Prevalence of RPGR-Mediated Retinal Dystrophy in an Unselected Cohort of Over 5000 Patients.

Purpose: Comprehensive genetic testing for inherited retinal dystrophy (IRD) is challenged by difficult-to-sequence genomic regions, which are often mutational hotspots, such as RPGR ORF15. The purpose of this study was to evaluate the diagnostic contribution of RPGR variants in an unselected IRD patient cohort referred for testing in a clinical diagnostic laboratory. Methods: A total of 5201 consecutive patients were analyzed with a clinically validated next-generation sequencing (NGS)-based assay, including the difficult-to-sequence RPGR ORF15 region. Copy number variant (CNV) detection from NGS data was included. Variant interpretation was performed per the American College of Medical Genetics and Genomics guidelines. Results: A confirmed molecular diagnosis in RPGR was found in 4.5% of patients, 24.0% of whom were females. Variants in ORF15 accounted for 74% of the diagnoses; 29% of the diagnostic variants were in the most difficult-to-sequence central region of ORF15 (c.2470-3230). Truncating variants made up the majority (91%) of the diagnostic variants. CNVs explained 2% of the diagnostic cases, of which 80% were one- or two-exon deletions outside of ORF15. Conclusions: Our findings indicate that high-throughput, clinically validated NGS-based testing covering the difficult-to-sequence region of ORF15, in combination with high-resolution CNV detection, can help to maximize the diagnostic yield for patients with IRD. Translational Relevance: These results demonstrate an accurate and scalable method for the detection of RPGR-related variants, including the difficult-to-sequence ORF15 hotspot, which is relevant given current and emerging therapeutic opportunities.

Western Diet Deregulates Bile Acid Homeostasis, Cell Proliferation, and Tumorigenesis in Colon.

Western-style diets (WD) high in fat and scarce in fiber and vitamin D increase risks of colorectal cancer. Here, we performed a long-term diet study in mice to follow tumorigenesis and characterize structural and metabolic changes in colon mucosa associated with WD and predisposition to colorectal cancer. WD increased colon tumor numbers, and mucosa proteomic analysis indicated severe deregulation of intracellular bile acid (BA) homeostasis and activation of cell proliferation. WD also increased crypt depth and colon cell proliferation. Despite increased luminal BA, colonocytes from WD-fed mice exhibited decreased expression of the BA transporters FABP6, OSTß, and ASBT and decreased concentrations of secondary BA deoxycholic acid and lithocholic acid, indicating reduced activity of the nuclear BA receptor FXR. Overall, our results suggest that WD increases cancer risk by FXR inactivation, leading to BA deregulation and increased colon cell proliferation. Cancer Res; 77(12); 3352-63. ©2017 AACR.

Western diet enhances intestinal tumorigenesis in Min/+ mice, associating with mucosal metabolic and inflammatory stress and loss of Apc heterozygosity.

Western-type diet (WD) is a risk factor for colorectal cancer, but the underlying mechanisms are poorly understood. We investigated the interaction of WD and heterozygous mutation in the Apc gene on adenoma formation and metabolic and immunological changes in the histologically normal intestinal mucosa of ApcMin/+ (Min/+) mice. The diet used was high in saturated fat and low in calcium, vitamin D, fiber and folate. The number of adenomas was twofold higher in the WD mice compared to controls, but adenoma size, proliferation or apoptosis did not differ. The ratio of the Min to wild-type allele was higher in the WD mice, indicating accelerated loss of Apc heterozygosity (LOH). Densities of intraepithelial CD3ε+ T lymphocytes and of mucosal FoxP3+ regulatory T cells were higher in the WD mice, implying inflammatory changes. Western blot analyses from the mucosa of the WD mice showed suppressed activation of the ERK and AKT pathways and a tendency for reduced activation of the mTOR pathway as measured in phosphoS6/S6 levels. The expression of pyruvate dehydrogenase kinase 4 was up-regulated in both mRNA and protein levels. Gene expression analyses showed changes in oxidation/reduction, fatty acid and monosaccharide metabolic pathways, tissue organization, cell fate and regulation of apoptosis. Together, our results suggest that the high-risk Western diet primes the intestine to tumorigenesis through synergistic effects in energy metabolism, inflammation and oxidative stress, which culminate in the acceleration of LOH of the Apc gene.

Inherited cancer predisposition sensitizes colonic mucosa to address Western diet effects and putative cancer-predisposing changes on mouse proteome.

Human epidemiological evidence and previous studies on mice have shown that Western-style diet (WD) may predispose gut mucosa to colorectal cancer (CRC). The mechanisms that mediate the effects of diet on tumorigenesis are largely unknown. To address putative cancer-predisposing events available for early detection, we quantitatively analyzed the proteome of histologically normal colon of a wild-type (Mlh1(+/+)) and an Mlh1(+/-) mouse after a long-term feeding experiment with WD and AIN-93G control diet. The Mlh1(+/-) mouse carries susceptibility to colon cancer analogous to a human CRC syndrome (Lynch syndrome). Remarkably, WD induced expression changes reflecting metabolic disturbances especially in the cancer-predisposed colon, while similar changes were not significant in the wild-type proteome. Overall, the detected changes constitute a complex interaction network of proteins involved in ATP synthesis coupled proton transport, oxidoreduction coenzyme and nicotinamide nucleotide metabolic processes, important in cell protection against reactive oxygen species toxicity. Of these proteins, selenium binding protein 1 and galectin-4, which directly interact with MutL homolog 1, are underlined in neoplastic processes, suggesting that sensitivity to WD is increased by an Mlh1 mutation. The significance of WD on CRC risk is highlighted by the fact that five out of six mice with neoplasias were fed with WD.

Cancer-predicting gene expression changes in colonic mucosa of Western diet fed Mlh1+/- mice.

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world and interactions between genetic and environmental factors, including diet, are suggested to play a critical role in its etiology. We conducted a long-term feeding experiment in the mouse to address gene expression and methylation changes arising in histologically normal colonic mucosa as putative cancer-predisposing events available for early detection. The expression of 94 growth-regulatory genes previously linked to human CRC was studied at two time points (5 weeks and 12 months of age) in the heterozygote Mlh1(+/-) mice, an animal model for human Lynch syndrome (LS), and wild type Mlh1(+/+) littermates, fed by either Western-style (WD) or AIN-93G control diet. In mice fed with WD, proximal colon mucosa, the predominant site of cancer formation in LS, exhibited a significant expression decrease in tumor suppressor genes, Dkk1, Hoxd1, Slc5a8, and Socs1, the latter two only in the Mlh1(+/-) mice. Reduced mRNA expression was accompanied by increased promoter methylation of the respective genes. The strongest expression decrease (7.3 fold) together with a significant increase in its promoter methylation was seen in Dkk1, an antagonist of the canonical Wnt signaling pathway. Furthermore, the inactivation of Dkk1 seems to predispose to neoplasias in the proximal colon. This and the fact that Mlh1 which showed only modest methylation was still expressed in both Mlh1(+/-) and Mlh1(+/+) mice indicate that the expression decreases and the inactivation of Dkk1 in particular is a prominent early marker for colon oncogenesis.

Pathogenicity of MSH2 missense mutations is typically associated with impaired repair capability of the mutated protein.

BACKGROUND & AIMS: Inherited deleterious mutations in mismatch repair genes MLH1, MSH2, and MSH6 predispose to hereditary nonpolyposis colorectal cancer. A major diagnostic challenge is the difficulty in evaluating the pathogenicity of missense mutations. Previously we showed that most missense variants in MSH6 do not impair MMR capability and are associated with no or low cancer susceptibility, whereas in MLH1, functional studies distinguished nontruncating mutations with severe defects from those not or slightly impaired in protein expression or function. The present study was undertaken to evaluate the pathogenicity of inherited missense mutations in MSH2. METHODS: Fifteen mutated MSH2 proteins including 14 amino acid substitutions and one in-frame deletion were tested for expression/stability, MSH2/MSH6 interaction, and repair efficiency. The genetic and biochemical data were correlated with the clinical data. Comparative sequence analysis was performed to assess the value of sequence homology as a tool for predicting functional results. RESULTS: None of the studied MSH2 mutations destroyed the protein or abolished MSH2/MSH6 interaction, whereas 12 mutations impaired the repair capability of the protein. Comparative sequence analysis correctly predicted functional studies for 13 of 14 amino acid substitutions. CONCLUSIONS: Interpretation was pathogenic for 12, nonpathogenic for 2, and contradictory for 1 mutation. The pathogenicity could not be distinguished unambiguously by phenotypic characteristics, although correlation between the absence of staining for MSH2 and pathogenicity of the missense mutation was notable. Unlike in MSH6 and MLH1, the pathogenicity of missense mutations in MSH2 was always associated with impaired repair capability of the mutated protein.

The importance of functional testing in the genetic assessment of Muir-Torre syndrome, a clinical subphenotype of HNPCC.

A majority of families with hereditary nonpolyposis colorectal cancer (HNPCC) are attributable to germline mutations in three DNA mismatch repair (MMR) genes, MLH1, MSH2 and MSH6. However, the clinical phenotype appears to reflect a complex interplay between the predisposing mutation and putative constitutional and somatic modifiers. Certain MMR gene mutations predispose to combined occurrence of cutaneous sebaceous gland neoplasms and visceral malignancies, which is known as Muir-Torre syndrome (MTS) and regarded as a phenotypic variant of HNPCC. The sebaceous tumors associated with MTS appear in many patients before visceral malignancies providing important predictability of HNPCC-related integral cancers in mutation carriers. Since most sebaceous skin tumors are, however, sporadic, the contribution of non-truncating mutations found in skin cancer patients is difficult to interpret and genetic assessment of MTS requires a functional test. Here, we studied the repair efficiency of the two MSH2 missense mutations, L187P and C697F, found in HNPCC families including a few mutation carriers with sebaceous skin tumors. Both mutations were completely deficient in an MMR assay, which together with tumor findings suggested their predisposing role in both internal and skin malignancies in the families.

Genome-wide scanning for linkage in Finnish breast cancer families.

Only a proportion of breast cancer families has germline mutations in the BRCA1 or BRCA2 genes, suggesting the presence of additional susceptibility genes. Finding such genes by linkage analysis has turned out to be difficult due to the genetic heterogeneity of the disease, phenocopies and incomplete penetrance of the mutations. Isolated populations may be helpful in reducing the level of genetic heterogeneity and in providing useful starting points for further genetic analyses. Here, we report results from a genome-wide linkage analysis of 14 high-risk breast cancer families from Finland. These families tested negative for BRCA1 and BRCA2 germline mutations and showed no linkage to the 13q21 region, recently proposed as an additional susceptibility locus. Suggestive linkage was seen at marker D2S364 (2q32) with a parametric two-point LOD score of 1.61 (theta=0), and an LOD score of 2.49 in nonparametric analyses. Additional genotyping of a 40 cM chromosomal region surrounding the region of interest yielded a maximum parametric two-point LOD score of 1.80 (theta=0) at D2S2262 and a nonparametric LOD score of 3.11 at an adjacent novel marker 11291M1 in BAC RP11-67G7. A nonparametric multipoint LOD score of 3.20 was seen at 11291M1 under the assumption of dominant inheritance. While not providing proof of linkage considering the small number of families and large number of laboratory and statistical analyses performed, these results warrant further studies of the 2q32 chromosomal region as a candidate breast cancer susceptibility locus. Both linkage and association studies are likely to be useful, particularly in other isolated populations.