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Background: Vascular Ehlers-Danlos syndrome (vEDS) is a rare connective tissue disorder with a high risk for arterial, bowel, and uterine rupture, caused by heterozygous pathogenic variants in COL3A1 . The aim of this cohort study is to provide further insights into the natural history of vEDS and describe genotype-phenotype correlations in a Dutch multicenter cohort to optimize patient care and increase awareness of the disease., Methods: Individuals with vEDS throughout the Netherlands were included. The phenotype was charted by retrospective analysis of molecular and clinical data, combined with a one-time physical examination., Results: A total of 142 individuals (50% female) participated the study, including 46 index patients (32%). The overall median age at genetic diagnosis was 41.0 years. More than half of the index patients (54.3%) and relatives (53.1%) had a physical appearance highly suggestive of vEDS. In these individuals, major events were not more frequent ( P =0.90), but occurred at a younger age ( P =0.01). A major event occurred more often and at a younger age in men compared with women ( P <0.001 and P =0.004, respectively). Aortic aneurysms ( P =0.003) and pneumothoraces ( P =0.029) were more frequent in men. Aortic dissection was more frequent in individuals with a COL3A1 variant in the first quarter of the collagen helical domain ( P =0.03)., Conclusions: Male sex, type and location of the COL3A1 variant, and physical appearance highly suggestive of vEDS are risk factors for the occurrence and early age of onset of major events. This national multicenter cohort study of Dutch individuals with vEDS provides a valuable basis for improving guidelines for the diagnosing, follow-up, and treatment of individuals with vEDS., Competing Interests: Disclosures None.

Familial adenomatous polyposis is most frequently caused by pathogenic variants in either the APC gene or the MUTYH gene. The detection rate of pathogenic variants depends on the severity of the phenotype and sensitivity of the screening method, including sensitivity for mosaic variants. For 171 patients with multiple colorectal polyps without previously detectable pathogenic variant, APC was reanalyzed in leukocyte DNA by one uniform technique: high-resolution melting (HRM) analysis. Serial dilution of heterozygous DNA resulted in a lowest detectable allelic fraction of 6% for the majority of variants. HRM analysis and subsequent sequencing detected pathogenic fully heterozygous APC variants in 10 (6%) of the patients and pathogenic mosaic variants in 2 (1%). All these variants were previously missed by various conventional scanning methods. In parallel, HRM APC scanning was applied to DNA isolated from polyp tissue of two additional patients with apparently sporadic polyposis and without detectable pathogenic APC variant in leukocyte DNA. In both patients a pathogenic mosaic APC variant was present in multiple polyps. The detection of pathogenic APC variants in 7% of the patients, including mosaics, illustrates the usefulness of a complete APC gene reanalysis of previously tested patients, by a supplementary scanning method. HRM is a sensitive and fast pre-screening method for reliable detection of heterozygous and mosaic variants, which can be applied to leukocyte and polyp derived DNA.

SPG8 is a rare autosomal dominant hereditary spastic paraplegia (AD-HSP), with only six SPG8 families described so far. Our purpose was to screen for KIAA0196 (SPG8) mutations in AD-HSP patients and to investigate their phenotype. Extensive family investigation was performed after positive KIAA0196 mutation analysis, which was part of an on-going mutation screening effort in AD-HSP patients. A novel pathogenic KIAA0196 mutation p.(Gly696Ala) was identified in two AD-HSP patients, who subsequently were shown to belong to a single large Dutch pedigree with more than 10 affected family members. The phenotype consisted of a pure HSP with ages at onset between 20 and 60 years, distally reduced vibration sense in the legs in all, and urinary urgency in seven out of 10 patients. Frequent features were exercise- or emotion-induced increase of spasticity and gait problems and chronic nonspecific lower back and joint pains. We have identified a fourth pathogenic KIAA0196 mutation in a Dutch HSP-family, the seventh family worldwide, with a less severe clinical course than described before.

Lynch syndrome patients are susceptible to colorectal and endometrial cancers owing to inactivating germline mutations in mismatch repair genes, including MSH2 (ref. 1). Here we describe patients from Dutch and Chinese families with MSH2-deficient tumors carrying heterozygous germline deletions of the last exons of TACSTD1, a gene directly upstream of MSH2 encoding Ep-CAM. Due to these deletions, transcription of TACSTD1 extends into MSH2. The MSH2 promoter in cis with the deletion is methylated in Ep-CAM positive but not in Ep-CAM negative normal tissues, thus revealing a correlation between activity of the mutated TACSTD1 allele and epigenetic inactivation of the corresponding MSH2 allele. Gene silencing by transcriptional read-through of a neighboring gene in either sense, as demonstrated here, or antisense direction, could represent a general mutational mechanism. Depending on the expression pattern of the neighboring gene that lacks its normal polyadenylation signal, this may cause either generalized or mosaic patterns of epigenetic inactivation.

In the present investigation, the localization of proteins involved in ovarian apoptosis were studied throughout the estrous cycle in the presence of fluctuating hormone levels. Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA expression and proteins were detected in all ovarian tissue extracts, though the amount of protein varied with the phase of the estrous cycle. Fas, Bax and caspase-3 protein levels were highest at diestrus and decreased thereafter towards metestrus. In contrast, Fas ligand and Bcl-2 protein levels were lowest at diestrus and increased toward metestrus. Immunohistochemistry revealed that the staining of the anti-apoptotic protein Bcl-2 was more pronounced in healthy preantral follicles than in atretic follicles. In contrast, the pro-apoptotic proteins Fas, Fas ligand, Bax and active caspase-3 were more predominantly present in atretic follicles. In the ovarian surface epithelium (OSE), Fas, procaspase-3 and Bcl-2 immunostaining appeared independent of the phase of the estrous cycle. Fas ligand and Bax staining was detected particularly during proestrus in OSE cells surrounding the ovulatory follicles, while active caspase-3 was observed only in OSE cells at the postovulatory site during estrus. The proportion of luteal cells that stained positively for Fas, Bax and caspase-3 increased with the age of the corpus luteum, while Fas ligand and Bcl-2 immunostaining was strongest in newly formed corpora lutea and decreased thereafter. In conclusion, the components of the Fas signalling pathway were differentially expressed throughout the estrous cycle in a variety of ovarian cell types, which may correspond to hormone dependent survival mechanisms.

Human ovarian cancers are thought to arise from sequestered ovarian surface epithelial (OSE) cells that line the wall of inclusion cysts. Nevertheless, the early events toward neoplasia are not well understood. In this study, immunoreactivity for apoptotic proteins in human OSE of control and tumor ovarian sections was examined. Ki67, a marker for cell proliferation, was generally absent in the flat-to-cuboidal OSE cells on the ovarian surface and in regularly shaped inclusion cysts. Fas, Fas ligand, and caspase-3, components of the apoptotic pathway, were also largely absent. Ki67, Fas, Fas ligand, and procaspase-3 expression, though not active caspase-3 expression, was more frequently observed in epithelial cells lining irregularly shaped inclusion cysts, particularly in the columnar and Müllerian-like OSE cell types that resembled ovarian tumor OSE cells. Immunoreactivity for these factors as well as active caspase-3 was found frequently in ovarian tumors. We postulate that the appearance of the Fas system and its related proteins in sequestered columnar OSE cells of irregularly shaped inclusion cysts may contribute to balance cell growth with cell death, although little active caspase-3 expression was observed. Further studies are required to identify whether inhibition of apoptosis in inclusion cysts is an early event in ovarian carcinogenesis.