Uncovering frequent motives of action by which variants impair 3′ splice site (3′ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini‐gene experiments we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3( NF1):c.1722‐11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG‐exclusion zone (AGEZ) between the authentic 3′ss AG and the branch point. Evaluation of 90 additional non‐canonical intronic NF1 3′ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single‐nucleotide variants upstream of positions ‐3 interrupt the AGEZ. Of these AGEZ‐interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3′ss selection already in the initial steps of splicing. The analysis of 91 non‐canonical NF1 3′ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position ‐3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice‐neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing.

Colorectal cancer (CRC) in adolescents and young adults (AYA) is very rare. Known predisposition syndromes include Lynch syndrome (LS) due to highly penetrant MLH1 and MSH2 alleles, familial adenomatous polyposis (FAP), constitutional mismatch-repair deficiency (CMMRD), and polymerase proofreading-associated polyposis (PPAP). Yet, 60% of AYA-CRC cases remain unexplained. In two teenage siblings with multiple adenomas and CRC, we identified a maternally inherited heterozygous PMS2 exon 12 deletion, NM_000535.7:c.2007-786_2174+493del1447, and a paternally inherited POLD1 variant, NP_002682.2:p.Asp316Asn. Comprehensive molecular tumor analysis revealed ultra-mutation (>100 Mut/Mb) and a large contribution of COSMIC signature SBS20 in both siblings’ CRCs, confirming their predisposition to AYA-CRC results from a high propensity for somatic MMR deficiency (MMRd) compounded by a constitutional Pol δ proofreading defect. COSMIC signature SBS20 as well as SBS26 in the index patient’s CRC were associated with an early mutation burst, suggesting MMRd was an early event in tumorigenesis. The somatic second hits in PMS2 were through loss of heterozygosity (LOH) in both tumors, suggesting PPd-independent acquisition of MMRd. Taken together, these patients represent the first cases of cancer predisposition due to heterozygous variants in PMS2 and POLD1. Analysis of their CRCs supports that POLD1-mutated tumors acquire hypermutation only with concurrent MMRd.

Lynch syndrome (LS) and constitutional mismatch repair deficiency (CMMRD) are distinct cancer syndromes caused, respectively, by mono- and bi-allelic germline mismatch repair (MMR) variants. LS predisposes to mainly gastrointestinal and genitourinary cancers in adulthood. CMMRD predisposes to brain, haematological, and LS-spectrum cancers from childhood. Two suspected LS patients with first cancer diagnosis aged 27 or 38 years were found to be homozygous for an MMR (likely) pathogenic variant, MSH6 c.3226C>T (p.(Arg1076Cys)), or variant of uncertain significance (VUS), MLH1 c.306G>A (p.(Glu102=)). MLH1 c.306G>A was shown to cause leaky exon 3 skipping. The apparent genotype-phenotype conflict was resolved by detection of constitutional microsatellite instability in both patients, a hallmark feature of CMMRD. A hypomorphic effect of these and other variants found in additional late onset CMMRD cases, identified by literature review, likely explains a LS-like phenotype. CMMRD testing in carriers of compound heterozygous or homozygous MMR VUS may find similar cases and novel hypomorphic variants. Individualised management of mono- and bi-allelic carriers of hypomorphic MMR variants is needed until we better characterise the associated phenotypes.

Lynch syndrome (LS) is the most common hereditary colorectal cancer (CRC) syndrome, caused by heterozygous mutations in the mismatch repair (MMR) genes. Biallelic mutations in these genes lead however, to constitutive mismatch repair deficiency (CMMRD). In this study, we follow the diagnostic journey of a 12-year old patient with CRC, with a clinical phenotype overlapping CMMRD. We perform molecular and functional assays to discard a CMMRD diagnosis then identify by exome sequencing and validation in a cohort of 134 LS patients, a candidate variant in the MLH1 UTR region in homozygosis. We propose that this variant, together with other candidates, could be responsible for age-of-onset modulation. Our data support the idea that low-risk modifier alleles may influence early development of cancer in LS leading to a LS-to-CMMRD phenotypic continuum. Therefore, it is essential that larger efforts are directed to the identification and study of these genetic modifiers, in order to provide optimal cancer prevention strategies to these patients.

Background: Homologous recombination deficiency (HRD) has evolved into a major diagnostic marker in high-grade ovarian cancer (HGOC), predicting the response to poly (adenosine diphosphate-ribose) polymerase inhibitors (PARPi) and also platinum-based therapy. In addition to HRD, the type of peritoneal tumor spread influences the treatment response and patient survival; miliary type tumor spread has a poorer predicted outcome than non-miliary type tumor spread. Methods: Known methods for HRD assessment were adapted for our technical requirements and the predictive-value integrated genomic instability score (PIGIS) for HRD assessment evolved as an outcome. PIGIS was validated in HGOC samples from 122 patients. We used PIGIS to analyze whether the type of tumor spread correlated with HRD status and whether this had an impact on survival. Results: We demonstrated that PIGIS can discriminate HRD-positive from HRD-negative samples. Tumors with a miliary tumor spread are HRD-negative and have a very bad prognosis with a progression-free survival (PFS) of 15.6 months and an overall survival (OS) of 3.9 years. However, HRD-negative non-miliary spreading tumors in our cohort had a much better prognosis (PFS 35.4 months, OS 8.9 years); similar to HRD-positive tumors (PFS 34.7 months, OS 8.9 years). Conclusions: Our results indicate that in a predominantly PARPi naïve cohort, the type of tumor spread and concomitant cytoreduction efficiency is a better predictor of survival than HRD and that HRD may be an accidental surrogate marker for tumor spread and concomitant cytoreduction efficiency. It remains to be determined whether this also applies for sensitivity to PARPi.

Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer. Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the Plakophilin-3 (PKP3) gene was functionally analysed regarding promoter and enhancer activity in presence and absence of methylation using luciferase reporter assays. We confirmed in a unique cohort that monozygotic twins, even if clinically discordant, have only minor differences in global DNA methylation patterns and blood cell composition. Further, we determined the most differentially methylated positions, a high proportion of which are blood cell-type-specific, whereas others may be acquired and thus have potential relevance in the context of inflammation as lung cancer risk factors. We identified a sequence in the gene body of PKP3 which is hypermethylated in blood from CF twins with severe phenotype and highly variably methylated in lung cancer patients and controls, independent of known clinical parameters, and showed that this region exhibits methylation-dependent promoter activity in lung epithelial cells.