High yield of surveillance in patients diagnosed with constitutional mismatch repair deficiency.

BACKGROUND: Constitutional mismatch repair deficiency (CMMRD) is a rare autosomal recessively inherited syndrome that is caused by biallelic pathogenic variants of the mismatch repair genes. It is characterised by the development of multiple tumours in the first and second decade of life including brain, gastrointestinal and haematological tumours often resulting in early death. In order to improve the prognosis of these patients, the European collaborative group 'care for CMMRD' developed a surveillance programme in 2014 and established a registry of patients with CMMRD in Paris. The aim of the study was to evaluate the outcome of this programme. METHODS: Twenty-two patients with a definitive diagnosis of CMMRD and with at least one follow-up study were selected from the registry. Medical data on the outcome of surveillance were collected from these patients. RESULTS: During a mean follow-up of 4 years, the programme detected eight malignant tumours including three brain tumours, three upper gastrointestinal cancers and two colorectal cancers. Most tumours could successfully be treated. In addition, many adenomas were detected in the duodenum, and colorectum and subsequently removed. Seven patients developed a symptomatic malignancy, including two brain tumours, one small bowel cancer and four haematological malignancies. At the end of the follow-up, 16 out of 22 patients (73%) who participated in the surveillance programme were still alive. CONCLUSION: The study suggests a beneficial effect of surveillance of the digestive tract and brains.

Diagnostic chest X-rays and breast cancer risk among women with a hereditary predisposition to breast cancer unexplained by a BRCA1 or BRCA2 mutation.

BACKGROUND: Diagnostic ionizing radiation is a risk factor for breast cancer (BC). BC risk increases with increased dose to the chest and decreases with increased age at exposure, with possible effect modification related to familial or genetic predisposition. While chest X-rays increase the BC risk of BRCA1/2 mutation carriers compared to non-carriers, little is known for women with a hereditary predisposition to BC but who tested negative for a BRCA1 or BRCA2 (BRCA1/2) mutation. METHODS: We evaluated the effect of chest X-rays from diagnostic medical procedures in a dataset composed of 1552 BC cases identified through French family cancer clinics and 1363 unrelated controls. Participants reported their history of X-ray exposures in a detailed questionnaire and were tested for 113 DNA repair genes. Logistic regression and multinomial logistic regression models were used to assess the association with BC. RESULTS: Chest X-ray exposure doubled BC risk. A 3% increased BC risk per additional exposure was observed. Being 20 years old or younger at first exposure or being exposed before first full-term pregnancy did not seem to modify this risk. Birth after 1960 or carrying a rare likely deleterious coding variant in a DNA repair gene other than BRCA1/2 modified the effect of chest X-ray exposure. CONCLUSION: Ever/never chest X-ray exposure increases BC risk 2-fold regardless of age at first exposure and, by up to 5-fold when carrying 3 or more rare variants in a DNA repair gene. Further studies are needed to evaluate other DNA repair genes or variants to identify those which could modify radiation sensitivity. Identification of subpopulations that are more or less susceptible to ionizing radiation is important and potentially clinically relevant.

Familial breast cancer and DNA repair genes: Insights into known and novel susceptibility genes from the GENESIS study, and implications for multigene panel testing.

Pathogenic variants in BRCA1 and BRCA2 only explain the underlying genetic cause of about 10% of hereditary breast and ovarian cancer families. Because of cost-effectiveness, multigene panel testing is often performed even if the clinical utility of testing most of the genes remains questionable. The purpose of our study was to assess the contribution of rare, deleterious-predicted variants in DNA repair genes in familial breast cancer (BC) in a well-characterized and homogeneous population. We analyzed 113 DNA repair genes selected from either an exome sequencing or a candidate gene approach in the GENESIS study, which includes familial BC cases with no BRCA1 or BRCA2 mutation and having a sister with BC (N = 1,207), and general population controls (N = 1,199). Sequencing data were filtered for rare loss-of-function variants (LoF) and likely deleterious missense variants (MV). We confirmed associations between LoF and MV in PALB2, ATM and CHEK2 and BC occurrence. We also identified for the first time associations between FANCI, MAST1, POLH and RTEL1 and BC susceptibility. Unlike other associated genes, carriers of an ATM LoF had a significantly higher risk of developing BC than carriers of an ATM MV (ORLoF = 17.4 vs. ORMV = 1.6; p Het = 0.002). Hence, our approach allowed us to specify BC relative risks associated with deleterious-predicted variants in PALB2, ATM and CHEK2 and to add MAST1, POLH, RTEL1 and FANCI to the list of DNA repair genes possibly involved in BC susceptibility. We also highlight that different types of variants within the same gene can lead to different risk estimates.

Ovarian Clear Cell Carcinoma in Cowden Syndrome.

Cowden syndrome (CS) is an autosomal dominant mendelian disease related to germline pathogenic variants affecting the PTEN-gene. CS is characterized by macrocephaly, mucocutaneous lesions, and an increased risk of breast and thyroid cancers. Rare ovarian cancer cases (mostly embryonic tumors) associated with PTEN have been described in the literature, but no current CS guidelines are available for ovarian cancer risk management. We report on a woman diagnosed with ovarian clear cell carcinoma (OCCC) at 28 years of age. The patient displayed macrocephaly, trichilemmomas, oral papillomatosis, and acral keratosis. A family history of multiple cancer cases within the PTEN-related tumor spectrum was identified. In addition, PET scan and fine-needle biopsy results led to a diagnosis of thyroid follicular neoplasia. PTEN sequencing revealed that she carried a germline inherited pathogenic variant in exon 5 c.388C>T, p.(Arg130*) (NM_000314). Somatic mismatch repair immunohistochemistry analysis showed normal expression, and germline BRCA1/2 sequencing did not reveal pathogenic or likely pathogenic variants. An ovarian cell immunohistochemistry analysis reported total loss of PTEN expression, which strongly suggested the role of PTEN in the oncogenesis of this cancer. Hence, a total thyroid resection was performed instead of thyroid lobectomy and a risk-reducing bilateral mastectomy was discussed. Co-occurrence of this pathogenic germline mutation in PTEN in this patient, early development of OCCC at age 28 years, and total loss of PTEN expression in the tumor might support the involvement of PTEN in the carcinogenesis of her ovarian cancer. We describe a new ovarian cancer case with an atypical histologic type-clear cell carcinoma-in CS. This observation might be the first indication of the need to expand the PTEN-related tumor spectrum to incorporate OCCC. The CS diagnosis significantly changed the therapeutic outcome of this patient.

Diagnosis of Constitutional Mismatch Repair-Deficiency Syndrome Based on Microsatellite Instability and Lymphocyte Tolerance to Methylating Agents.

BACKGROUND & AIMS: Patients with bi-allelic germline mutations in mismatch repair (MMR) genes (MLH1, MSH2, MSH6, or PMS2) develop a rare but severe variant of Lynch syndrome called constitutional MMR deficiency (CMMRD). This syndrome is characterized by early-onset colorectal cancers, lymphomas or leukemias, and brain tumors. There is no satisfactory method for diagnosis of CMMRD because screens for mutations in MMR genes are noninformative for 30% of patients. MMR-deficient cancer cells are resistant to genotoxic agents and have microsatellite instability (MSI), due to accumulation of errors in repetitive DNA sequences. We investigated whether these features could be used to identify patients with CMMRD. METHODS: We examined MSI by PCR analysis and tolerance to methylating or thiopurine agents (functional characteristics of MMR-deficient tumor cells) in lymphoblastoid cells (LCs) from 3 patients with CMMRD and 5 individuals with MMR-proficient LCs (controls). Using these assays, we defined experimental parameters that allowed discrimination of a series of 14 patients with CMMRD from 52 controls (training set). We then used the same parameters to assess 23 patients with clinical but not genetic features of CMMRD. RESULTS: In the training set, we identified parameters, based on MSI and LC tolerance to methylation, that detected patients with CMMRD vs controls with 100% sensitivity and 100% specificity. Among 23 patients suspected of having CMMRD, 6 had MSI and LC tolerance to methylation (CMMRD highly probable), 15 had neither MSI nor LC tolerance to methylation (unlikely to have CMMRD), and 2 were considered doubtful for CMMRD based on having only 1 of the 2 features. CONCLUSION: The presence of MSI and tolerance to methylation in LCs identified patients with CMMRD with 100% sensitivity and specificity. These features could be used in diagnosis of patients.

GENESIS: a French national resource to study the missing heritability of breast cancer.

BACKGROUND: Less than 20% of familial breast cancer patients who undergo genetic testing for BRCA1 and BRCA2 carry a pathogenic mutation in one of these two genes. The GENESIS (GENE SISter) study was designed to identify new breast cancer susceptibility genes in women attending cancer genetics clinics and with no BRCA1/2 mutation. METHODS: The study involved the French national network of family cancer clinics. It was based on enrichment in genetic factors of the recruited population through case selection relying on familial criteria, but also on the consideration of environmental factors and endophenotypes like mammary density or tumor characteristics to assess potential genetic heterogeneity. One of the initial aims of GENESIS was to recruit affected sibpairs. Siblings were eligible when index cases and at least one affected sister were diagnosed with infiltrating mammary or ductal adenocarcinoma, with no BRCA1/2 mutation. In addition, unrelated controls and unaffected sisters were recruited. The enrolment of patients, their relatives and their controls, the collection of the clinical, epidemiological, familial and biological data were centralized by a coordinating center. RESULTS: Inclusion of participants started in February 2007 and ended in December 2013. A total of 1721 index cases, 826 affected sisters, 599 unaffected sisters and 1419 controls were included. 98% of participants completed the epidemiological questionnaire, 97% provided a blood sample, and 76% were able to provide mammograms. Index cases were on average 59 years old at inclusion, were born in 1950, and were 49.7 years of age at breast cancer diagnosis. The mean age at diagnosis of affected sisters was slightly higher (51.4 years). The representativeness of the control group was verified. CONCLUSIONS: The size of the study, the availability of biological specimens and the clinical data collection together with the detailed and complete epidemiological questionnaire make this a unique national resource for investigation of the missing heritability of breast cancer, by taking into account environmental and life style factors and stratifying data on endophenotypes to decrease genetic heterogeneity.

The UMD-APC database, a model of nation-wide knowledge base: update with data from 3,581 variations.

Familial adenomatous polyposis (FAP) is a rare autosomal-inherited disease that highly predisposes to colorectal cancer, characterized by a diffuse duodenal and colorectal polyposis associated with various extradigestive tumors and linked to germline mutations within the APC gene. A French consortium of laboratories involved in APC mutation screening has progressively improved the description of the variation spectrum, inferred functional significance of nontruncating variations, and delineated phenotypic characteristics of the disease. The current version of the UMD-APC database is described here. The total number of variations has risen to 5,453 representing 1,473 distinct variations. The published records initially registered into the database were extended with 3,581 germline variations found through genetic testing performed by the eight licensed laboratories belonging to the French APC network. Sixty six of 149 variations of previously unknown significance have now been classified as (likely) causal or neutral. The database is available on the Internet (http://www.umd.be/APC/) and updated twice per year according to the consensus rules of the network. The UMD-APC database is thus expected to facilitate functional classification of rare synonymous, nonsynonymous, and intronic mutations and consequently improve genetic counseling and medical care in FAP families.

Germline mutations of inhibins in early-onset ovarian epithelial tumors.

To identify novel genetic bases of early-onset epithelial ovarian tumors, we used the trio exome sequencing strategy in a patient without familial history of cancer who presented metastatic serous ovarian adenocarcinomas at 21 years of age. We identified a single de novo mutation (c.1157A>G/p.Asn386Ser) within the INHBA gene encoding the ßA-subunit of inhibins/activins, which play a key role in ovarian development. In vitro, this mutation alters the ratio of secreted activins and inhibins. In a second patient with early-onset serous borderline papillary cystadenoma, we identified an unreported germline mutation (c.179G>T/p.Arg60Leu) of the INHA gene encoding the α-subunit, the partner of the ßA-subunit. This mutation also alters the secreted activin/inhibin ratio, by disrupting both inhibin A and inhibin B biosynthesis. In a cohort of 62 cases, we detected an additional unreported germline mutation of the INHBA gene (c.839G>A/p.Gly280Glu). Our results strongly suggest that inhibin mutations contribute to the genetic determinism of epithelial ovarian tumors.

Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants.

BACKGROUND: CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype. METHODS: All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL). Phenotypic data related to primary melanoma and pigmentation characteristics were collected. The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced. RESULTS: Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation. The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years. Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM). A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects. CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (p<0.001). MPM subjects had a higher frequency of MC1R red hair colour variants compared with subjects with one tumour (p=0.010). CONCLUSION: Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM. In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.

Variation in breast cancer risk associated with factors related to pregnancies according to truncating mutation location, in the French National BRCA1 and BRCA2 mutations carrier cohort (GENEPSO).

INTRODUCTION: Mutations in BRCA1 and BRCA2 confer a high risk of breast cancer (BC), but the magnitude of this risk seems to vary according to the study and various factors. Although controversial, there are data to support the hypothesis of allelic risk heterogeneity. METHODS: We assessed variation in BC risk according to factors related to pregnancies by location of mutation in the homogeneous risk region of BRCA1 and BRCA2 in 990 women in the French study GENEPSO by using a weighted Cox regression model. RESULTS: Our results confirm the existence of the protective effect of an increasing number of full-term pregnancies (FTPs) toward BC among BRCA1 and BRCA2 mutation carriers (≥3 versus 0 FTPs: hazard ratio (HR) = 0.51, 95% confidence interval (CI) = 0.33 to 0.81). Additionally, the HR shows an association between incomplete pregnancies and a higher BC risk, which reached 2.39 (95% CI = 1.28 to 4.45) among women who had at least three incomplete pregnancies when compared with women with zero incomplete pregnancies. This increased risk appeared to be restricted to incomplete pregnancies occurring before the first FTP (HR = 1.77, 95% CI = 1.19 to 2.63). We defined the TMAP score (defined as the Time of Breast Mitotic Activity during Pregnancies) to take into account simultaneously the opposite effect of full-term and interrupted pregnancies. Compared with women with a TMAP score of less than 0.35, an increasing TMAP score was associated with a statistically significant increase in the risk of BC (P trend = 0.02) which reached 1.97 (95% CI = 1.19 to 3.29) for a TMAP score >0.5 (versus TMAP ≤0.35). All these results appeared to be similar in BRCA1 and BRCA2. Nevertheless, our results suggest a variation in BC risk associated with parity according to the location of the mutation in BRCA1. Indeed, parity seems to be associated with a significantly decreased risk of BC only among women with a mutation in the central region of BRCA1 (low-risk region) (≥1 versus 0 FTP: HR = 0.27, 95% CI = 0.13 to 0.55) (Pinteraction <10-3). CONCLUSIONS: Our findings show that, taking into account environmental and lifestyle modifiers, mutation position might be important for the clinical management of BRCA1 and BRCA2 mutation carriers and could also be helpful in understanding how BRCA1 and BRCA2 genes are involved in BC.

TUMOSPEC: A Nation-Wide Study of Hereditary Breast and Ovarian Cancer Families with a Predicted Pathogenic Variant Identified through Multigene Panel Testing.

Assessment of age-dependent cancer risk for carriers of a predicted pathogenic variant (PPV) is often hampered by biases in data collection, with a frequent under-representation of cancer-free PPV carriers. TUMOSPEC was designed to estimate the cumulative risk of cancer for carriers of a PPV in a gene that is usually tested in a hereditary breast and ovarian cancer context. Index cases are enrolled consecutively among patients who undergo genetic testing as part of their care plan in France. First- and second-degree relatives and cousins of PPV carriers are invited to participate whether they are affected by cancer or not, and genotyped for the familial PPV. Clinical, family and epidemiological data are collected, and all data including sequencing data are centralized at the coordinating centre. The three-year feasibility study included 4431 prospective index cases, with 19.1% of them carrying a PPV. When invited by the coordinating centre, 65.3% of the relatives of index cases (5.7 relatives per family, on average) accepted the invitation to participate. The study logistics were well adapted to clinical and laboratory constraints, and collaboration between partners (clinicians, biologists, coordinating centre and participants) was smooth. Hence, TUMOSPEC is being pursued, with the aim of optimizing clinical management guidelines specific to each gene.

Coexistence of schwannomatosis and glioblastoma in two families.

Schwannomatosis is a rare affection predisposing to multiple peripheral neurologic tumors development. Approximatively, one third of patients with schwannomatosis are carriers of a germline mutation in LZTR1 (Leucin Zipper Transcription Regulator 1). Tumorigenesis in schwannomatosis responds to a somatic 5-hit/3-step mechanism resulting in a loss of function (LOF) of LZTR1 and the contiguous genes of locus 22q11.2q12.2. Effectively, LZTR1 is mapped on 22q11.2 and centromeric to SMARCB1 also implicated in the determinism of schwannomatosis and NF2, responsible for neurofibromatosis type 2. On a somatic point of view, LZTR1 mutations are known to drive with a significant frequency glioblastoma (GB) development. We report here two families in which segregate both multiple schwannomas and GB. In the first family, the proband received a diagnosis with of schwannomatosis after a surgery for a lumbar schwannoma at age 43, molecularly confirmed by identification of a germline heterozygous mutation in LZTR1. Her father, having unremarkable medical history deceased from an apparently isolated GB at age 59. In the second family, LZTR1-related schwannomatosis was diagnosed in the index case at age 70 after multiple schwannomas surgeries. Her elder sister had no neurological medical history before occurrence of a lethal GB at age 78. Molecular analysis of GB sample from both affected relatives showed the presence of the familial mutation. These observations hypothesize a potential link between schwannomatosis and the GB development.

Optimization of the diagnosis of inherited colorectal cancer using NGS and capture of exonic and intronic sequences of panel genes.

We have developed and validated for the diagnosis of inherited colorectal cancer (CRC) a massive parallel sequencing strategy based on: (i) fast capture of exonic and intronic sequences from ten genes involved in Mendelian forms of CRC (MLH1, MSH2, MSH6, PMS2, APC, MUTYH, STK11, SMAD4, BMPR1A and PTEN); (ii) sequencing on MiSeq and NextSeq 500 Illumina platforms; (iii) a bioinformatic pipeline that includes BWA-Picard-GATK (Broad Institute) and CASAVA (Illumina) in parallel for mapping and variant calling, Alamut Batch (Interactive BioSoftware) for annotation, CANOES for CNV detection and finally, chimeric reads analysis for the detection of other types of structural variants (SVs). Analysis of 1644 new index cases allowed the identification of 323 patients with class 4 or 5 variants, corresponding to a 20% disease-causing variant detection rate. This rate reached 37% in patients with Lynch syndrome, suspected on the basis of tumour analyses. Thanks to this strategy, we detected overlapping phenotypes (e.g., MUTYH biallelic mutations mimicking Lynch syndrome), mosaic alterations and complex SVs such as a genomic deletion involving the last BMPR1A exons and PTEN, an Alu insertion within MSH2 exon 8 and a mosaic deletion of STK11 exons 3-10. This strategy allows, in a single step, detection of all types of CRC gene alterations including SVs and provides a high disease-causing variant detection rate, thus optimizing the diagnosis of inherited CRC.

BRCA1 allele-specific expression in genetic predisposed breast/ovarian cancer.

Germline allele specific expression (ASE), resulting in a lowered expression of one of the BRCA1 alleles, has been described as a possible predisposition marker in Hereditary Breast or Ovarian Cancer (HBOC), usable for molecular diagnosis in HBOC. The main objective of this prospective case-control study was to compare the proportion of ASE between controls without familial history of breast or ovarian cancer, and HBOC cases without BRCA1 or BRCA2 deleterious mutation. BRCA1 ASE evaluated on three SNPs among controls and HBOC patients without deleterious mutation were assessed by pyrosequencing. The allelic ratios and the proportion of ASE were compared between controls and cases using a Student's t test and a Fisher exact test, respectively. The linearity and reproducibility of the ASE dosage was demonstrated with R2 > 0.99 and a coefficient of variation below 10 %, and ASE was detected in two positive controls harbouring BRCA1 truncated mutations. In the heterozygote population, composed of 99/264 controls (37.5 %) and 96/227 patients (42.3 %), we detected a 5 % ASE without truncated mutations, in each population. We failed to detect any significant difference of ASE between controls and patients. So far, BRCA1 Allelic specific expression is not usable in routine diagnosis as a possible predisposition marker in HBOC patients except for the detection of truncated mutations.

Revisiting Li-Fraumeni Syndrome From TP53 Mutation Carriers.

PURPOSE: The aim of the study was to update the description of Li-Fraumeni syndrome (LFS), a remarkable cancer predisposition characterized by extensive clinical heterogeneity. PATIENTS AND METHODS: From 1,730 French patients suggestive of LFS, we identified 415 mutation carriers in 214 families harboring 133 distinct TP53 alterations and updated their clinical presentation. RESULTS: The 322 affected carriers developed 552 tumors, and 43% had developed multiple malignancies. The mean age of first tumor onset was 24.9 years, 41% having developed a tumor by age 18. In childhood, the LFS tumor spectrum was characterized by osteosarcomas, adrenocortical carcinomas (ACC), CNS tumors, and soft tissue sarcomas (STS) observed in 30%, 27%, 26%, and 23% of the patients, respectively. In adults, the tumor distribution was characterized by the predominance of breast carcinomas observed in 79% of the females, and STS observed in 27% of the patients. The TP53 mutation detection rate in children presenting with ACC or choroid plexus carcinomas, and in females with breast cancer before age 31 years, without additional features indicative of LFS, was 45%, 42% and 6%, respectively. The mean age of tumor onset was statistically different (P < .05) between carriers harboring dominant-negative missense mutations (21.3 years) and those with all types of loss of function mutations (28.5 years) or genomic rearrangements (35.8 years). Affected children, except those with ACC, harbored mostly dominant-negative missense mutations. CONCLUSION: The clinical gradient of the germline TP53 mutations, which should be validated by other studies, suggests that it might be appropriate to stratify the clinical management of LFS according to the class of the mutation.

Breast Cancer Risk Associated with Estrogen Exposure and Truncating Mutation Location in BRCA1/2 Carriers.

BACKGROUND: Mutations in BRCA1/2 confer a high risk of breast cancer, but literature values of this risk vary. A genotype-phenotype correlation has been found in both genes, and the effect of reproductive factors differs according to mutation location. Therefore, we hypothesize that such a variation may exist for other factors related to estrogen exposure. METHODS: We used a weighted Cox regression model to assess variation in breast cancer risk with these factors using location of mutation in homogeneous breast cancer risk region of BRCA1/2 in the GENEPSO study. RESULTS: We found that late age at menarche reduced breast cancer risk by 31% and that among BRCA1 carriers, a long or a short menstrual cycle increased risk (by 65% and 73%, respectively). Among premenopausal women, overweight was associated with a 45% decrease in risk whereas underweight was associated with an increased risk (HR, 2.40). A natural menopause, mainly after age 50, was associated with a high breast cancer risk (HR, 2.46), and a significant interaction between menopause status and the location of mutations was found leading up to 10% variation in absolute risk according to the age at menopause. CONCLUSIONS: As observed in the general population, a late menarche, a long or a short menstrual cycle, over- or underweight, and being postmenopausal were associated with breast cancer risk in BRCA1/2 carriers. The association with the menopause was observed only when the mutation was located in the "high-risk" zones. IMPACT: Taking into account modifier factors, location of mutation might be important for the clinical management of BRCA1/2 mutation carriers.

Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes.

To optimize the molecular diagnosis of hereditary breast and ovarian cancer (HBOC), we developed a next-generation sequencing (NGS)-based screening based on the capture of a panel of genes involved, or suspected to be involved in HBOC, on pooling of indexed DNA and on paired-end sequencing in an Illumina GAIIx platform, followed by confirmation by Sanger sequencing or MLPA/QMPSF. The bioinformatic pipeline included CASAVA, NextGENe, CNVseq and Alamut-HT. We validated this procedure by the analysis of 59 patients' DNAs harbouring SNVs, indels or large genomic rearrangements of BRCA1 or BRCA2. We also conducted a blind study in 168 patients comparing NGS versus Sanger sequencing or MLPA analyses of BRCA1 and BRCA2. All mutations detected by conventional procedures were detected by NGS. We then screened, using three different versions of the capture set, a large series of 708 consecutive patients. We detected in these patients 69 germline deleterious alterations within BRCA1 and BRCA2, and 4 TP53 mutations in 468 patients also tested for this gene. We also found 36 variations inducing either a premature codon stop or a splicing defect among other genes: 5/708 in CHEK2, 3/708 in RAD51C, 1/708 in RAD50, 7/708 in PALB2, 3/708 in MRE11A, 5/708 in ATM, 3/708 in NBS1, 1/708 in CDH1, 3/468 in MSH2, 2/468 in PMS2, 1/708 in BARD1, 1/468 in PMS1 and 1/468 in MLH3. These results demonstrate the efficiency of NGS in performing molecular diagnosis of HBOC. Detection of mutations within other genes than BRCA1 and BRCA2 highlights the genetic heterogeneity of HBOC.

Diversity of the clinical presentation of the MMR gene biallelic mutations.

Constitutional mismatch repair-deficiency, due to biallelic mutations of MMR genes, results in a tumour spectrum characterized by leukaemias, lymphomas, brain tumours and adenocarcinomas of the gastro-intestinal tract, occurring mostly in childhood. We report here two families illustrating the phenotypic diversity associated with biallelic MMR mutations. In the first family, two siblings developed six malignancies including glioblastoma, lymphoblastic T cell lymphoma, rectal and small bowel adenocarcinoma with onset as early as 6 years of age. We showed that this dramatic clinical presentation was due to the presence of two complex genomic PMS2 deletions in each patient predicted to result into complete PMS2 inactivation. In the second family, the index case presented with an early form of Lynch syndrome with colorectal adenocarcinomas at ages 17 and 20 years, and urinary tract tumours at the age of 25 years. We identified in this patient two MSH6 mutations corresponding to a frameshift deletion and an in frame deletion. The latter was not predicted to result into complete inactivation of MSH6. These reports show that the clinical expression of biallelic MMR mutations depends on the biological impact of the second MMR mutation and that, in clinical practice, the presence of a second MMR mutation located in trans should also be considered in patients suspected to present a Lynch syndrome with an unusual early-onset of tumours.