Hereditary Colorectal Cancer and Polyposis Syndromes Caused by Variants in Uncommon Genes.

A substantial number of hereditary colorectal cancer (CRC) and colonic polyposis cannot be explained by alteration in confirmed predisposition genes, such as mismatch repair (MMR) genes, APC and MUTYH. Recently, a certain number of potential predisposition genes have been suggested, involving each a small number of cases reported so far. Here, we describe the detection of rare variants in the NTLH1, AXIN2, RNF43, BUB1, and TP53 genes in nine unrelated patients who were suspected for inherited CRC and/or colonic polyposis. Seven of them were classified as pathogenic or likely pathogenic variants (PV/LPV). Clinical manifestations of carriers were largely consistent with reported cases with, nevertheless, distinct characteristics. PV/LPV in these uncommon gene can be responsible for up to 2.7% of inherited CRC or colonic polyposis syndromes. Our findings provide supporting evidence for the role of these genes in cancer predisposition, and contribute to the determination of related cancer spectrum and cancer risk for carriers, allowing for the establishment of appropriate screening strategy and genetic counseling in affected families.

BRCA1 Intragenic Duplication Combined with a Likely Pathogenic TP53 Variant in a Patient with Triple-Negative Breast Cancer: Clinical Risk and Management.

For patients with hereditary breast and ovarian cancer, the probability of carrying two pathogenic variants (PVs) in dominant cancer-predisposing genes is rare. Using targeted next-generation sequencing (NGS), we investigated a 49-year-old Caucasian woman who developed a highly aggressive breast tumor. Our analyses identified an intragenic germline heterozygous duplication in BRCA1 with an additional likely PV in the TP53 gene. The BRCA1 variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints were characterized at the nucleotide level (c.135-2578_442-1104dup). mRNA extracted from lymphocytes was amplified by RT-PCR and then Sanger sequenced, revealing a tandem duplication r.135_441dup; p.(Gln148Ilefs*20). This duplication results in the synthesis of a truncated and, most likely, nonfunctional protein. Following functional studies, the TP53 exon 5 c.472C > T; p.(Arg158Cys) missense variant was classified as likely pathogenic by the Li-Fraumeni Syndrome (LFS) working group. This type of unexpected association will be increasingly identified in the future, with the switch from targeted BRCA sequencing to hereditary breast and ovarian cancer (HBOC) panel sequencing, raising the question of how these patients should be managed. It is therefore important to record and investigate these rare double-heterozygous genotypes.

AXIN2 germline testing in a French cohort validates pathogenic variants as a rare cause of predisposition to colorectal polyposis and cancer.

Only a few patients with germline AXIN2 variants and colorectal adenomatous polyposis or cancer have been described, raising questions about the actual contribution of this gene to colorectal cancer (CRC) susceptibility. To assess the clinical relevance for AXIN2 testing in patients suspected of genetic predisposition to CRC, we collected clinical and molecular data from the French Oncogenetics laboratories analyzing AXIN2 in this context. Between 2004 and June 2020, 10 different pathogenic/likely pathogenic AXIN2 variants were identified in 11 unrelated individuals. Eight variants were from a consecutive series of 3322 patients, which represents a frequency of 0.24%. However, loss-of-function AXIN2 variants were strongly associated with genetic predisposition to CRC as compared with controls (odds ratio: 11.89, 95% confidence interval: 5.103-28.93). Most of the variants were predicted to produce an AXIN2 protein devoid of the SMAD3-binding and DIX domains, but preserving the ß-catenin-binding domain. Ninety-one percent of the AXIN2 variant carriers who underwent colonoscopy had adenomatous polyposis. Forty percent of the variant carriers developed colorectal or/and other digestive cancer. Multiple tooth agenesis was present in at least 60% of them. Our report provides further evidence for a role of AXIN2 in CRC susceptibility, arguing for AXIN2 testing in patients with colorectal adenomatous polyposis or cancer.

Medulloblastomas with ELP1 pathogenic variants: A weakly penetrant syndrome with a restricted spectrum in a limited age window.

Background: ELP1 pathogenic variants (PV) have been recently identified as the most frequent variants predisposing to Sonic Hedgehog (SHH) medulloblastomas (MB); however, guidelines are still lacking for genetic counseling in this new syndrome. Methods: We retrospectively reviewed clinical and genetic data of a French series of 29 ELP1-mutated MB. Results: All patients developed SHH-MB, with a biallelic inactivation of PTCH1 found in 24 tumors. Other recurrent alterations encompassed the TP53 pathway and activation of MYCN/MYCL signaling. The median age at diagnosis was 7.3 years (range: 3-14). ELP1-mutated MB behave as sporadic cases, with similar distribution within clinical and molecular risk groups and similar outcomes (5 y - OS = 86%); no unusual side effect of treatments was noticed. Remarkably, a germline ELP1 PV was identified in all patients with available constitutional DNA (n = 26); moreover, all tested familial trio (n = 11) revealed that the PVs were inherited. Two of the 26 index cases from the French series had a family history of MB; pedigrees from these patients and from 1 additional Dutch family suggested a weak penetrance. Apart from MB, no cancer was associated with ELP1 PVs; second tumors reported in 4 patients occurred within the irradiation fields, in the usual time-lapse for expected radiotherapy-induced neoplasms. Conclusions: The low penetrance, the "at risk' age window limited to childhood and the narrow tumor spectrum, question the actual benefit of genetic screening in these patients and their family. Our results suggest restricting ELP1 germline sequencing to patients with SHH-MB, depending on the parents" request.

From variant of unknown significance to likely pathogenic: Characterization and pathogenicity determination of a large genomic deletion in the MLH1 gene.

BACKGROUND: The MLH1 gene is one of the DNA mismatch repair genes (MMR), implicated in Lynch syndrome (LS), an autosomal dominant hereditary tumor susceptibility disease. The advent of next-generation sequencing (NGS) technologies has accelerated the diagnosis of inherited diseases and increased the percentage of diagnosis of inherited cancers. However, some complex genomic alterations require the combination of several analytical strategies to allow correct biological interpretations. Here, we describe a novel MLH1 deletion and its pathogenicity determination in a patient suspected of LS. METHODS: The index case was a French 73-year-old man diagnosed with colorectal cancer displaying microsatellite instability and the loss of MLH1 and PMS2 expression. NGS analysis was used as the primary method for MMR genes screening. Long-range PCR and reverse transcriptase polymerase chain reaction (RT-PCR) were used for breakpoints and pathogenicity determinations. RESULTS: A large genomic deletion was detected which removed the last six nucleotides of MLH1 exon 11 together with a large part of intron 11. It was initially considered as a variant of unknown significance (VUS). Genomic breakpoints were subsequently characterized defining the deletion as c.1033_1039-248del. Further RNA analysis demonstrated that this variant activated a cryptic donor splice site at the 5' of the breakpoint, leading to a premature truncated protein: p.Thr345Alafs*13. CONCLUSION: Our finding suggested that although NGS technologies have increased variant detection yield, combined approaches were still needed for complex variant characterization and pathogenicity assessment.

A PMS2 non-canonical splicing site variant leads to aberrant splicing in a patient suspected for lynch syndrome.

The PMS2 gene is one of the DNA mismatch repair genes (MMR) implicated in Lynch syndrome (LS). A subset of PMS2 pathogenic variants (PVs) are splice variants mostly affecting canonical GT/AG splicing sequences. However, the majority of the intronic variants outside canonical splice sites remained as variants of unknown significance, even though some of them would alter the splicing process. In this report, we describe the analysis of such an intronic variant (c.251-5T > C) detected in an 82-year-old patient diagnosed with endometrial cancer displaying microsatellite instability and the loss of PMS2 expression displayed. RNA analysis demonstrated that this variant lead to the complete exon 4 skipping, resulting in the synthesis of a truncated protein. This finding shows the relevance of functional RNA analysis in the non-canonical intronic variant assessment and the importance of systematic evaluation of MSI/loss of expression of MMR genes for LS screening in patients with endometrial cancers.

A novel POLD1 pathogenic variant identified in two families with a cancer spectrum mimicking Lynch syndrome.

The POLD1 gene is involved in DNA proofreading to ensure accurate DNA replication. Some germline alterations in its exonuclease domain are associated with predisposition to cancers and colonic polyps. Only a few pathogenic variants have been clearly identified so far. Here we report a novel variant: c.1458G>T p.(Lys486Asn) that we classified as pathogenic, detected in two putatively unrelated families. The cancer spectrum was very similar to Lynch syndrome, implying an overlapped tissue susceptibility. The common presence of colonic polyps in carriers and the MMR proficient phenotype in tumors were distinctive features suggesting POLD1 implication. Some clinical characteristics observed in the carriers of this variant differed from those reported previously, suggesting a potential genotype/phenotype correlation, and very likely in relation to the functional importance of affected residues. Our findings provide further insight into understanding the role of POLD1 in cancer-related risk.

Transformation of a low-grade follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing Terminal deoxynucleotidyl Transferase: a case report.

BACKGROUND: High-grade B-cell lymphoma with rearrangements of MYC and BCL2 and/or BCL6 is an aggressive mature B-cell neoplasm, whereas B-lymphoblastic lymphoma is immature cell proliferation, with a frequent positivity for terminal deoxynucleotidyl transferase. The transformation of a low-grade follicular lymphoma into a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase is a very rare event. CASE PRESENTATION: A 55-year-old Caucasian man was followed for a grade 1-2 follicular lymphoma carrying a t(14;18) IGH/BCL2+ and was initially treated with R-CHOP. The follicular lymphoma presented two relapses. In the third relapse, the patient had multiple lymphadenopathy and ascites, which motivated a retroperitoneal biopsy and an ascitic tap. These samples were analyzed by histological, cytological, flow cytometric, cytogenetic, and molecular assessments. The patient died of a multiple organ dysfunction syndrome 2 weeks after his third relapse. The biopsy revealed a diffuse proliferation made up of two types of tumor cells: centroblasts (Bcl-6-positive) and immature cells (terminal deoxynucleotidyl transferase-positive). Flow cytometric analysis confirmed the immature phenotype, with an expression of terminal deoxynucleotidyl transferase, combined with a loss of membrane immunoglobulins. The cytogenetic analysis performed on the ascites revealed a clonal evolution characterized by a t(8;22)(q24;q11) MYC+ translocation not previously detected in follicular lymphoma. Fluorescence in situ hybridization confirmed the double rearrangement of the BCL2 and MYC genes. Polymerase chain reactions and sequencing were used to study the clonal relationship between follicular lymphoma and the secondary tumors. The IGVH gene rearrangement revealed a unique clonal rearrangement involving an IGVH4-59 subset in all three specimens. CONCLUSION: These findings suggest a clonal relationship between the two types of lymphoma cells. Furthermore, they support the transformation of an acute follicular lymphoma into a composite lymphoma combining a high-grade B-cell lymphoma and a lymphoblastic neoplasm expressing terminal deoxynucleotidyl transferase. This case report highlights the possible transformation of follicular lymphoma into a highly aggressive and immature proliferation.

Mutations in CFAP43 and CFAP44 cause male infertility and flagellum defects in Trypanosoma and human.

Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.