The clinical value of optical genome mapping in the rapid characterization of RB1 duplication and 15q23q24.2 triplication, for more appropriate prenatal genetic counselling.

BACKGROUND: Despite recent advances in prenatal genetic diagnosis, medical geneticists still face considerable difficulty in interpreting the clinical outcome of copy-number-variant duplications and defining the mechanisms underlying the formation of certain chromosomal rearrangements. Optical genome mapping (OGM) is an emerging cytogenomic tool with proved ability to identify the full spectrum of cytogenetic aberrations. METHODS: Here, we report on the use of OGM in a prenatal diagnosis setting. Detailed breakpoint mapping was used to determine the relative orientations of triplicated and duplicated segments in two unrelated foetuses harbouring chromosomal aberrations: a de novo 15q23q24.2 triplication and a paternally inherited 13q14.2 duplication that overlapped partially with the RB1 gene. RESULTS: OGM enabled us to suggest a plausible mechanism for the triplication and confirmed that the RB1 duplication was direct oriented and in tandem. This enabled us to predict the pathogenic consequences, refine the prognosis and adapt the follow-up and familial screening appropriately. CONCLUSION: Along with an increase in diagnostic rates, OGM can rapidly highlight genotype-phenotype correlations, improve genetic counselling and significantly influence prenatal management.

Male breast cancer: No evidence for mosaic BRCA1 promoter methylation involvement.

Breast cancers (BC) are rare in men and are often caused by constitutional predisposing factors. In women, mosaic BRCA1 promoter methylations (MBPM) are frequent events, detected in 4-8% of healthy subjects. This constitutional epimutation increases risk of early-onset and triple-negative BC. However, the role of MBPM in male BC predisposition has never been assessed. We screened 40 blood samples from men affected by BC, and performed extensive tumour analysis on MBPM-positive patients. We detected two patients carrying MBPM. Surprisingly, tumour analysis revealed that neither of these two male BCs were caused by the constitutional BRCA1 epimutations carried by the patients.

Diffuse Infiltrating Retinoblastoma with Anterior Chamber Involvement: Conservative Management and Identification of RB1 Alterations in Aqueous Humor.

Introduction: The aim of the study was to describe the successful conservative management of diffuse infiltrating retinoblastoma (DIR). Identification of RB1 pathogenic variant was done after cell-free DNA (cfDNA) analysis in aqueous humor. Case presentation: Herein, we report 2 patients with unilateral, non-familial DIR with anterior and posterior involvement. Both patients underwent liquid biopsy for tumor cfDNA analysis in aqueous humor. Treatment consisted of a combination of systemic and intra-arterial chemotherapy, with consecutive intracameral and intravitreal injections of melphalan. One patient also required iodine-125 brachytherapy. In both cases, tumor cfDNA analysis revealed biallelic somatic alterations of the RB1 gene. These alterations were not found in germline DNA. Both patients retained their eyes and had a useful vision after a follow-up of 2 years. Conclusion: In selected cases, conservative management of DIR is safe and effective. Tumor cfDNA analysis in aqueous humor is an effective technique to disclose RB1 somatic alterations that guide the germline molecular explorations and improve genetic counseling after conservative treatment.

Germline HPF1 retrogene insertion in RB1 gene involved in cancer predisposition.

About half of the human genome is composed of repeated sequences derived from mobile elements, mainly retrotransposons, generally without pathogenic effect. Familial forms of retinoblastoma are caused by germline pathogenic variants in RB1 gene. Here, we describe a family with retinoblastoma affecting a father and his son. No pathogenic variant was identified after DNA analysis of RB1 gene coding sequence and exon-intron junctions. However, RB1 mRNA analysis showed a chimeric transcript with insertion of 114 nucleotides from HPF1 gene inside RB1 gene. This chimeric transcript led to an insertion of 38 amino acids in functional domain of retinoblastoma protein. Subsequent DNA analysis in RB1 intron 17 revealed the presence of a full-length HPF1 retrogene insertion in opposite orientation. Functional assay shows that this insertion has a deleterious impact on retinoblastoma protein function. This is the first report of a full-length retrogene insertion involved in human Mendelian disease leading to a chimeric transcript and a non-functional chimeric protein. Some retrogene insertions may be missed by standard diagnostic genetic testing, so contribution of retrogene insertions to human disease may be underestimated. The increasing use of whole genome sequencing in diagnostic settings will help to get a more comprehensive view of retrogenes.

Hereditary cancer predispositions: Comparison of multigene panel sequencing on fresh-frozen breast/ovarian tumor versus blood.

In breast or ovarian cancer (BC/OC) patients with evocative personal and/or family history, multigene panel sequencing is performed on blood to diagnose hereditary predispositions. Additionally, BRCA1/BRCA2 testing can be performed on tumor sample for therapeutic purpose. The accuracy of multigene panel tumor analysis on BC/OC to detect predisposing germline pathogenic variants (gPV) has not been precisely assessed. By comparing sequencing data from blood and fresh-frozen tumor we show that tumor genomic instability causes pitfalls to consider when performing tumor testing to detect gPV. Even if loss of heterozygosity increases germline signal in most cases, somatic copy number variants (CNV) can mask germline CNV and collapse point gPV variant allele frequency (VAF). Moreover, VAF does not allow an accurate distinction between germline and somatic pathogenic variants.

APC germline pathogenic variants and epithelial ovarian cancer: causal or coincidental findings?

APC germline pathogenic variants result in predisposition to familial adenomatous polyposis and extraintestinal tumours such as desmoid fibromatosis, medulloblastomas and thyroid cancers. They have also been recently involved in ovarian microcystic stromal tumours. APC inactivation has been described at the tumour level in epithelial ovarian cancers (EOCs). Here, we report the identification of APC germline pathogenic variants in two patients diagnosed with premenopausal EOC in early 30s, with no other pathogenic variant detected in the known ovarian cancer predisposing genes. Subsequent tumour analysis showed neither a second hit of APC inactivation nor ß-catenin activation. Both tumours did not have a homologous recombination (HR) deficiency, pointing towards the implication of other genes than those involved in HR. APC may contribute to the carcinogenesis of EOC in a multifactorial context. Further studies are required to clarify the role of APC in predisposition to EOC.

Highly Sensitive Detection Method of Retinoblastoma Genetic Predisposition and Biomarkers.

Retinoblastoma is a malignant tumor of the infant retina. Nearly half of patients are predisposed to retinoblastoma by a germline RB1 pathogenic variant. Nonhereditary retinoblastoma is mainly caused by inactivation of both RB1 alleles at a somatic level. Several polymorphisms have been reported as biomarkers of retinoblastoma risk, aggressiveness, or invasion. The most informative genetic testing is obtained from tumor DNA. Historically, access to tumor DNA has been warranted by the frequent indication of enucleation, which has decreased because of advances in conservative approaches. Recent studies showed that tumor cell-free DNA can be analyzed in aqueous humor from retinoblastoma patients. This report describes a next-generation sequencing method relying on unique molecular identifiers for a highly sensitive detection of retinoblastoma genetic predisposition and biomarkers in a single analysis. It is the first use of unique molecular identifiers for retinoblastoma genetics. This gene panel enables the detection of RB1 point variants, large genome rearrangements, and loss of heterozygosity. It is adapted for genomic DNA extracted from blood or tumor DNA extracted from tumor fragment, aqueous humor, or plasma. The access to tumor cell-free DNA improves the diagnosis of genetic predisposition in case of conservative ocular therapy and provides access to biomarkers guiding the treatment strategy. The analysis of a gene panel is cost-effective and can be easily implemented in diagnostic laboratories.

Molecular diagnosis of retinoblastoma by circulating tumor DNA analysis.

PURPOSE: The analysis of circulating tumor DNA (ctDNA), a fraction of total cell-free DNA (cfDNA), might be of special interest in retinoblastoma patients. Because the accessibility to tumor tissue is very limited in these patients, either for histopathological diagnosis of suspicious intraocular masses (biopsies are proscribed) or for somatic RB1 studies and genetic counseling (due to current successful conservative approaches), we aim to validate the detection of ctDNA in plasma of non-hereditary retinoblastoma patients by molecular analysis of RB1 gene. EXPERIMENTAL DESIGN: In a cohort of 19 intraocular unilateral non-hereditary retinoblastoma patients for whom a plasma sample was available at diagnosis, we performed high-deep next-generation sequencing (NGS) of RB1 in cfDNA. Two different bioinformatics/statistics approaches were applied depending on whether the somatic RB1 status was available or not. RESULTS: Median plasma sample volume was 600 µL [100-1000]; median cfDNA plasma concentration was 119 [38-1980] and 27 [11-653] ng/mL at diagnosis and after complete remission, respectively. In the subgroup of patients with known somatic RB1 alterations (n = 11), seven of nine somatic mutations were detected (median allele fraction: 6.7%). In patients without identified somatic RB1 alterations (n = 8), six candidate variants were identified for seven patients. CONCLUSIONS: Despite small tumor size, blood-ocular barrier, poor ctDNA blood release and limited plasma sample volumes, we confirm that it is possible to detect ctDNA with high-deep NGS in plasma from patients with intraocular non-hereditary retinoblastoma. This may aid in diagnosis of suspicious cases, family genetic counseling or follow-up of residual intraocular disease.

Expanding the phenotype of the X-linked BCOR microphthalmia syndromes.

Two distinct syndromes arise from pathogenic variants in the X-linked gene BCOR (BCL-6 corepressor): oculofaciocardiodental (OFCD) syndrome, which affects females, and a severe microphthalmia ('Lenz'-type) syndrome affecting males. OFCD is an X-linked dominant syndrome caused by a variety of BCOR null mutations. As it manifests only in females, it is presumed to be lethal in males. The severe male X-linked recessive microphthalmia syndrome ('Lenz') usually includes developmental delay in addition to the eye findings and is caused by hypomorphic BCOR variants, mainly by a specific missense variant c.254C > T, p.(Pro85Leu). Here, we detail 16 new cases (11 females with 4 additional, genetically confirmed, affected female relatives; 5 male cases each with unaffected carrier mothers). We describe new variants and broaden the phenotypic description for OFCD to include neuropathy, muscle hypotonia, pituitary underdevelopment, brain atrophy, lipoma and the first description of childhood lymphoma in an OFCD case. Our male X-linked recessive cases show significant new phenotypes: developmental delay (without eye anomalies) in two affected half-brothers with a novel BCOR variant, and one male with high myopia, megalophthalmos, posterior embryotoxon, developmental delay, and heart and bony anomalies with a previously undescribed BCOR splice site variant. Our female OFCD cases and their affected female relatives showed variable features, but consistently had early onset cataracts. We show that a mosaic carrier mother manifested early cataract and dental anomalies. All female carriers of the male X-linked recessive cases for whom genetic confirmation was available showed skewed X-inactivation and were unaffected. In view of the extended phenotype, we suggest a new term of X-linked BCOR-related syndrome.

Sex chromosome aneuploidies and copy-number variants: a further explanation for neurodevelopmental prognosis variability?

Sex chromosome aneuploidies (SCA) is a group of conditions in which individuals have an abnormal number of sex chromosomes. SCA, such as Klinefelter's syndrome, XYY syndrome, and Triple X syndrome are associated with a large range of neurological outcome. Another genetic event such as another cytogenetic abnormality may explain a part of this variable expressivity. In this study, we have recruited fourteen patients with intellectual disability or developmental delay carrying SCA associated with a copy-number variant (CNV). In our cohort (four patients 47,XXY, four patients 47,XXX, and six patients 47,XYY), seven patients were carrying a pathogenic CNV, two a likely pathogenic CNV and five a variant of uncertain significance. Our analysis suggests that CNV might be considered as an additional independent genetic factor for intellectual disability and developmental delay for patients with SCA and neurodevelopmental disorder.