A de novo germline pathogenic BRCA1 variant identified following an osteosarcoma pangenomic molecular analysis.

De novo germline pathogenic variants (gPV) of the BReast CAncer 1 (BRCA1) gene are very rare. Only a few have been described up to date, usually in patients with a history of ovarian or breast cancer. Here, we report the first case of an incidental de novo BRCA1 germline pathogenic variant which was identified within the framework of the Plan France Médecine Génomique (PFMG) 2025 French national tumor sequencing program. The proband was a 29-year-old man diagnosed with metastatic osteosarcoma. Tumor whole exome sequencing identified a BRCA1 c.3756_3759del p.(Ser1253Argfs*10) pathogenic variant without loss-of-heterozygosity. A low genomic instability score and the absence of single base substitution signatures of homologous recombination deficiency suggested that the BRCA1 variant was not driver in the osteosarcoma tumorigenesis. Germline whole genome sequencing asserted the germline nature of this variant, with a 36% allele frequency, suggesting a mosaicism caused by a post-zygotic mutational event. The proband's family (parents and siblings) were not carriers of this variant confirming the de novo occurrence. Tumor sequencing programs like the French PFMG 2025 have been implemented worldwide and may help identify new gPV, including de novo variants.

Mosaic BRCA1 promoter methylation contribution in hereditary breast/ovarian cancer pedigrees.

PURPOSE: Mosaic BRCA1 promoter methylation (BRCA1meth) increases the risk of early-onset breast cancer, triple-negative breast cancer and ovarian cancer. As mosaic BRCA1meth are believed to occur de novo, their role in family breast/ovarian cancer has not been assessed. PATIENTS: Blood-derived DNA from 20 unrelated affected cases from families with aggregation of breast/ovarian cancer, but with no germline pathogenic variants in BRCA1/2, PALB2 or RAD51C/D, were screened by methylation-sensitive high-resolution melting. CpG analysis was performed by pyrosequencing on blood and buccal swab. Two probands carried a pathogenic variant in a moderate-penetrance gene (ATM and BARD1), and 8 of 18 others (44%) carried BRCA1meth (vs none of the 20 age-matched controls). Involvement of BRCA1 in tumourigenesis in methylated probands was demonstrated in most tested cases by detection of a loss of heterozygosity and a homologous recombination deficiency signature. Among the eight methylated probands, two had relatives with breast cancer with detectable BRCA1meth in blood, including one with high methylation levels in two non-tumour tissues. CONCLUSIONS: The high prevalence of mosaic BRCA1meth in patients with breast/ovarian cancer with affected relatives, as well as this first description of a family aggregation of mosaic BRCA1meth, shows how this de novo event can contribute to hereditary breast/ovarian cancer pedigrees.

Lynch syndrome: influence of additional susceptibility variants on cancer risk.

Some patients with Lynch syndrome (LS) have extreme phenotypes, i.e. cancer before the recommended screening age, or cancer for which there are no screening guidelines. We made the hypothesis that additional germline variants in cancer susceptibility genes (CSG) could explain some of these phenotypes. We compared the prevalence of additional CSG variants in LS patients with a cancer diagnosis before age 30 (early-onset, EO group) and after 40 (usual-onset, UO group). While there was no overall difference, we did find an excess of pathogenic variants and variants of unknown significance in EO cases when only gastrointestinal CSG were considered (OR 2.25; 95% CI: 1.01-5.06, p value = 0.04). Four EO cases stood out: two with POLE/POLD1 variants in the key exonuclease domain, one with a BMPR1A duplication and one with an EPCAM deletion. Additional germline variants should be considered in future screening recommendations, as they might influence cancer risk.

Contribution of whole genome sequencing in the molecular diagnosis of mosaic partial deletion of the NF1 gene in neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disease with complete penetrance but highly variable expressivity. In most patients, Next Generation Sequencing (NGS) technologies allow the identification of a loss-of-function pathogenic variant in the NF1 gene, a negative regulator of the RAS-MAPK pathway. We describe the 5-year diagnosis wandering of a patient with a clear NF1 clinical diagnosis, but no molecular diagnosis using standard molecular technologies. The patient presented with a typical NF1 phenotype but NF1 targeted NGS, NF1 transcript analysis, MLPA, and array comparative genomic hybridization failed to reveal a genetic aberration. After 5 years of unsuccessful investigations, trio WGS finally identified a de novo mosaic (VAF ~ 14%) 24.6 kb germline deletion encompassing the promoter and first exon of NF1. This case report illustrates the relevance of WGS to detect structural variants including copy number variants that would be missed by alternative approaches. The identification of the causal pathogenic variant allowed a tailored genetic counseling with a targeted non-invasive prenatal diagnosis by detecting the deletion in plasmatic cell-free DNA from the proband's pregnant partner. This report clearly highlights the need to make WGS a clinically accessible test, offering a tremendous opportunity to identify a molecular diagnosis for otherwise unsolved cases.

Usefulness of anti-factor VIII IgG ELISA in acquired hemophilia A follow-up.

Acquired hemophilia A (AHA) is a rare autoimmune bleeding disorder due to the presence of neutralizing autoantibodies directed against the coagulation factor VIII (FVIII). The reference method to detect and quantify anti-FVIII antibodies is the Bethesda assay (BA), but it presents some limitations such as a lack of sensitivity for low titers of inhibitor and the need for experienced laboratory. A commercially available ELISA detecting anti-FVIII antibodies has demonstrated excellent sensitivity and specificity. The aim of our study was to assess the performance of this ELISA for the detection of anti-FVIII IgG in AHA patients during the follow-up. In total, 11 acquired hemophilia A patients were recruited, and anti-FVIII antibody levels were monitored by BA and ELISA. Anti-FVIII IgG ELISA showed 100% sensitivity and 100% specificity, and it correlated with the BA. Discrepancies observed in 13.3% of cases were consistent with patients' biological evolution. All these data suggest the possible use of anti-FVIII IgG ELISA for both diagnosis and follow-up of AHA patients.

Noninvasive Prenatal Diagnosis of a Paternally Inherited MEN1 Pathogenic Splicing Variant.

CONTEXT: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease caused by mutations in the tumor suppressor gene MEN1. The uncertainty of pathogenicity of MEN1 variants complexifies the selection of the patients likely to benefit from specific care. OBJECTIVE: MEN1-mutated patients should be offered tailored tumor screening and genetic counseling. We present a patient with hyperparathyroidism for whom genetic analysis identified a variant of uncertain significance in the MEN1 gene (NM_130799.2): c.654G > T p.(Arg218=). Additional functional genetic tests were performed to classify the variant as pathogenic and allowed prenatal testing. DESIGN: Targeted next generation sequencing identified a synonymous variant in the MEN1 gene in a 26-year-old male with symptomatic primary hyperparathyroidism. In silico and in vitro genetic tests were performed to assess variant pathogenicity. RESULTS: Genetic testing of the proband's unaffected parents showed the variant occurred de novo. Transcript study showed a splicing defect leading to an in-frame deletion. The classification of the MEN1 variant as pathogenic confirmed the diagnosis of MEN1 and recommended an adapted medical care and follow-up. Pathogenic classification also allowed to propose a genetic counseling to the proband and his wife. Noninvasive prenatal diagnosis was performed with a personalized medicine-based protocol by detection of the paternally inherited variant in maternal plasmatic cell free DNA, using digital PCR. CONCLUSION: We showed that functional genetic analysis can help to assess the pathogenicity of a MEN1 variant with crucial consequences for medical care and genetic counseling decisions.

Identification of rare defective allelic variants in cases of thiopurine S-methyltransferase deficient activity.

Aim: To assess rare TPMT variants in patients carrying a deficient phenotype not predicted by the four more frequent genotypes (*2, *3A, *3B and *3C). Materials & methods: Next-generation sequencing of TPMT in 39 patients with a discordant genotype. Results: None of the variants identified explained the discordances assuming that they are of uncertain significance according to the Clinical Pharmacogenetics Implementation Consortium classification. Two unknown variants were detected and predicted to result in a splicing defect. We show that TPMT*16 and TMPT*21 are defective alleles, and TPMT*8 and TPMT*24 are associated with a normal activity. Conclusion: Whole-exon sequencing for rare TPMT mutations has a low diagnostic yield. A reassessment of the functional impact of rare variants of uncertain significance is a critical issue.

Cost-effectiveness analysis of pretreatment screening for NUDT15 defective alleles.

BACKGROUND: Nucleotide triphosphate diphosphatase (NUDT15) genetic testing in addition to thiopurine methyl transferase (TPMT) is recommended to reduce the incidence of adverse severe myelotoxicity episodes induced by thiopurines. OBJECTIVE: We assessed the cost-effectiveness ratio of combined screening for TMPT and NUDT15 defective alleles by genotyping or next-generation sequencing (NGS) using TPMT genotyping as the reference. Because of the genetic differences in thiopurine toxicity, we tested the screening strategies on individuals of Caucasian and Asian descent. METHODS: A decision tree compared conventional TPMT genotyping with combined TPMT/NUDT15 genotyping or NGS using a Monte-Carlo microsimulation model of patients with inflammatory bowel disease. The main outcome was the incremental cost-effectiveness ratios (ICER) with effectiveness being one averted severe myelotoxicity requiring hospitalization. RESULTS: The mean estimated cost of the TPMT genotyping for one year is twice in Asian compared with Caucasian patients (980 euro/patient versus 488 euro/patient), and the effectiveness of TPMT genotyping in Caucasian avoided 43 severe myelosuppressions per 10 000 patients over a year compared with 3.6 per 10 000 patients in Asian. Combined TPMT/NUDT15 genotyping compared with TPMT genotyping had an ICER of 7 491 281 euro per severe myelotoxicity averted in Caucasian, compared to 619 euro in Asian. The ICER of the NGS-based screening strategy is disproportionally high compared with genotyping, irrespective of ethnic descent. CONCLUSION: With a low cost-effectiveness threshold, combined screening for NUDT15 and TPMT defective alleles is cost-effective compared to TMPT screening alone in patients of Asian descent, but is unrealistic from a cost-effectiveness point of view in Caucasians.

Design, Synthesis, and Evaluation of 2,9-Bis[(substituted-aminomethyl)phenyl]-1,10-phenanthroline Derivatives as G-Quadruplex Ligands.

Genomic sequences able to form guanine quadruplexes (G4) are found in oncogene promoters, in telomeres, and in 5'- and 3'-untranslated regions as well as introns of messenger RNAs. These regions are potential targets for drugs designed to treat cancer. Herein, we present the design and syntheses of ten new phenanthroline derivatives and characterization of their interactions with G4-forming oligonucleotides. We evaluated ligand-induced stabilization and specificity and selectivity of ligands for various G4 conformations using FRET-melting experiments. We investigated the interaction of compound 1 a (2,9-bis{4-[(3-dimethylaminopropyl)aminomethyl]phenyl}-1,10-phenanthroline), which combined the greatest stabilizing effect and specificity for G4, with human telomeric sequences using FRET, circular dichroism, and ESI-MS. In addition, we showed that compound 1 a interferes with the G4 helicase activity of Saccharomyces cerevisiae Pif1. Interestingly, compound 1 a was significantly more cytotoxic toward two human leukemic cell lines than to normal human blood mononuclear cells. These novel phenanthroline derivatives will be a starting point for further development and optimization of potent G4 ligands that have potential as anticancer agents.