Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Integrative analysis of hereditary nonpolyposis colorectal cancer: the contribution of allele-specific expression and other assays to diagnostic algorithms.

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

Mutation analysis of CCM1, CCM2 and CCM3 genes in a cohort of Italian patients with cerebral cavernous malformation.

Cerebral cavernous malformations (CCMs) are vascular lesions of the CNS characterized by abnormally enlarged capillary cavities. CCMs can occur as sporadic or familial autosomal dominant form. Familial cases are associated with mutations in CCM1[K-Rev interaction trapped 1 (KRIT1)], CCM2 (MGC4607) and CCM3 (PDCD10) genes. In this study, a three-gene mutation screening was performed by direct exon sequencing, in a cohort of 95 Italian patients either sporadic or familial, as well as on their at-risk relatives. Sixteen mutations in 16 unrelated CCM patients were identified,nine mutations are novel: c.413T > C; c.601C > T; c.846 + 2T > G; c.1254delA; c.1255-4delGTA; c.1682-1683 delTA in CCM1; c.48A > G; c.82-83dupAG in CCM2; and c.395 + 1G > A in CCM3 genes [corrected].The samples, negative to direct exon sequencing, were investigated by MLPA to search for intragenic deletions or duplications. One deletion in CCM1 exon 18 was detected in a sporadic patient. Among familial cases 67% had a mutation in CCM1, 5.5% in CCM2, and 5.5% in CCM3, whereas in the remaining 22% no mutations were detected, suggesting the existence of either undetectable mutations or other CCM genes. This study represents the first extensive research program for a comprehensive molecular screening of the three known genes in an Italian cohort of CCM patients and their at-risk relatives.

Prognostic relevance of MLH1 and MSH2 mutations in hereditary non-polyposis colorectal cancer patients.

AIMS AND BACKGROUND: Colorectal carcinoma patients from hereditary non-polyposis colorectal cancer families are suggested to have a better prognosis than sporadic colorectal carcinoma cases. Since the majority of hereditary non-polyposis colorectal cancer-related colorectal carcinomas are characterized by microsatellite instability due to germline mutations in DNA mismatch repair genes, this is consistent with the prolonged survival observed in sporadic microsatellite instability-positive colorectal carcinoma compared to microsatellite stable cases. However, a fraction of colorectal carcinoma cases belongs to families that, despite fulfilling the clinical criteria for hereditary non-polyposis colorectal cancer, do not carry mismatch repair gene mutations. Our aim was to verify to what extent the genotypic heterogeneity influences the prognosis of hereditary non-polyposis colorectal cancer patients. METHODS: A survival analysis was performed on 526 colorectal carcinoma cases from 204 Amsterdam Criteria-positive hereditary non-polyposis colorectal cancer families. Enrolled cases were classified as MLH1-positive, MSH2-positive and mutation-negative, according to the results of genetic testing in each family. RESULTS: Five-year survival rates were 0.73 (95% CI, 0.66-0.80), 0.75 (95% CI, 0.66-0.84) and 0.62 (95% CI, 0.55-0.68) for MLH1-positive, MSH2-positive and mutation-negative groups, respectively (logrank test, P = 0.01). Hazard ratio, computed using Cox regression analysis and adjusted for age, sex, tumor site and stage, was 0.71 (95% CI, 0.51-0.98) for the mutation-positive compared to the mutation-negative group. Moreover, in the latter group, patients with microsatellite instability-positive colorectal carcinomas showed a better outcome than microsatellite stable cases (5-year survival rates, 0.81 and 0.60, respectively; logrank test, P = 0.006). CONCLUSIONS: Our results suggest that the prognosis of hereditary non-polyposis colorectal cancer-related colorectal carcinoma patients depends on the associated constitutional mismatch repair genotype.

Mutation analysis of oxisterol-binding-protein gene in patients with age-related macular degeneration.

Allelic variants of several genes are increasingly recognized as susceptibility factors in age-related macular degeneration (AMD). Because of its metabolic characteristics the macula is sensitive to oxidative damage, and supplementation with antioxidants has been shown to be effective in slowing the progression of disease in AMD patients. The oxisterol-binding-protein (OSBP2) gene is expressed mainly in the retinal pigmented epithelium underlying the macular region. Its product specifically binds and transports oxisterols, the cytotoxic effects of which may be involved in macular damage. The aim of this study was to search for allelic variants of OSBP2 gene, as well as to evaluate several risk factors in 24 patients with AMD; 17 with nonexudative (NE) and 7 with neovascular (NV) form. Total cholesterol was elevated in 66% of the patients, high-density lipoprotein (HDL) cholesterol was reduced in 12%; vitamin A or vitamin E deficiency was not observed. OSBP2 gene analysis was performed in AMD patients and in 110 control subjects by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Six allelic variants were detected: 2 nonpolymorphic unique exonic variants in 2 AMD subjects and 4 polymorphic variants (2 exonic and 2 intronic). These data indicate a possible role of OSBP2 gene in the pathogenesis of oxidative damage to the macula induced by oxysterols in AMD patients.

Combined use of MLPA and nonfluorescent multiplex PCR analysis by high performance liquid chromatography for the detection of genomic rearrangements.

Large genomic rearrangements are recognized as playing a pathogenic role in an increasing number of human genetic diseases. It is important to develop efficient methods for the routine detection and confirmation of these germline defects. Multiplex ligation-dependent probe amplification (MLPA) is considered an early step for molecular diagnosis of several genetic disorders. However, artifacts might hamper the interpretation of MLPA analysis, especially when rearrangements involve a single exon. Therefore, rearrangements must be verified by two independent methods. In this study, we developed nonfluorescent multiplex-PCR coupled to high-performance liquid chromatography (NFMP-HPLC) and analyzed whether the use of this method combined with MLPA could be helpful in the detection and confirmation of gross MSH2 and MLH1 genomic rearrangements. A total of nine nonfluorescent multiplex-PCRs were developed to analyze the 16 MSH2 and 19 MLH1 exons. Reliable multiplex amplifications and nonfluorescent peak quantitation were obtained with a limited number of cycles (< or = 25) using a denaturing HPLC (DHPLC) instrument under nondenaturing conditions. The results obtained by NFMP-HPLC were highly reproducible. The combined use of MLPA and NFMP-HPLC identified and independently confirmed putative MLH1 and MSH2 deletions in eight out of 50 unrelated patients with hereditary nonpolyposis colorectal cancer (HNPCC). In five cases, the deletions affected a single exon and in three cases multiple contiguous exons. These results were in agreement with breakpoint and complementary DNA (cDNA) analyses. Considering that MLPA and NFMP-HPLC are unlikely to be affected by the same artifacts, their combined use could also provide a robust and cost-effective strategy for routine screening and confirmation of putative rearrangements in other genes, especially when a single exon is involved or a precise characterization of breakpoints is not achieved.

Prevalence of the Y165C, G382D and 1395delGGA germline mutations of the MYH gene in Italian patients with adenomatous polyposis coli and colorectal adenomas.

Biallelic germline mutations in the base excision repair gene MYH have been reported in patients with multiple colorectal adenomas and cancer and in sporadic FAP patients not showing a detectable APC germline mutation. In this study, the prevalence of the common Y165C and G382D germline variants of the MYH gene was examined in 70 FAP/AAPC patients with no detectable APC mutation and a family history compatible with recessive inheritance. In addition, 141 normal-population adenoma patients (mean number of adenomas, 2.8; range, 1-9) and 52 clean colon controls were studied. The entire coding region of the MYH gene was analyzed in Y165C or G382D heterozygous patients. Since the same second mutational event (a 3 bp deletion in exon 14, 1395delGGA) was detected in 3 patients, the prevalence of this variant was also examined in all groups. In all, 14 of 70 patients in the FAP/AAPC group (20%; 95% CI = 11.7-31.6%) had biallelic germline MYH variants and 3 were heterozygotes (4.3%). None of the 141 normal-population adenoma patients carried biallelic germline MYH variants (95% CI = 0.06-4.1%) and 3 were heterozygotes (2.1%). In the control group, no MYH variants were detected. These results indicated that MYH-associated polyposis (MAP) is present in about 20% of Italian FAP/AAPC patients, in whom no germline APC mutation is detectable and showing a family history compatible with recessive inheritance, and in a small fraction of patients with colorectal adenomas in the general population. In addition, our data suggest that mutation 1395delGGA is a subpolymorphic MYH mutational event in some Caucasian populations.

Identification of a novel KRIT1 mutation in an Italian family with cerebral cavernous malformation by the protein truncation test.

Familial cerebral cavernous malformation (CCM) exhibits autosomal dominant inheritance and is characterized by vascular disorders of the brain, which can lead to seizures, focal neurological deficits, hemorrhagic stroke, and migraine. Three CCM loci have been mapped, but the gene for only one locus--KRIT1 coding for Krev-1/rap1 interaction trapped 1 (KRIT1) protein, which is responsible for more than 40% of familial cases--has been identified. To date, a total of 72 mutations have been described, with one founder effect in the Mexican/Hispanic community. We report the case of an Italian family with CCM that has a novel KRIT1 gene mutation leading to a truncated KRIT1 protein. The protein truncation test (PTT) has been used as a rapid method of identifying germline mutations in the KRIT1 gene.