Objective: Unclassified variants (UVs, variants of uncertain clinical significance) are found in 13% of all BRCA1/2 mutation analyses. Little is known about the counsellees' recall and interpretation of a UV, and its psychosocial/medical impact.Method: Retrospective semi-structured interviews with open questions and five-point Likert scales were carried out in 24 counsellees who received a UV result 3 years before (sd ¼ 1:9).Results: Sixty-seven percent (16/24) recalled the UV result as a non-informative DNA result; 29% recalled a pathogenic result. However, 79% of all counsellees interpreted the UV result as a genetic predisposition for cancer. Variations in recall and interpretation were unexplained by demographics, cancer history of themselves and relatives, and communication aspects of UV disclosure. Sixty-seven percent perceived genetic counselling as completed, whereas 71% expected to receive new DNA information. Although most counsellees reported that UV disclosure had changed their lives in general little, one in three counsellees reported large changes in specific life domains, especially in surveillance behavior and medical decisions. Ten out of 19 participants who interpreted the UV as pathogenic had undergone preventive surgery against none of the 5 counsellees who interpreted the UV as non-informative.Conclusion: Counsellors and researchers need to address discrepancies between the counsellees' factual recall and their subjective interpretation of non-informative BRCA1/2-test results.

Two lipid membrane sculpting BAR domain proteins, PICK1 and ICA69, play a key role early in the biogenesis of peptide hormone secretory vesicles and are critical for normal growth and metabolic homeostasis.

Many suspected Lynch Syndrome (sLS) patients who lack mismatch repair (MMR) germline gene variants and MLH1 or MSH2 hypermethylation are currently explained by somatic MMR gene variants or, occasionally, by germline POLE variants. To further investigate unexplained sLS patients, we analyzed leukocyte and tumor DNA of 62 sLS patients using gene panel sequencing including the POLE, POLD1 and MMR genes. Forty tumors showed either one, two or more somatic MMR variants predicted to affect function. Nine sLS tumors showed a likely ultramutated phenotype and were found to carry germline (n=2) or somatic variants (n=7) in the POLE/POLD1 exonuclease domain (EDM). Six of these POLE/POLD1-EDM mutated tumors also carried somatic MMR variants. Our findings suggest that faulty proofreading may result in loss of MMR and thereby in microsatellite instability.

Genetic counselors need to explicitly address the counselee's interpretations and intended medical decisions. In case of misinterpretations, additional counseling might be offered. Communication of unclassified variants needs special attention given the pitfall of overestimation of risk.

A subset of DNA variants causes genetic disease through aberrant splicing. Experimental splicing assays, either RT-PCR analyses of patient RNA or functional splicing reporter minigene assays, are required to evaluate the molecular nature of the splice defect. Here, we present minigene assays performed for 17 variants in the consensus splice site regions, 14 exonic variants outside these regions, and two deep intronic variants, all in the DNA mismatch-repair (MMR) genes MLH1, MSH2, MSH6, and PMS2, associated with Lynch syndrome. We also included two deep intronic variants in APC and PKD2. For one variant (MLH1 c.122A>G), our minigene assay and patient RNA analysis could not confirm the previously reported aberrant splicing. The aim of our study was to further investigate the concordance between minigene splicing assays and patient RNA analyses. For 30 variants results from patient RNA analyses were available, either performed by our laboratory or presented in literature. Some variants were deliberately included in this study because they resulted in multiple aberrant transcripts in patient RNA analysis, or caused a splice effect other than the prevalent exon skip. While both methods were completely concordant in the assessment of splice effects, four variants exhibited major differences in aberrant splice patterns. Based on the present and earlier studies, together showing an almost 100% concordance of minigene assays with patient RNA analyses, we discuss the weight given to minigene splicing assays in the current criteria proposed by InSiGHT for clinical classification of MMR variants.