Clinical versus molecular diagnosis of Gorlin syndrome: Relevance of diagnostic criteria depends on the age of patients.

BACKGROUND: Gorlin Syndrome (GS) is an autosomal dominant disorder characterised by a predisposition to basal cell carcinoma and developmental defects, and caused by pathogenic variants in PTCH1 or SUFU genes. OBJECTIVES: To ascertain the efficiency of molecular screening in a cohort of patients with a suspicion of GS and to describe patients' clinical and genetic characteristics. METHODS: 110 patients with a suspicion of GS, addressed to the Genetic Department of Bichat University Hospital for molecular screening were studied. Patients' clinical and paraclinical data were collected and analysed according to Evan's diagnosis criteria, and compared to molecular information. RESULTS: Among 110 probands, only 56% fulfill Evan's diagnosis criteria. 75% of patients who fulfill those criteria carry a PTCH1/SUFU pathogenic variation. We compared clinical and paraclinical data of 54 probands carrying a PTCH1/SUFU mutation with 56 probands without identified mutation. Among patients carrying a pathogenic variation in PTCH1 or SUFU genes, 30 years of age appears to be the cut off age after which all patients have a clear clinical GS. Indeed, after 30 years, all patients carrying a PTCH1/SUFU mutation fulfill the diagnosis criteria of Evans (82% meet the clinical criteria, and we reach 100% with complementary exams such as X-rays and ultrasound). Before 30 years of age, only 37% of mutated patients fulfilled the clinical diagnosis criteria's and we only reach 62% with simple complementary exams. Furthermore, we report 22 new mutations in PTCH1. CONCLUSIONS: Molecular screening of patients with GS who do not fulfill Evan's diagnostic criteria should only be offered in first intention to patients under 30 years of age. After 30 years, a careful clinical examination and complementary radiological exams should be enough to eliminate the diagnosis of GS among patients who do not fulfill diagnostic criteria.

The BRCA2 R2645G variant increases DNA binding and induces hyper-recombination.

BRCA2 tumor suppressor protein ensures genome integrity by mediating DNA repair via homologous recombination (HR). This function is executed in part by its canonical DNA binding domain located at the C-terminus (BRCA2CTD), the only folded domain of the protein. Most germline pathogenic missense variants are located in this highly conserved region which binds to single-stranded DNA (ssDNA) and to the acidic protein DSS1. These interactions are essential for the HR function of BRCA2. Here, we report that the variant R2645G, identified in breast cancer and located at the DSS1 interface, unexpectedly increases the ssDNA binding activity of BRCA2CTDin vitro. Human cells expressing this variant display a hyper-recombination phenotype, chromosomal instability in the form of chromatid gaps when exposed to DNA damage, and increased PARP inhibitor sensitivity. In mouse embryonic stem cells (mES), this variant alters viability and confers sensitivity to cisplatin and Mitomycin C. These results suggest that BRCA2 interaction with ssDNA needs to be tightly regulated to limit HR and prevent chromosomal instability and we propose that this control mechanism involves DSS1. Given that several missense variants located within this region have been identified in breast cancer patients, these findings might have clinical implications for carriers.

Value of the loss of heterozygosity to BRCA1 variant classification.

At least 10% of the BRCA1/2 tests identify variants of uncertain significance (VUS) while the distinction between pathogenic variants (PV) and benign variants (BV) remains particularly challenging. As a typical tumor suppressor gene, the inactivation of the second wild-type (WT) BRCA1 allele is expected to trigger cancer initiation. Loss of heterozygosity (LOH) of the WT allele is the most frequent mechanism for the BRCA1 biallelic inactivation. To evaluate if LOH can be an effective predictor of BRCA1 variant pathogenicity, we carried out LOH analysis on DNA extracted from 90 breast and seven ovary tumors diagnosed in 27 benign and 55 pathogenic variant carriers. Further analyses were conducted in tumors with PVs yet without loss of the WT allele: BRCA1 promoter hypermethylation, next-generation sequencing (NGS) of BRCA1/2, and BRCAness score. Ninety-seven tumor samples were analyzed from 26 different BRCA1 variants. A relatively stable pattern of LOH (65.4%) of WT allele for PV tumors was observed, while the allelic balance (63%) or loss of variant allele (15%) was generally seen for carriers of BV. LOH data is a useful complementary argument for BRCA1 variant classification.

Combining Homologous Recombination and Phosphopeptide-binding Data to Predict the Impact of BRCA1 BRCT Variants on Cancer Risk.

BRCA1 mutations have been identified that increase the risk of developing hereditary breast and ovarian cancers. Genetic screening is now offered to patients with a family history of cancer, to adapt their treatment and the management of their relatives. However, a large number of BRCA1 variants of uncertain significance (VUS) are detected. To better understand the significance of these variants, a high-throughput structural and functional analysis was performed on a large set of BRCA1 VUS. Information on both cellular localization and homology-directed DNA repair (HR) capacity was obtained for 78 BRCT missense variants in the UMD-BRCA1 database and measurement of the structural stability and phosphopeptide-binding capacities was performed for 42 mutated BRCT domains. This extensive and systematic analysis revealed that most characterized causal variants affect BRCT-domain solubility in bacteria and all impair BRCA1 HR activity in cells. Furthermore, binding to a set of 5 different phosphopeptides was tested: all causal variants showed phosphopeptide-binding defects and no neutral variant showed such defects. A classification is presented on the basis of mutated BRCT domain solubility, phosphopeptide-binding properties, and VUS HR capacity. These data suggest that HR-defective variants, which present, in addition, BRCT domains either insoluble in bacteria or defective for phosphopeptide binding, lead to an increased cancer risk. Furthermore, the data suggest that variants with a WT HR activity and whose BRCT domains bind with a WT affinity to the 5 phosphopeptides are neutral. The case of variants with WT HR activity and defective phosphopeptide binding should be further characterized, as this last functional defect might be sufficient per se to lead to tumorigenesis. IMPLICATIONS: The analysis of the current study on BRCA1 structural and functional defects on cancer risk and classification presented may improve clinical interpretation and therapeutic selection.

Azathioprine induction of tumors with microsatellite instability: risk evaluation using a mouse model.

Mismatch-repair (MMR)-deficient cells show increased in vitro tolerance to thiopurines because they escape apoptosis resulting from MMR-dependent signaling of drug-induced DNA damage. Prolonged treatment with immunosuppressants including azathioprine (Aza), a thiopurine prodrug, has been suggested as a risk factor for the development of late onset leukemias/lymphomas displaying a microsatellite instability (MSI) phenotype, the hallmark of a defective MMR system. We performed a dose effect study in mice to investigate the development of MSI lymphomas associated with long term Aza treatment. Over two years, Aza was administered to mice that were wild type, null or heterozygous for the MMR gene Msh2. Ciclosporin A, an immunosuppressant with an MMR-independent signaling, was also administered to Msh2(wt) mice as controls. Survival, lymphoma incidence and MSI tumor phenotype were investigated. Msh2(+/-) mice were found more tolerant than Msh2(wt) mice to the cytotoxicity of Aza. In Msh2(+/-) mice, Aza induced a high incidence of MSI lymphomas in a dose-dependent manner. In Msh2(wt) mice, a substantial lifespan was only observed at the lowest Aza dose. It was associated with the development of lymphomas, one of which displayed the MSI phenotype, unlike the CsA-induced lymphomas. Our findings define Aza as a risk factor for an MSI-driven lymphomagenesis process.

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.