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.

A draft genome sequence of an invasive mosquito: an Italian Aedes albopictus.

The draft genome sequence of Italian specimens of the Asian tiger mosquito Aedes (Stegomyia) albopictus (Diptera: Culicidae) was determined using a standard NGS (next generation sequencing) approach. The size of the assembled genome is comparable to that of Aedes aegypti; the two mosquitoes are also similar as far as the high content of repetitive DNA is concerned, most of which is made up of transposable elements. Although, based on BUSCO (Benchmarking Universal Single-Copy Orthologues) analysis, the genome assembly reported here contains more than 99% of protein-coding genes, several of those are expected to be represented in the assembly in a fragmented state. We also present here the annotation of several families of genes (tRNA genes, miRNA genes, the sialome, genes involved in chromatin condensation, sex determination genes, odorant binding proteins and odorant receptors). These analyses confirm that the assembly can be used for the study of the biology of this invasive vector of disease.

A genetic-demographic approach reveals a gender-specific association of SLC6A3/DAT1 40 bp-VNTR with life-expectancy.

Several recent lines of evidence are proving an important role for dopamine in the aging process and in the determination of life span. Components of the dopaminergic system may represent good candidates for longevity studies. Herein, we tested the possible association of the functional SLC6A3/DAT1 40-bp VNTR with life-expectancy in a healthy population of Central Italy (N = 993) by applying a genetic-demographic approach that takes into account the demographic information and different survival rates between sexes for modeling the survival of specific allele carriers in the population. Male carriers of S*/S* genotype showed a lower survival chance across most of the lifespan respect to the survival of DAT1*L-carriers (P = 0.021). The same analyses gave non-significant results in females. Several studies already reported significant sex differences in dopamine metabolism and its related biological pathways. Thus, we can hypothesize that the SLC6A3/DAT1 40 bp-VNTR may affect life expectancy in a sex-specific way. Moreover, it is conceivable that DAT1 S*/S* carriers, who are prone to assume "risk" type behaviors, may be dropped out of the "healthy" population by a sort of "demographic selection".

MLH1 constitutional and somatic methylation in patients with MLH1 negative tumors fulfilling the revised Bethesda criteria.

Lynch syndrome (LS) is a tumor predisposing condition caused by constitutional defects in genes coding for components of the mismatch repair (MMR) apparatus. While hypermethylation of the promoter of the MMR gene MLH1 occurs in about 15% of colorectal cancer samples, it has also been observed as a constitutional alteration, in the absence of DNA sequence mutations, in a small number of LS patients. In order to obtain further insights on the phenotypic characteristics of MLH1 epimutation carriers, we investigated the somatic and constitutional MLH1 methylation status of 14 unrelated subjects with a suspicion of LS who were negative for MMR gene constitutional mutations and whose tumors did not express the MLH1 protein. A novel case of constitutional MLH1 epimutation was identified. This patient was affected with multiple primary tumors, including breast cancer, diagnosed starting from the age of 55 y. Investigation of her offspring by allele specific expression revealed that the epimutation was not stable across generations. We also found MLH1 hypermethylation in cancer samples from 4 additional patients who did not have evidence of constitutional defects. These patients had some characteristics of LS, namely early age at onset and/or positive family history, raising the possibility of genetic influences in the establishment of somatic MLH1 methylation.

Duodenal carcinoma in a 37-year-old man with Cowden/Bannayan syndrome.

A 37-year-old man was hospitalised because of anaemia and fatigue due to an infiltrating adenocarcinoma of the Treitz angle (duodenum), together with gastric, duodenal and colorectal polyps. After the operation, removal of colorectal lesions revealed the presence of ganglioneuromatosis of the large bowel. Further investigations showed lack of MLH1 protein expression and microsatellite instability in the duodenal neoplasm, while the gene was normally expressed in the polyps. MLH1 sequence and Multiple Ligation-dependent Probes Amplification analysis (from constitutional DNA) were normal. Analysis of the PTEN gene revealed the presence of a constitutional mutation (c.510 T>A; p.Ser170Arg) which had been associated with the Cowden phenotype. Further detailed clinical investigations revealed macrocephaly (63 cm), melanotic spots of the penis, small angiomas, millimetric trichilemmomas in the nose and multiple lipomas, which led to the diagnosis of Cowden/Bannayan disease. The unusual appearance of a duodenal carcinoma as the first symptom rendered the identification of the syndrome extremely difficult.

High resolution melting analysis for a rapid identification of heterozygous and homozygous sequence changes in the MUTYH gene.

BACKGROUND: MUTYH-associated polyposis (MAP) is an autosomal recessive form of intestinal polyposis predisposing to colorectal carcinoma. High resolution melting analysis (HRMA) is a mutation scanning method that allows detection of heterozygous sequence changes with high sensitivity, whereas homozygosity for a nucleotide change may not lead to significant curve shape or melting temperature changes compared to homozygous wild-type samples. Therefore, HRMA has been mainly applied to the detection of mutations associated with autosomal dominant or X-linked disorders, while applications to autosomal recessive conditions are less common. METHODS: MUTYH coding sequence and UTRs were analyzed by both HRMA and sequencing on 88 leukocyte genomic DNA samples. Twenty-six samples were also examined by SSCP. Experiments were performed both with and without mixing the test samples with wild-type DNA. RESULTS: The results show that all MUTYH sequence variations, including G > C and A > T homozygous changes, can be reliably identified by HRMA when a condition of artificial heterozygosity is created by mixing test and reference DNA. HRMA had a sensitivity comparable to sequencing and higher than SSCP. CONCLUSIONS: The availability of a rapid and inexpensive method for the identification of MUTYH sequence variants is relevant for the diagnosis of colorectal cancer susceptibility, since the MAP phenotype is highly variable.