abcodia, Ltd., a company formed to develop academic and commercial development of biomarkers for screening and risk prediction. IJ is a member of the board of Abcodia Ltd, a Director of Women’s Health Specialists Ltd and received consultancy from Beckton Dickinson. RM declares research funding from The Eve Appeal and Cancer Research UK into population testing and from Barts & the London Charity and Rose Trees Trust outside this work, an honorarium for grant review from Israel National Institute for Health Policy Research and honorarium for advisory board membership from Astrazeneca/MSD. RM is supported by an NHS Innovation Accelerator (NIA) Fellowship for population testing. The other authors declare no conflict of interest.

Purpose To describe a snapshot of international genetic testing practices, specifically regarding the use of multigene panels, for hereditary breast/ovarian cancers. We conducted a survey through the Evidence-Based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium, covering questions about 16 non- BRCA1/ 2 genes. Methods Data were collected via in-person and paper/electronic surveys. ENIGMA members from around the world were invited to participate. Additional information was collected via country networks in the United Kingdom and in Italy. Results Responses from 61 cancer genetics practices across 20 countries showed that 16 genes were tested by > 50% of the centers, but only six ( PALB2, TP53, PTEN, CHEK2, ATM, and BRIP1) were tested regularly. US centers tested the genes most often, whereas United Kingdom and Italian centers with no direct ENIGMA affiliation at the time of the survey were the least likely to regularly test them. Most centers tested the 16 genes through multigene panels; some centers tested TP53, PTEN, and other cancer syndrome–associated genes individually. Most centers reported (likely) pathogenic variants to patients and would test family members for such variants. Gene-specific guidelines for breast and ovarian cancer risk management were limited and differed among countries, especially with regard to starting age and type of imaging and risk-reducing surgery recommendations. Conclusion Currently, a small number of genes beyond BRCA1/ 2 are routinely analyzed worldwide, and management guidelines are limited and largely based on expert opinion. To attain clinical implementation of multigene panel testing through evidence-based management practices, it is paramount that clinicians (and patients) participate in international initiatives that share panel testing data, interpret sequence variants, and collect prospective data to underpin risk estimates and evaluate the outcome of risk intervention strategies.

Background Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumour predisposition syndrome characterised by predisposition to cutaneous and uterine leiomyomata and renal cell carcinoma (RCC). Objective To define the clinical findings, molecular genetics, and prognosis in a cohort of 69 families with a fumarate hydratase (FH) pathogenic variant and/or clinical features of HLRCC. Design, setting, and participants Clinical and molecular findings were obtained for 185 individuals from 69 families from four UK regional genetics clinics. Outcome measurements and statistical analysis Ages at confirmed diagnoses, last dates of follow-up, and molecular results were attained for probands and relatives. To study the effect of potential ascertainment bias, phenotypes of probands and their affected relatives were compared. Results and limitations A germline FH variant (19 novel and 21 known, >50% missense variants) was identified in 68/69 probands and 90 relatives. Cutaneous leiomyomata occurred in 90/185 (48.6%) individuals (mean age 45.9 yr) and uterine leiomyomata in 33/107 (30.8%) females (mean age 35.0 yr). Of 185 individuals, 23 (12.4%) had a confirmed renal tumour, and histopathology where known (n = 18) was variable: seven clear cell RCCs, nine papillary RCCs (six of type 2), one collecting duct tumour, and one tumour with oncocytic cystic morphology. Mean age at symptomatic RCC diagnosis was 44.0 yr and median survival was 21.0 mo. Eighty-one individuals underwent 187 renal imaging surveillance scans; three stage 1 RCCs were detected. Mean survival of individuals diagnosed with stage 1/2 RCC was significantly longer than those diagnosed with stage 3/4 RCC (p = 0.0004). Conclusions Management of HLRCC is challenging as RCC occurs in a minority of cases but is highly aggressive. This large multicentre series has identified novel features and evidence that renal screening in HLRCC detects early-stage RCCs. Patient summary We show that hereditary leiomyomatosis and renal cell cancer is associated with a 21% lifetime risk of renal cell carcinoma (RCC; 95% confidence interval 8.2–37.1), and renal imaging screening detects early-stage RCC.

Objectives To evaluate the factors affecting interest, intention, uptake, and attitude towards unselected population-based BRCA-testing in the Ashkenazi Jewish (AJ) population. Methods Design: Cohort-study set within recruitment to the GCaPPS-trial (ISRCTN73338115). AJ women/men >18-years, from the North-London AJ-population were recruited through self-referral. AJ-women/men underwent pre-test counselling for BRCA-testing through recruitment clinics (clusters). Consenting individuals provided blood-sample for BRCA-testing. Socio-demographic/family-history/knowledge/psychological well-being data along-with benefits/risks/cultural-influences (18-item-questionnaire measuring ‘attitude’) were collected. 4-item likert-scales analysed initial ‘interest’ and ‘intention-to-test’ pre-counselling. Uni-&-multivariable logistic-regression-models evaluated factors affecting uptake/interest/intention-to undergo BRCA-testing. Statistical inference was based on cluster robust standard-errors and joint Wald-tests for significance. Item-Response-Theory and graded-response-models modelled responses to 18-item questionnaire. Main Outcome Measures: Interest, intention, uptake, attitude towards BRCA-testing Results 935 AJ women (67%) and men (33%) underwent pre-test genetic-counselling (mean-age=53.8(S.D=15.02) years). Pre-counselling 96% expressed interest but 60% had clear intention-to undergo BRCA-testing. Subsequently 88% opted for BRCA-testing. BRCA-related knowledge (p=0.013) and degree-level education(p=0.01) were positively and negatively (respectively) associated with intention-to-test. Being married/cohabiting had four-fold higher-odds for BRCA-testing uptake (p=0.009). Perceived benefits were associated with higher pre-counselling odds for interest and intention-to undergo BRCA-testing. Reduced uncertainty/reassurance were the most important factors contributing to decision-making. Increased importance/concern towards risks/limitations (confidentiality/insurance/emotional-impact/inability to prevent cancer/marriage-ability/ethnic-focus/stigmatization) were significantly associated with lower-odds of uptake-of BRCA-testing, and discriminated between acceptors and decliners. Having children had stronger (p=0.005) while male-gender/degree-level-education (p=0.001) had weaker, attitudes towards BRCA-testing. Conclusions BRCA testing in the AJ population has high acceptability. Pre-test counselling increases awareness of disadvantages/limitations of BRCA-testing, influencing the final cost-benefit perception and decision-making on undergoing testing.

International audience; GJA8 encodes connexin 50 (Cx50), a transmembrane protein involved in the formation of lens gap junctions. GJA8 mutations have been linked to early onset cataracts in humans and animal models. In mice, missense mutations and homozygous Gja8 deletions lead to smaller lenses and microphthalmia in addition to cataract, suggesting that Gja8 may play a role in both lens development and ocular growth. Following screening of GJA8 in a cohort of 426 individuals with severe congenital eye anomalies, primarily anophthalmia, microphthalmia and coloboma, we identified four known [p.(Thr39Arg), p.(Trp45Leu), p.(Asp51Asn), and p.(Gly94Arg)] and two novel [p.(Phe70Leu) and p.(Val97Gly)] likely pathogenic variants in seven families. Five of these co-segregated with cataracts and microphthalmia, whereas the variant p.(Gly94Arg) was identified in an individual with congenital aphakia, sclerocornea, microphthalmia and coloboma. Four missense variants of unknown or unlikely clinical significance were also identified. Furthermore, the screening of GJA8 structural variants in a subgroup of 188 individuals identified heterozygous 1q21 microdeletions in five families with coloboma and other ocular and/or extraocular findings. However, the exact genotype-phenotype correlation of these structural variants remains to be established. Our data expand the spectrum of GJA8 variants and associated phenotypes, confirming the importance of this gene in early eye development.

OBJECTIVE To evaluate factors affecting unselected population-based BRCA testing in Ashkenazi Jews (AJ). DESIGN Cohort-study set within recruitment to the GCaPPS trial (ISRCTN73338115). SETTING North London AJ population. POPULATION OR SAMPLE Ashkenazi Jews women/men >18 years, recruited through self-referral. METHODS Ashkenazi Jews women/men underwent pre-test counselling for BRCA testing through recruitment clinics (clusters). Consenting individuals provided blood samples for BRCA testing. Data were collected on socio-demographic/family history/knowledge/psychological well-being along with benefits/risks/cultural influences (18-item questionnaire measuring 'attitude'). Four-item Likert-scales analysed initial 'interest' and 'intention-to-test' pre-counselling. Uni- and multivariable logistic regression models evaluated factors affecting uptake/interest/intention to undergo BRCA testing. Statistical inference was based on cluster robust standard errors and joint Wald tests for significance. Item-Response Theory and graded-response models modelled responses to 18-item questionnaire. MAIN OUTCOME MEASURES Interest, intention, uptake, attitude towards BRCA testing. RESULTS A total of 935 individuals (women = 67%/men = 33%; mean age = 53.8 (SD = 15.02) years) underwent pre-test genetic-counselling. During the pre-counselling, 96% expressed interest in and 60% indicated a clear intention to undergo BRCA testing. Subsequently, 88% opted for BRCA testing. BRCA-related knowledge (P = 0.013) and degree-level education (P = 0.01) were positively and negatively (respectively) associated with intention-to-test. Being married/cohabiting had four-fold higher odds for BRCA testing uptake (P = 0.009). Perceived benefits were associated with higher pre-counselling odds for interest in and intention to undergo BRCA testing. Reduced uncertainty/reassurance were the most important factors contributing to decision-making. Increased importance/concern towards risks/limitations (confidentiality/insurance/emotional impact/inability to prevent cancer/marriage ability/ethnic focus/stigmatisation) were significantly associated with lower odds of uptake of BRCA testing, and discriminated between acceptors and decliners. Male gender/degree-level education (P = 0.001) had weaker correlations, whereas having children showed stronger (P = 0.005) associations with attitudes towards BRCA testing. CONCLUSIONS BRCA testing in the AJ population has high acceptability. Pre-test counselling increases awareness of disadvantages/limitations of BRCA testing, influencing final cost-benefit perception and decision-making on undergoing testing. TWEETABLE ABSTRACT BRCA testing in Ashkenazi Jews has high acceptability and uptake. Pre-test counselling facilitates informed decision-making.

Anophthalmia, microphthalmia, and coloboma are a genetically heterogeneous spectrum of developmental eye disorders and affect around 30 per 100,000 live births. OLFM2 encodes a secreted glycoprotein belonging to the noelin family of olfactomedin domain-containing proteins that modulate the timing of neuronal differentiation during development. OLFM2 SNPs have been associated with open angle glaucoma in a case-control study, and knockdown of Olfm2 in zebrafish results in reduced eye size. From a cohort of 258 individuals with developmental eye anomalies, we identified two with heterozygous variants in OLFM2: an individual with bilateral microphthalmia carrying a de novo 19p13.2 microdeletion involving OLFM2 and a second individual with unilateral microphthalmia and contralateral coloboma who had a novel single base change in the 5' untranslated region. Dual luciferase assays demonstrated that the latter variant causes a significant decrease in expression of OLFM2. Furthermore, RNA in situ hybridisation experiments using human developmental tissue revealed expression in relevant structures, including the lens vesicle and optic cup. Our study indicates that OLFM2 is likely to be important in mammalian eye development and disease and should be considered as a gene for human ocular anomalies.

Previous studies have documented the serotonin transporter linked polymorphic region (5-HTTLPR) as a genetic susceptibility variant that contributes to variability in outcomes related to affective psychopathology, with the short allele associated with negative affectivity and the long allele associated with positive affectivity. In a separate but related line of research, extensive evidence suggests that frontal electroencephalography (EEG) hemispheric asymmetry in the alpha band is also associated with risk for affective psychopathologies, with leftward asymmetry associated with approach-related behavior patterns and rightward frontal EEG asymmetry associated with withdrawn behavioral tendencies. We examined frontal EEG hemispheric asymmetries in relation to 5-HTTLPR genotyping in 70 children between 4 and 6 years of age. Analyses revealed that frontal EEG lateralization interacted with genotype such that children homozygous for the short allele exhibited rightward frontal EEG asymmetries, children who were homozygous for the long allele consistently exhibited a positive pattern of leftward asymmetry, and heterozygotes exhibited equivalent left and right frontal activity. These findings suggest that the 5-HTTLPR short allele may provide a degree of susceptibility for later affective psychopathology in adolescence and adulthood, through mediation of frontal brain activity that is associated with cognitive–behavioral withdrawal tendencies and negative affectivity.

Pheochromocytomas and paragangliomas are notable for a high frequency of inherited cases, many of which present as apparently sporadic tumors.The objective of this study was to establish a comprehensive next generation sequencing (NGS)-based strategy for the diagnosis of patients with pheochromocytoma and paraganglioma by testing simultaneously for mutations in MAX, RET, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, and VHL.After the methodology for the assay was designed and established, it was validated on DNA samples with known genotype and then patients were studied prospectively.The study was performed in a diagnostic genetics laboratory.DNA samples from 205 individuals affected with adrenal or extraadrenal pheochromocytoma/head and neck paraganglioma (PPGL/HNPGL) were analyzed. A proof-of-principle study was performed using 85 samples known to contain a variant in 1 or more of the genes to be tested, followed by prospective analysis of an additional 120 samples.We assessed the ability to use an NGS-based method to perform comprehensive analysis of genes implicated in inherited PPGL/HNPGL.The proof-of-principle study showed that the NGS assay and analysis gave a sensitivity of 98.7%. A pathogenic mutation was identified in 16.6% of the prospective analysis cohort of 120 patients.A comprehensive NGS-based strategy for the analysis of genes associated with predisposition to PPGL and HNPGL was established, validated, and introduced into diagnostic service. The new assay provides simultaneous analysis of 9 genes and allows more rapid and cost-effective mutation detection than the previously used conventional Sanger sequencing-based methodology.

The sensitivity of testing BRCA1 and BRCA2 remains unresolved as the frequency of deep intronic splicing variants has not been defined in high-risk familial breast/ovarian cancer families. This variant category is reported at significant frequency in other tumour predisposition genes, including NF1 and MSH2. We carried out comprehensive whole gene RNA analysis on 45 high-risk breast/ovary and male breast cancer families with no identified pathogenic variant on exonic sequencing and copy number analysis of BRCA1/2. In addition, we undertook variant screening of a 10-gene high/moderate risk breast/ovarian cancer panel by next-generation sequencing. DNA testing identified the causative variant in 50/56 (89%) breast/ovarian/male breast cancer families with Manchester scores of ≥50 with two variants being confirmed to affect splicing on RNA analysis. RNA sequencing of BRCA1/BRCA2 on 45 individuals from high-risk families identified no deep intronic variants and did not suggest loss of RNA expression as a cause of lost sensitivity. Panel testing in 42 samples identified a known RAD51D variant, a high-risk ATM variant in another breast ovary family and a truncating CHEK2 mutation. Current exonic sequencing and copy number analysis variant detection methods of BRCA1/2 have high sensitivity in high-risk breast/ovarian cancer families. Sequence analysis of RNA does not identify any variants undetected by current analysis of BRCA1/2. However, RNA analysis clarified the pathogenicity of variants of unknown significance detected by current methods. The low diagnostic uplift achieved through sequence analysis of the other known breast/ovarian cancer susceptibility genes indicates that further high-risk genes remain to be identified.

Familial medullary thyroid cancer (MTC) and its precursor, C cell hyperplasia (CCH), is associated with germline RET mutations causing multiple endocrine neoplasia type 2. However, some rare families with apparent MTC/CCH predisposition do not have a detectable RET mutation. To identify novel MTC/CCH predisposition genes we undertook exome resequencing studies in a family with apparent predisposition to MTC/CCH and no identifiable RET mutation. We identified a novel ESR2 frameshift mutation, c.948delT, which segregated with histological diagnosis following thyroid surgery in family members and demonstrated loss of ESR2-encoded ERβ expression in the MTC tumour. ERα and ERβ form heterodimers binding DNA at specific oestrogen-responsive elements (EREs) to regulate gene transcription. ERβ represses ERα-mediated activation of the ERE and the RET promoter contains three EREs. In vitro, we showed that ESR2 c.948delT results in unopposed ERα mediated increased cellular proliferation, activation of the ERE and increased RET expression. In vivo, immunostaining of CCH and MTC using an anti-RET antibody demonstrated increased RET expression. Together these findings identify germline ESR2 mutation as a novel cause of familial MTC/CCH and provide important insights into a novel mechanism causing increased RET expression in tumourigenesis.

BACKGROUND: Newer approaches to genetic counselling are required for population-based testing. We compare traditional face-to-face genetic counselling with a DVD-assisted approach for population-based BRCA1/2 testing. METHODS: A cluster-randomised non-inferiority trial in the London Ashkenazi Jewish population. INCLUSION CRITERIA: Ashkenazi Jewish men/women >18 years; exclusion criteria: (a) known BRCA1/2 mutation, (b) previous BRCA1/2 testing and (c) first-degree relative of BRCA1/2 carrier. Ashkenazi Jewish men/women underwent pre-test genetic counselling prior to BRCA1/2 testing in the Genetic Cancer Prediction through Population Screening trial (ISRCTN73338115). Genetic counselling clinics (clusters) were randomised to traditional counselling (TC) and DVD-based counselling (DVD-C) approaches. DVD-C involved a DVD presentation followed by shorter face-to-face genetic counselling. Outcome measures included genetic testing uptake, cancer risk perception, increase in knowledge, counselling time and satisfaction (Genetic Counselling Satisfaction Scale). Random-effects models adjusted for covariates compared outcomes between TC and DVD-C groups. One-sided 97.5% CI was used to determine non-inferiority. SECONDARY OUTCOMES: relevance, satisfaction, adequacy, emotional impact and improved understanding with the DVD; cost-minimisation analysis for TC and DVD-C approaches. RESULTS: 936 individuals (clusters=256, mean-size=3.6) were randomised to TC (n=527, clusters=134) and DVD-C (n=409, clusters=122) approaches. Groups were similar at baseline, mean age=53.9 (SD=15) years, women=66.8%, men=33.2%. DVD-C was non-inferior to TC for increase in knowledge (d=-0.07; lower 97.5% CI=-0.41), counselling satisfaction (d=-0.38, 97.5% CI=1.2) and risk perception (d=0.08; upper 97.5% CI=3.1). Group differences and CIs did not cross non-inferiority margins. DVD-C was equivalent to TC for uptake of genetic testing (d=-3%; lower/upper 97.5% CI -7.9%/1.7%) and superior for counselling time (20.4 (CI 18.7 to 22.2) min reduction (p

Background: Colorectal adenomatous and, probably, hyperplastic polyp development requires epithelial remodelling and stratification, with loss of E-cadherin expression implicated in adenoma formation. We have shown that P-cadherin, normally expressed in stratified epithelia and placenta, is aberrantly expressed in disturbed epithelial architecture associated with colitis. Aims: (i) To investigate the role of P-cadherin in colonic polyp formation. (ii) To ascertain whether expression of P-cadherin is independent of or correlated with expression of its associated proteins— E-cadherin, β-catenin, and γ-catenin. (iii) To determine if P-cadherin is functional regarding catenin binding in polyps. Methods: Expression and localisation of cadherins (E- and P-) and their associated catenins (β- and γ-) were determined in aberrant crypt foci (ACF), in polyps with hyperplastic morphology (hyperplastic polyps and serrated adenomas), and in adenomatous polyps by immunohistochemistry, western blotting, and mRNA in situ hybridisation. Assessment of cadherin-catenin binding was evaluated by co-immunoprecipitation. Adenomatous polyposis coli (APC) mutation was assessed in adenomatous polyps. Results: P-cadherin was expressed from ACF through to hyperplastic and adenomatous polyps. Alterations in E-cadherin and catenin expression occurred later, with variant patterns in (i) ACF, (ii) hyperplastic polyps and serrated adenomas, and (iii) adenomatous polyps. P-cadherin present in adenomas was functional with regard to catenin binding, and its expression was independent of APC mutational status. Conclusions: P-cadherin is aberrantly expressed from the earliest morphologically identifiable stage of colonocyte transformation, prior to changes in E-cadherin, catenin, and APC expression/mutation. P-cadherin expression alone does not predict tissue morphology, and such expression is independent of that of associated cadherins and catenins.

The metabotropic glutamate receptor 1 (mGluR1) is abundantly expressed in the mammalian central nervous system, where it regulates intracellular calcium homeostasis in response to excitatory signaling. Here, we describe heterozygous dominant mutations in GRM1, which encodes mGluR1, that are associated with distinct disease phenotypes: gain-of-function missense mutations, linked in two different families to adult-onset cerebellar ataxia, and a de novo truncation mutation resulting in a dominant-negative effect that is associated with juvenile-onset ataxia and intellectual disability. Crucially, the gain-of-function mutations could be pharmacologically modulated in vitro using an existing FDA-approved drug, Nitazoxanide, suggesting a possible avenue for treatment, which is currently unavailable for ataxias.

Fluorescent cycle sequencing of PCR products is a multistage process and several methodologies are available to perform each stage. This chapter will describe the more commonly utilised dye-terminator cycle sequencing approach using BigDye® terminator chemistry (Applied Biosystems) ready for analysis on a 3730 DNA genetic analyzer. Even though DNA sequencing is one of the most common and robust techniques performed in molecular laboratories it may not always produce desirable results. The causes of the most common problems will also be discussed in this chapter.

Fluorescent cycle sequencing of PCR products is a multistage process and several methodologies are available to perform each stage. This chapter will describe the more commonly utilised dye-terminator cycle sequencing approach using BigDye® terminator chemistry (Applied Biosystems) ready for analysis on a 3730 DNA genetic analyzer. Even though DNA sequencing is one of the most common and robust techniques performed in molecular laboratories it may not always produce desirable results. The causes of the most common problems will also be discussed in this chapter.

Regions of the short arm of chromosome 8 are deleted frequently in a range of solid tumors, indicating that tumor suppressor genes reside at these loci. In this study, we have examined the properties of the Wnt signaling antagonist secreted frizzled-related protein (sFRP) 1 as a candidate for this role at c8p11.2. An initial survey of 10 colorectal tumors, selected by the presence of isolated short deletions of the 8p11.2 region, identified three chain-terminating mutations, all within the first exon, which encodes the cysteine-rich domain. None of these tumors exhibited microsatellite instability, indicating intact mismatch repair gene function. The preserved sFRP1 alleles in the remaining seven tumors each contained a polymorphic three-base insertion in the signal sequence, but in a broader study, no association was found between this and the development of colorectal cancer. Epigenetic inhibition of sFRP1 transcription was investigated, and increased methylation of the promotor region was demonstrated in an additional cohort of 51 locally advanced colorectal cancers. Hypermethylation was identified in 40 of 49 (82%) cancers and in only 11 of 36 (30%) matched normal mucosal samples (P < 0.001). Semiquantitative analysis, by real-time PCR, of mRNA expression in 37 of the same cohort of 51 cancers revealed that sFRP1 mRNA expression was down-regulated in 28 (76%) cases compared with matched normal large bowel mucosa. The 3′ end of the sFRP1 mRNA also was found to be alternatively spliced, compared with the prototype liver and lung forms, in the colon and a number of other tissues, yielding an extended COOH terminus, which may influence its activity in a tissue-specific manner. The inactivation and down-regulation of sFRP1 observed are consistent with it acting as a tumor suppressor gene in colorectal carcinogenesis. Because β-catenin is constitutively active in the majority of colorectal tumors, it is unlikely that sFRP1 can act in the canonical Wnt response pathway. Therefore, we propose that the reduced activity or absence of sFRP1 allows the transduction of noncanonical Wnt signals, which contribute to tumor progression.

Butyrate is produced in the colon by fermentation of dietary fibre and induces apoptosis in colon adenoma and cancer cell lines, which may contribute to the protective effect of a high fibre diet against colorectal cancer (CRC). However, butyrate is present in the colon together with unconjugated bile acids, which are tumour promoters in the colon. We show here that bile acids deoxycholate (DCA) and chenodeoxycholate (CDCA), at levels present in the colon, gave a modest increase in cell proliferation and decreased spontaneous apoptosis in AA/C1 adenoma cells. Bile acids significantly inhibited the induction of apoptosis by butyrate in AA/C1 cells. However, the survival-inducing effects of bile acids on AA/C1 cells could be overcome by increasing the concentration of sodium butyrate. These results suggest that dysregulation of apoptosis in colonic epithelial cells by dietary factors is a key factor in the pathophysiology of CRC.

Mutations in the adenomatous polyposis coli (APC) gene are responsible for the disease familial adenomatous polyposis (FAP), a dominantly inherited predisposition to colorectal cancer. The most common extra-colonic manifestation is congenital hypertrophy of the retinal pigment epithelium (CHRPE), expressed in up to 90% of FAP kindreds. Chain-terminating APC mutations were characterised in 26 unrelated FAP patients. Results show that CHRPE expression is determined by the length of truncated protein product. CHRPE is therefore the first extracolonic manifestation of FAP to be shown to be under the control of the APC mutation site and should facilitate the detection of constitutional APC mutations in FAP kindreds.