Capucine Delnatte

High cumulative risks of cancer in patients with PTEN hamartoma tumour syndrome

Background PTEN hamartoma tumour syndrome (PHTS) encompasses several clinical syndromes with germline mutations in the PTEN tumour suppressor gene, including Cowden syndrome which is characterised by an increased risk of breast and thyroid cancers. Because PHTS is rare, data regarding cancer risks and genotype–phenotype correlations are limited. The objective of this study was to better define cancer risks in this syndrome with respect to the type and location of PTEN mutations. Methods 154 PHTS individuals with a deleterious germline PTEN mutation were recruited from the activity of the Institut Bergonié genetic laboratory. Detailed phenotypic information was obtained for 146 of them. Age and sex adjusted standardised incidence ratio (SIR) calculations, cumulative cancer risk estimations, and genotype–phenotype analyses were performed. Results Elevated SIRs were found mainly for female breast cancer (39.1, 95% CI 24.8 to 58.6), thyroid cancer in women (43.2, 95% CI 19.7 to 82.1) and in men (199.5, 95% CI 106.39 to 342.03), melanoma in women (28.3, 95% CI 7.6 to 35.4) and in men (39.4, 95% CI 10.6 to 100.9), and endometrial cancer (48.7, 95% CI 9.8 to 142.3). Cumulative cancer risks at age 70 were 85% (95% CI 70% to 95%) for any cancer, 77% (95% CI 59% to 91%) for female breast cancer, and 38% (95% CI 25% to 56%) for thyroid cancer. The risk of cancer was two times greater in women with PHTS than in men with PHTS (p<0.05). Conclusions This study shows a considerably high cumulative risk of cancer for patients with PHTS, mainly in women without clear genotype–phenotype correlation for this cancer risk. New recommendations for the management of PHTS patients are proposed.

Contiguous Gene Deletion within Chromosome Arm 10q Is Associated with Juvenile Polyposis of Infancy, Reflecting Cooperation between the BMPR1A and PTEN Tumor-Suppressor Genes

We describe four unrelated children who were referred to two tertiary referral medical genetics units between 1991 and 2005 and who are affected with juvenile polyposis of infancy. We show that these children are heterozygous for a germline deletion encompassing two contiguous genes, PTEN and BMPR1A. We hypothesize that juvenile polyposis of infancy is caused by the deletion of these two genes and that the severity of the disease reflects cooperation between these two tumor-suppressor genes.

Evidence for SMAD3 as a modifier of breast cancer risk in BRCA2 mutation carriers.

IntroductionCurrent attempts to identify genetic modifiers of BRCA1 and BRCA2 associated risk have focused on a candidate gene approach, based on knowledge of gene functions, or the development of large genome-wide association studies. In this study, we evaluated 24 SNPs tagged to 14 candidate genes derived through a novel approach that analysed gene expression differences to prioritise candidate modifier genes for association studies.MethodsWe successfully genotyped 24 SNPs in a cohort of up to 4,724 BRCA1 and 2,693 BRCA2 female mutation carriers from 15 study groups and assessed whether these variants were associated with risk of breast cancer in BRCA1 and BRCA2 mutation carriers.ResultsSNPs in five of the 14 candidate genes showed evidence of association with breast cancer risk for BRCA1 or BRCA2 carriers (P < 0.05). Notably, the minor alleles of two SNPs (rs7166081 and rs3825977) in high linkage disequilibrium (r2 = 0.77), located at the SMAD3 locus (15q22), were each associated with increased breast cancer risk for BRCA2 mutation carriers (relative risk = 1.25, 95% confidence interval = 1.07 to 1.45, Ptrend = 0.004; and relative risk = 1.20, 95% confidence interval = 1.03 to 1.40, Ptrend = 0.018).ConclusionsThis study provides evidence that the SMAD3 gene, which encodes a key regulatory protein in the transforming growth factor beta signalling pathway and is known to interact directly with BRCA2, may contribute to increased risk of breast cancer in BRCA2 mutation carriers. This finding suggests that genes with expression associated with BRCA1 and BRCA2 mutation status are enriched for the presence of common genetic modifiers of breast cancer risk in these populations.

Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers

IntroductionCis-acting regulatory single nucleotide polymorphisms (SNPs) at specific loci may modulate penetrance of germline mutations at the same loci by introducing different levels of expression of the wild-type allele. We have previously reported that BRCA2 shows differential allelic expression and we hypothesize that the known variable penetrance of BRCA2 mutations might be associated with this mechanism.MethodsWe combined haplotype analysis and differential allelic expression of BRCA2 in breast tissue to identify expression haplotypes and candidate cis-regulatory variants. These candidate variants underwent selection based on in silico predictions for regulatory potential and disruption of transcription factor binding, and were functionally analyzed in vitro and in vivo in normal and breast cancer cell lines. SNPs tagging the expression haplotypes were correlated with the total expression of several genes in breast tissue measured by Taqman and microarray technologies. The effect of the expression haplotypes on breast cancer risk in BRCA2 mutation carriers was investigated in 2,754 carriers.ResultsWe identified common haplotypes associated with differences in the levels of BRCA2 expression in human breast cells. We characterized three cis-regulatory SNPs located at the promoter and two intronic regulatory elements which affect the binding of the transcription factors C/EBPα, HMGA1, D-binding protein (DBP) and ZF5. We showed that the expression haplotypes also correlated with changes in the expression of other genes in normal breast. Furthermore, there was suggestive evidence that the minor allele of SNP rs4942440, which is associated with higher BRCA2 expression, is also associated with a reduced risk of breast cancer (per-allele hazard ratio (HR) = 0.85, 95% confidence interval (CI) = 0.72 to 1.00, P-trend = 0.048).ConclusionsOur work provides further insights into the role of cis-regulatory variation in the penetrance of disease-causing mutations. We identified small-effect genetic variants associated with allelic expression differences in BRCA2 which could possibly affect the risk in mutation carriers through altering expression levels of the wild-type allele.

Complete sex reversal in a WAGR syndrome patient.

The WAGR contiguous gene deletion syndrome is a combination of Wilms tumor, aniridia, genitourinary abnormalities, and mental retardation. Children with WAGR syndrome invariably have a constitutional chromosomal deletion at 11p13. WT1 haploinsufficiency is associated with a significant risk of Wilms tumor while PAX6 haploinsufficiency lead to aniridia, both genes located in the deleted region. The 46,XY patients with WAGR syndrome are often born with genital abnormalities such as cryptorchidism or hypospadias but more rarely ambiguous genitalia. To our knowledge, complete sex reversal has never been observed in WAGR syndrome patients. Here, we report on the clinical, cytogenetic, and molecular characterization of a child with WAGR syndrome and complete sex reversal. The young girl had female external and internal genitalia with normal uterus and fallopian tubes while the ovaries were not observed. Chromosomal analysis showed a 46,XY,del(11)(p12p14.1) karyotype. A 1‐Mb resolution array CGH experiment estimated the size of the interstitial deletion at ≈10 Mb encompassing WT1 and PAX6. The entire coding regions of WT1 and SRY have been sequenced and no mutation has been identified. Frasier syndrome (FS) and Denys‐Drash syndrome (DDS) are two disorders associated with mutations in the WT1 gene. Complete sex reversal is a feature usually present in FS and sometimes in DDS, but until now never observed in WAGR syndrome. The present report suggests that these conditions may be considered as part of the spectrum of disease due to WT1 gene alterations.

Breast and ovarian cancer predisposition due to de novo BRCA1 and BRCA2 mutations.

BRCA1 and BRCA2 are the two major genes predisposing to breast and ovarian cancer. Whereas high de novo mutation rates have been demonstrated for several genes, only 11 cases of de novo BRCA1/2 mutations have been reported to date and the BRCA1/2 de novo mutation rate remains unknown. The present study was designed to fill this gap based on a series of 12 805 consecutive unrelated patients diagnosed with breast and/or ovarian cancer who met the inclusion criteria for BRCA1/2 gene analysis according to French guidelines. BRCA1/2 mutations were detected in 1527 (12%) patients, and three BRCA1 mutations and one BRCA2 mutation were de novo. The BRCA1/2 de novo mutation rate was estimated to be 0.3% (0.1%; 0.7%). Although rare, it may be useful to take the possibility of de novo BRCA1/2 mutation into account in genetic counseling of relatives and to improve the understanding of complex family histories of breast and ovarian cancers.

Reply to Salviati et al.

To the Editor: In this issue, Salviati and colleagues report the case of an 8-year-old girl whose mild developmental delay and dysmorphic features led them to perform a high-resolution karyotype.1 A 12-Mb deletion located in 10q23 and encompassing the PTEN and BMPR1A loci was detected. The child presented with rectal bleeding at age 5 years. Colonoscopy at age 6 years uncovered >15 hamartomatous polyps throughout the entire colon, allowing the diagnosis of juvenile polyposis (JP). By operational definition, JP in patients younger than 6 years may be classified as “JP of infancy.” Thus, the rectal bleeding in this child at age 5 years was almost certainly due to the juvenile polyps uncovered only the following year; therefore, this case corroborates our report.2 However, the presentation and the disease course of the patient reported by Salviati et al.1 were not as severe as those of the four patients reported in our recent study2 and about which we hypothesize that the cooperation of the deletions of both tumor-suppressor genes PTEN and BMPR1A leads to severe JP and, hence, to cases of JP of infancy. The report by Salviati and colleagues1 is very interesting, illustrating that a very large deletion (12 Mb, compared with 2 Mb in patients 1 and 2 in our report2 and ∼4.8 Mb in the patient reported by Tsuchiya et al.3) may be, paradoxically, associated with a less severe disease course than a small deletion or a point mutation. As proposed by Salviati and colleagues,1 the protective effect of a modifier allele of a gene unlinked to the PTEN and BMPR1A genes may explain this phenomenon even if, at the present time, no candidate gene is suggested. Another explanation for this milder phenotype may be the increased expression level of the PTEN and BMPR1A genes by the remaining allele, which thus allows correction of the haploinsufficiency. Indeed, evidence is accumulating that allelic-specific expression is relatively common among nonimprinted autosomal genes and may be due to cis-acting, genetically inherited variations.4 Because of the very large size of the 10q22.3-q23 deletion in the patient reported by Salviati and colleagues,1 we speculate that, without the increased expression level by the remaining chromosome of some critical genes other than PTEN and BMPR1A located in the deleted region, this condition would have been lethal. Thus, we suggest that the affected child may have some cis-acting–specific sequences in the remaining allele that correct the haploinsufficiency in the 10q22.3-q23 region and lead, paradoxically, to an attenuated phenotype. The study by Salviati et al.1 illustrates how meticulous analyses of cases presenting with a similar disease are useful. Together with our original publication, this study should spur the international community to cooperatively and systematically study all JP syndrome diagnoses, the frequency of deletion of either or both genes, and their precise phenotypic manifestations, including age at onset and severity.

Performance of multiplicom's BRCA MASTR Dx kit on the detection of BRCA1 and BRCA2 mutations in fresh frozen ovarian and breast tumor samples

Next-generation sequencing (NGS) has enabled new approaches for detection of mutations in the BRCA1 and BRCA2 genes responsible for hereditary breast and ovarian cancer (HBOC). The search for germline mutations in the BRCA1 and BRCA2 genes is of importance with respect to oncogenetic and surgical (bilateral mastectomy, ovariectomy) counselling. Testing tumor material for BRCA mutations is of increasing importance for therapeutic decision making as the poly ADP ribose polymerase (PARP) inhibitor, olaparib, is now available to treat patients with specific forms of ovarian cancer and BRCA mutations. Molecular genetics laboratories should develop reliable and sensitive techniques for the complete analysis of the BRCA1 and BRCA2 genes. This is a challenge due to the size of the coding sequence of the BRCA1/2 genes, the absence of hot spot mutations, and particularly by the lower DNA quality obtained from Formalin-Fixed Paraffin-Embedded (FFPE) tissue. As a result, a number of analyses are uninterpretable and do not always provide a result to the clinician, limiting the optimal therapeutic management of patients. The availability of Fresh Frozen Tissue (FFT) for some laboratories and the excellent quality of the DNA extracted from it offers an alternative. For this reason, we evaluated Multiplicoms BRCA MASTR Dx assay on a set of 97 FFT derived DNA samples, in combination with the MID for Illumina MiSeq for BRCA1 and BRCA2 mutation detection. We obtained interpretable NGS results for all tested samples and showed > 99,7% sensitivity, specificity and accuracy.

Germline CDKN2A/P16INK4A mutations contribute to genetic determinism of sarcoma

Background Sarcomas are rare mesenchymal malignancies whose pathogenesis is poorly understood; both environmental and genetic risk factors could contribute to their aetiology. Methods and results We performed whole-exome sequencing (WES) in a familial aggregation of three individuals affected with soft-tissue sarcoma (STS) without TP53 mutation (Li-Fraumeni-like, LFL) and found a shared pathogenic mutation in CDKN2A tumour suppressor gene. We searched for individuals with sarcoma among 474 melanoma-prone families with a CDKN2A-/+ genotype and for CDKN2A mutations in 190 TP53-negative LFL families where the index case was a sarcoma. Including the initial family, eight independent sarcoma cases carried a germline mutation in the CDKN2A/p16INK4A gene. In five out of seven formalin-fixed paraffin-embedded sarcomas, heterozygosity was lost at germline CDKN2A mutations sites demonstrating complete loss of function. As sarcomas are rare in CDKN2A/p16INK4A carriers, we searched in constitutional WES of nine carriers for potential modifying rare variants and identified three in platelet-derived growth factor receptor (PDGFRA) gene. Molecular modelling showed that two never-described variants could impact the PDGFRA extracellular domain structure. Conclusion Germline mutations in CDKN2A/P16INK4A, a gene known to predispose to hereditary melanoma, pancreatic cancer and tobacco-related cancers, account also for a subset of hereditary sarcoma. In addition, we identified PDGFRA as a candidate modifier gene.

Novel diagnostic tool for prediction of variant spliceogenicity derived from a set of 395 combined in silico/in vitro studies: an international collaborative effort

Abstract Variant interpretation is the key issue in molecular diagnosis. Spliceogenic variants exemplify this issue as each nucleotide variant can be deleterious via disruption or creation of splice site consensus sequences. Consequently, reliable in silico prediction of variant spliceogenicity would be a major improvement. Thanks to an international effort, a set of 395 variants studied at the mRNA level and occurring in 5′ and 3′ consensus regions (defined as the 11 and 14 bases surrounding the exon/intron junction, respectively) was collected for 11 different genes, including BRCA1, BRCA2, CFTR and RHD, and used to train and validate a new prediction protocol named Splicing Prediction in Consensus Elements (SPiCE). SPiCE combines in silico predictions from SpliceSiteFinder-like and MaxEntScan and uses logistic regression to define optimal decision thresholds. It revealed an unprecedented sensitivity and specificity of 99.5 and 95.2%, respectively, and the impact on splicing was correctly predicted for 98.8% of variants. We therefore propose SPiCE as the new tool for predicting variant spliceogenicity. It could be easily implemented in any diagnostic laboratory as a routine decision making tool to help geneticists to face the deluge of variants in the next-generation sequencing era. SPiCE is accessible at (https://sourceforge.net/projects/spicev2-1/).