The Role of miRNA Expression Profile in Sudden Cardiac Death Cases.

Sudden cardiac death (SCD) is one of the leading causes of death in the world and for this reason it has attracted the attention of numerous researchers in the field of legal medicine. It is not easy to determine the cause in a SCD case and the available methods used for diagnosis cannot always give an exhaustive answer. In addition, the molecular analysis of genes does not lead to a clear conclusion, but it could be interesting to focus attention on the expression level of miRNAs, a class of non-coding RNA of about 22 nucleotides. The role of miRNAs is to regulate the gene expression through complementary binding to 3'-untraslated regions of miRNAs, leading to the inhibition of translation or to mRNA degradation. In recent years, several studies were performed with the aim of exploring the use of these molecules as biomarkers for SCD cases, and to also distinguish the causes that lead to cardiac death. In this review, we summarize experiments, evidence, and results of different studies on the implication of miRNAs in SCD cases. We discuss the different biological starting materials with their respective advantages and disadvantages, studying miRNA expression on tissue (fresh-frozen tissue and FFPE tissue), circulating cell-free miRNAs in blood of patients affected by cardiac disease at high risk of SCD, and exosomal miRNAs analyzed from serum of people who died from SCD.

Development and Validation of MPS-Based System for Human Appearance Prediction in Challenging Forensic Samples.

Forensic DNA phenotyping (FDP) provides the ability to predict the human external traits from unknown sample donors, directly from minute amounts of DNA found at the crime scene. We developed a MPS multiplex assay, with the aim of genotyping all 41 DNA markers included in the HIrisPlex-S system for simultaneous prediction of eye, hair and skin colours. Forensic samples such as blood, skeletal remains, touch DNA, saliva swab, artificially degraded samples together with individuals with known phenotypes and a set of 2800 M control DNA were sequenced on the Ion Torrent platform in order to evaluate the concordance testing results and the forensic suitability of the 41-plex MPS assay. The panel was evaluated by testing a different number of PCR cycles and the volume of reagents for library preparation. The study demonstrated that full and reliable profiles were obtained with 0.1-5 ng, even with high degraded DNA. The increment of the number of PCR cycles results in an improvement of correctly genotyping and phenotyping for samples with low amounts of degraded DNA but higher frequencies of artefacts were found. The high DNA degradation level did not influence the correct genotyping and phenotyping and the critical parameter affecting the result is the quantity of input DNA. Eye and hair colour was predicted in 92.60% of individuals and skin colour in 85.15% of individuals. The results suggest that this MPS assay is robust, highly sensitive and useful for human pigmentation prediction in the forensic genetic field.

Circulating SARS-CoV-2 variants in Italy, October 2020-March 2021.

A growing number of emerging SARS-CoV-2 variants is being identified worldwide, potentially impacting the effectiveness of current vaccines. We report the data obtained in several Italian regions involved in the SARS-CoV-2 variant monitoring from the beginning of the epidemic and spanning the period from October 2020 to March 2021.

Death following extreme temperature exposure: Histological, biochemical and immunohistochemical markers.

INTRODUCTION: Defining extreme temperatures as the cause of death remains challenging. It is mostly based on circumstantial, macroscopic and microscopic features. METHODS: We retrospectively compared groups of cases of fatal hypothermia, fatal hyperthermia and non-extreme temperature-related deaths. We analysed specific histological findings, focusing on samples from the liver, pancreas and kidney. RESULTS: Between 1 January 2013 and 31 December 2016, 15 autopsies were performed for deaths related to extreme temperatures. They included 11 cases of fatal hypothermia (group A), four cases of fatal hyperthermia (group B) and eight controls (group C). Perinuclear hepatocyte vacuolisation was observed in seven cases of hypothermia, one case of hyperthermia and four controls. Pancreatic cytoarchitecture was well preserved in two cases of hypothermia, one case of hyperthermia and two controls. No particular microscopic feature was found in pancreatic samples. Renal epithelial tubular cell vacuolisation was observed in seven cases of hypothermia and one case of hyperthermia, while it was absent in all controls. Chromogranin A (CgA) was markedly positive in the pancreatic tissue of five cases of fatal hypothermia and one control, and mildly positive in one case of fatal hyperthermia. No significant p-values were observed for any comparisons (p > 0.05), except when hypothermia cases group were compared to the control group for the Armanni-Ebstein phenomenon test (p = 0.0078). CONCLUSIONS: Although our study did not find a specific microscopic marker, hepatocyte vacuolisation, the Armanni-Ebstein phenomenon and pancreatic CgA positivity, taken together, may be useful tools to confirm hypo- and hyperthermia-related deaths, in addition to circumstantial and macroscopic findings.

Assessment of the Precision ID Identity Panel kit on challenging forensic samples.

The performance of the Precision ID Identity Panel (Thermo Fisher Scientific) was assessed on a set of 87 forensic samples with different levels of degradation for which a reference sample from the "same donor" or from a "first degree relative" was available. PCR-MPS analysis was performed with DNA input ranging from 1 ng to 12 pg and through 21-26 PCR cycles, in replicate tests, and a total number of 255 libraries were sequenced on the Ion Personal Genome Machine™ (PGM™) System. The evaluation of the molecular data allowed to set a fix threshold for locus call at 50 x which suitably worked even when low amounts of degraded DNA (12 pg) were investigated. In these analytical conditions, in fact, 25 PCR cycles allowed the genotyping of about 50 % and 35 % of the autosomal and the Y-specific markers on average, respectively, for each single amplification with a negligible frequency of drop ins (0.01 %). On the other hand, drop out artefacts reached 18-23 % when low copy number and degraded DNA samples were studied, with surviving alleles showing more than 600 reads in 2.9 % of the cases. Our data pointed out that the Precision ID Identity Panel allowed accurate typing of almost any amount of good quality/moderately degraded DNA samples, in duplicate tests. The analysis of low copy number DNAs evidenced that the same allele of a heterozygous genotype could be lost twice, thus suggesting that a third amplification could be useful for a correct genotype assignment in these peculiar cases. Using the consensus approach, a limited number of genotyping errors were computed and about 37 % of the autosomal markers was finally typed with a corresponding combined random match probability of at least 1.6 × 10-13, which can be considered an excellent result for this kind of challenging samples. In the end, the results presented in this study emphasize the crucial role of the expert opinion in the correct evaluation of artefacts arising from PCR-MPS technology that could potentially lead to genetic mistyping.

Searching the undetected mtDNA variants in forensic MPS data.

The efficiency of MPS in forensic mtDNA analysis has been thoroughly proven, although a reliable and well established data evaluation still remains a critical point. Numerous bioinformatics tools have been developed, but most of them require specific operating systems and high costs, while free open-source programs with user-friendly interfaces are few. In this study, 43 full mtGenomes were sequenced using the Ion Personal Genome Machine™ (PGM™) System and analyzed utilizing the plug-in Variant Caller (TVC) of the Ion Torrent Software Suite and the mtDNA-Server (mDS), a free web-based mitochondrial analysis tool for MPS data. The outcomes of these two different analysis tools were compared to variants noted after manual inspection of the aligned reads performed using Integrative Genomics Viewer (IGV). The comparison highlighted the presence of thirty-nine discordant variant calls, which were resolved by Sanger sequencing that confirmed the presence of all variants, except for 7 deletions. The combined adoption of IGV and Sanger type sequencing confirmatory steps, in addition of TVC and mDS analysis, resulted in a more accurate variants assignment with the detection of 32 additional true polymorphisms, which were noted in the final dataset. Regarding the heteroplasmy issue, out of a total of thirty heteroplasmic variants, twenty-eight were detected by the TVC, while the mDS detected twenty-two. Overall, none of the used bioinformatics tools were the perfect choice and a secondary analysis with an expert's opinion in complete mtGenome MPS data evaluation is still required in forensic genetic analysis.

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing.

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer's instructions.

Evaluation of a microhaplotypes panel for forensic genetics using massive parallel sequencing technology.

Massive parallel DNA sequencing (MPS) makes it possible to explore a new type of genetic marker, known as microhaplotypes or microhaps. These loci were recently introduced in the landscape of forensic genetic and appear to be useful for identification purposes, reconstruction of family relationships, ancestry prediction and DNA mixtures deconvolution. Microhaplotypes loci, based on 89 loci in ALFRED, were selected and their genetic variations in 100 Italian individuals were evaluated by using MPS, in order to make inference about utility of a set of microhaps in forensic genetics. After MPS, the panel was reduced to 87 microhaps, comprised of 266 different SNPs and spread across 22 human autosomes. Genotype and haplotype frequencies were estimated, as well as the effective number of alleles at each locus (Ae), which relates to the usefulness of the locus in resolution of relationships and deconvolution of DNA mixtures. Overall, the Ae values for the 87 microhaps range from 1.010 to 8.344, with about 80% showing values greater than 2.0. Noteworthy, 32 microhaps display Ae values greater than 3.0 and 18 loci Ae above 4.0. To explore the suitability of microhaplotypes in mixture deconvolution, the probability of detecting a mixture, as a function of Ae, was inferred for different groups of loci. Considering the fourteen loci with Ae between 3.0 and 3.999 the probability of detecting a mixture was at least 0.99973, while considering the ten loci with Ae between 4.0 and 4.999 the probability was at least 0.99998. Moreover, when considering just the six loci with Ae between 5.0 and 5.999 the probability of detecting a mixture was at least 0.99984, while when considering just the two loci with Ae above 6 the probability was 0.97228. Combining these 32 MH loci, the theoretical probability of detecting a mixture was 0.999999999999973. These results make the subset of 32 loci with Ae above three informative for mixture resolution. The individual matching probabilities (PI) of the 87 microhaps ranged from 0.032 to 0.9802. Considering the 32 microhap loci with Ae greater than 3.0, the cumulative PI value was 1.6 × 10-33, while considering the 18 microhap loci with Ae above 4.0, the cumulative PI value was 2.34 × 10-21. Overall the results of this study confirmed the utility of microhaps in forensic genetics.

The mitochondrial DNA makeup of Romanians: A forensic mtDNA control region database and phylogenetic characterization.

To evaluate the pattern of Romanian population from a mitochondrial perspective and to establish an appropriate mtDNA forensic database, we generated a high-quality mtDNA control region dataset from 407 Romanian subjects belonging to four major historical regions: Moldavia, Transylvania, Wallachia and Dobruja. The entire control region (CR) was analyzed by Sanger-type sequencing assays and the resulting 306 different haplotypes were classified into haplogroups according to the most updated mtDNA phylogeny. The Romanian gene pool is mainly composed of West Eurasian lineages H (31.7%), U (12.8%), J (10.8%), R (10.1%), T (9.1%), N (8.1%), HV (5.4%),K (3.7%), HV0 (4.2%), with exceptions of East Asian haplogroup M (3.4%) and African haplogroup L (0.7%). The pattern of mtDNA variation observed in this study indicates that the mitochondrial DNA pool is geographically homogeneous across Romania and that the haplogroup composition reveals signals of admixture of populations of different origin. The PCA scatterplot supported this scenario, with Romania located in southeastern Europe area, close to Bulgaria and Hungary, and as a borderland with respect to east Mediterranean and other eastern European countries. High haplotype diversity (0.993) and nucleotide diversity indices (0.00838±0.00426), together with low random match probability (0.0087) suggest the usefulness of this control region dataset as a forensic database in routine forensic mtDNA analysis and in the investigation of maternal genetic lineages in the Romanian population.

The molecular characterization of a depurinated trial DNA sample can be a model to understand the reliability of the results in forensic genetics.

The role of DNA damage in PCR processivity/fidelity is a relevant topic in molecular investigation of aged/forensic samples. In order to reproduce one of the most common lesions occurring in postmortem tissues, a new protocol based on aqueous hydrolysis of the DNA was developed in vitro. Twenty-five forensic laboratories were then provided with 3.0 µg of a trial sample (TS) exhibiting, in mean, the loss of 1 base of 20, and a molecular weight below 300 bp. Each participating laboratory could freely choose any combination of methods, leading to the quantification and to the definition of the STR profile of the TS, through the documentation of each step of the analytical approaches selected. The results of the TS quantification by qPCR showed significant differences in the amount of DNA recorded by the participating laboratories using different commercial kits. These data show that only DNA quantification "relative" to the used kit (probe) is possible, being the "absolute" amount of DNA inversely related to the length of the target region (r(2) = 0.891). In addition, our results indicate that the absence of a shared stable and certified reference quantitative standard is also likely involved. STR profiling was carried out selecting five different commercial kits and amplifying the TS for a total number of 212 multiplex PCRs, thus representing an interesting overview of the different analytical protocols used by the participating laboratories. Nine laboratories decided to characterize the TS using a single kit, with a number of amplifications varying from 2 to 12, obtaining only partial STR profiles. Most of the participants determined partial or full profiles using a combination of two or more kits, and a number of amplifications varying from 2 to 27. The performance of each laboratory was described in terms of number of correctly characterized loci, dropped-out markers, unreliable genotypes, and incorrect results. The incidence of unreliable and incorrect genotypes was found to be higher for participants carrying out a limited number of amplifications, insufficient to define the correct genotypes from damaged DNA samples such as the TS. Finally, from a dataset containing about 4500 amplicons, the frequency of PCR artifacts (allele dropout, allele drop-in, and allelic imbalance) was calculated for each kit showing that the new chemistry of the kits is not able to overcome the concern of template-related factors. The results of this collaborative exercise emphasize the advantages of using a standardized degraded DNA sample in the definition of which analytical parameters are critical for the outcome of the STR profiles.

Role of 5-HTTLPR polymorphism in the development of the inward/outward personality organization: a genetic association study.

Reciprocity with primary caregivers affects subjects' adaptive abilities toward the construction of the most useful personal meaning organization (PMO) with respect to their developmental environment. Within cognitive theory the post-rationalist approach has outlined two basic categories of identity construction and of regulation of cognitive and emotional processes: the Outward and the Inward PMO. The presence of different, consistent clinical patterns in Inward and Outward subjects is paralleled by differences in cerebral activation during emotional tasks on fMRI and by different expression of some polymorphisms in serotonin pathways. Since several lines of evidence support a role for the 5-HTTLPR polymorphism in mediating individual susceptibility to environmental emotional stimuli, this study was conducted to investigate its influence in the development of the Inward/Outward PMO. PMO was assessed and the 5-HTTLPR polymorphism investigated in 124 healthy subjects who were subdivided into an Inward (n = 52) and an Outward (n = 72) group. Case-control comparisons of short allele (S) frequencies showed significant differences between Inwards and Outwards (p = 0.036, χ2 test; p = 0.026, exact test). Genotype frequencies were not significantly different although values slightly exceeded p ≤ 0.05 (p = 0.056, χ2 test; p = 0.059, exact test). Analysis of the 5-HTTLPR genotypes according to the recessive inheritance model showed that the S/S genotype increased the likelihood of developing an Outward PMO (p = 0.0178, χ2 test; p = 0.0143, exact test; OR = 3.43, CI (95%) = 1.188-9.925). A logistic regression analysis confirmed the association between short allele and S/S genotypes with the Outward PMO also when gender and age were considered. However none of the differences remained significant after correction for multiple testing, even though using the recessive model they approach significance. Overall our data seem to suggest a putative genetic basis for interindividual differences in PMO development.

ADH4 intronic variations are associated with alcohol dependence: results from an Italian case-control association study.

OBJECTIVES: This study investigated the involvement of ADH4 gene polymorphisms in the susceptibility to alcohol use disorders. METHODS: Thirty-eight single-nucleotide polymorphisms (SNPs) in and around the ADH4 gene were investigated in 136 Italian alcoholics and 276 healthy controls. A new approach based on a bioinformatic method selected 26 SNPs that may affect the splicing sites, destroying or creating binding sites of splicing regulatory proteins. RESULTS: Case-control comparisons for allele and genotype frequencies showed that ADH4 SNPs were associated with alcohol dependence but not with alcohol abuse. The association signal was strongest for rs1009145, rs13148577 (both P=0.0008) and rs7689753 (P=0.0007), whose minor alleles were predicted to alter the target protein sequences involved in mRNA splicing. A pairwise linkage disequilibrium analysis showed that all SNPs except five were located in a single haplotype block. Six haplotype tag SNPs were selected to infer haplotypes and to estimate their frequency distributions. A logistic regression analysis confirmed the association between ADH4 variants and alcohol dependence when sex, age, years of education, marital status and the allele genotype, haplotype and diplotype data of the six haplotype tag SNP were considered. Haplotype ATAAAT, which contained the minor allele of rs10009145 and the major allele of rs7689753, increased the risk of alcohol dependence, whereas haplotype GGGGAT, bearing the major allele of rs10009145 and the minor allele of rs7689753, protected against it. Again, there was no evidence of an association with alcohol abuse. CONCLUSION: These data suggest that ADH4 intronic variants play a role in alcohol dependence susceptibility in Italian populations. Functional studies are needed to establish the role of the genetic variations that seem to affect the splicing mechanism.

An overview of the genetic susceptibility to alcoholism.

AIMS: Alcoholism is a multifactorial, genetically influenced disorder. It is a major health and social issue, a highly frequent disease and a cause of premature death. It is also the most expensive addictive disorder due to morbidity, mortality, societal and legal problems. Besides their involvement in alcohol-related fatalities, forensic scientists are also required to assess driving and working ability as well as permanent invalidity due to alcohol-related conditions. Greater knowledge of the genetic basis of alcoholism could improve prevention by identifying specific risk factors and mechanisms, leading to effective therapeutic strategies and eventually to personalized treatments. METHODS: This overview of the recent scientific literature on the genetic basis of alcoholism summarizes the analytical strategies currently applied to the identification of candidate genes involved in alcohol-use disorders (AUDs) and discusses some genes and related phenotypes that have been shown to influence the risk of alcoholism. RESULTS: Alcoholism is a complex heterogeneous genetic disease. It is a quantitative disorder, in which the combined incidence of multiple genetic factors and environmental factors varies from one subject to another. Family, twin and adoption studies indicate that 50-60% of the risk of alcoholism is due to genetic factors. Risk loci for AUDs include both genes involved in alcohol pharmacokinetics and pharmacodynamics as well as genes moderating neurophysiological responses such as impulsivity, disinhibition, sensation-seeking and externalizing behaviours. Alcoholism also co-exists with other addictions and psychiatric disorders. Such co-morbidity suggests the existence of shared aetiological factors. CONCLUSIONS: Despite several genes that influence the risk for AUDs having been identified, the genetic bases of alcoholism remain largely unknown. Particularly the mechanism of action or the understanding of the physiology of some genes, as well as the gene-environment interactions, is still unknown. Technological progress and advances in transcriptomics, epigenomics and proteomics are expected to enhance our knowledge of the genetic susceptibility to alcoholism.

GABRA2 and alcohol use disorders: no evidence of an association in an Italian case-control study.

BACKGROUND: Alcoholism is a major health and social issue, a highly frequent disease and a cause of premature death. It is also the most expensive addictive disorder being related to high morbidity and mortality, violence, accidents, and social and legal problems. It is a quantitative disorder, where the combined incidence of environmental and multiple genetic factors varies from 1 subject to another. Recent association studies have identified several genes as candidates for alcoholism, including GABAA receptor genes, due to their role in mediating several behavioral effects of alcohol, such as motor incoordination, anxiolysis, sedation, and withdrawal. The proposed association between the 3' half of the gene encoding the alpha-2 subunit of GABA receptor (3'-GABRA2) and alcohol use disorders (AUDs) has received several independent confirmations. METHODS: In this study, 10 single nucleotide polymorphisms (SNPs) of the 3'-GABRA2 gene, previously reported to be implicated in alcohol dependence, were used to evaluate the linkage between selected SNPs and AUDs in an Italian sample and to compare findings with those of previous studies. RESULTS: No evidence of an association was found at the allele, genotype, haplotype, or diplotype levels between the 3'-GABRA2 polymorphisms investigated and alcoholism in 149 Italian alcoholics (98 alcohol dependents and 51 alcohol abusers) and 278 controls. CONCLUSIONS: Despite previous reports, we did not find an association between AUDs and 3'-GABRA2 polymorphisms. This is probably due to the minimal comorbidity of our Italian sample suggesting that this gene is implicated in polysubstance dependence rather than in alcoholism alone.

Polymorphisms of mtDNA control region in Tunisian and Moroccan populations: an enrichment of forensic mtDNA databases with Northern Africa data.

Current forensic mitochondrial (mt)DNA databases are limited in representative population data of African origin. We investigated HVS-I/HVS-II sequences of 120 Tunisian and Moroccan healthy male donors applying stringent quality criteria to assure high quality of the data and phylogenetic alignment and notation of the sequences. Among 64 Tunisians, 56 different haplotypes were observed and the most common haplotype (16187T 16189C 16223T 16264T 16270T 16278T 16293G 16311C 73G 152C 182T 185T 195C 247A 263G 309.1C 315.1C; haplogroup (hg) L1b) was shared by four individuals. 56 Moroccans could be assigned to 52 different haplotypes where the most common haplotype was of West Eurasian origin with the hg H sequence motif 263G 315.1C and variations in the HVS-II polyC-stretch (309.1C 309.2C) shared by six samples. The majority of the observed haplotypes belong to the west Eurasian phylogeny (50% in Tunisians and 62.5% in Moroccans). Our data are consistent with the current phylogeographic knowledge displaying the occurrence of sub-Saharan haplogroup L sequences, found in 48.4% of Tunisians and 25% of Moroccans as well as the presence of the two re-migrated haplogroups U6 (7.8% and 1.8% in Tunisians and Moroccans, respectively) and M1 (1.6% in Tunisians and 8.9% in Moroccans).

Italian mitochondrial DNA database: results of a collaborative exercise and proficiency testing.

This work is a review of a collaborative exercise on mtDNA analysis undertaken by the Italian working group (Ge.F.I.). A total of 593 samples from 11 forensic genetic laboratories were subjected to hypervariable region (HVS-I/HVS-II) sequence analysis. The raw lane data were sent to MtDNA Population Database (EMPOP) for an independent evaluation. For the inclusion of data for the Italian database, quality assurance procedures were applied to the control region profiles. Only eight laboratories with a final population sample of 395 subjects passed the quality conformance test. Control region haplogroup (hg) assignments were confirmed by restriction fragment length polymorphism (RFLP) typing of the most common European hg-diagnostic sites. A total of 306 unique haplotypes derived from the combined analysis of control and coding region polymorphisms were found; the most common haplotype--CRS, 263, 309.1C, 315.1C/ not7025 AluI--was shared by 20 subjects. The majority of mtDNAs detected in the Italian population fell into the most common west Eurasian hgs: R0a (0.76%), HV (4.81%), H (38.99%), HV0 (3.55%), J (7.85%), T (13.42%), U (11.65%), K (10.13%), I (1.52%), X (2.78%), and W (1.01%).

Y-chromosome genetic structure in sub-Apennine populations of Central Italy by SNP and STR analysis.

To define the Y-chromosome genetic structure in Apennine populations, 17 Y-chromosome short tandem repeats (Y-STRs) and 37 Y-single nucleotide polymorphisms (Y-SNPs) were typed in 162 subjects living in the upland area of the Marches (Central Italy). A total number of 155 haplotypes (haplotype diversity was 0.9994) and 14 SNP haplogroups were observed. Testing high-resolution Y-chromosome data sets, e.g. using Yfiler and SNPs, increases the discriminatory capacity in individual identification for forensic purposes. It is also useful in autochthonous population and micro-population studies to highlight the most informative loci for evolutionary aims.

A missense germline mutation in exon 7 of the MSH2 gene in a HNPCC family from center-Italy.

INTRODUCTION: Hereditary Non-Polyposis Colorectal Cancer (HNPCC) is an autosomal dominant inherited disease predisposing to the development of colorectal cancers and several other malignancies (endometrium, ovaries, stomach, small bowel, hepatobiliary and urinary tract). HNPCC is caused by germline mutations in any of the MisMatch Repair (MMR) genes. Mutations in MLH1 and MSH2 account for almost 90% of all identified ones. About 15% of mutations identified in MSH2 are missense ones. PATIENTS AND METHODS: We studied one family, fulfilling Amsterdam II criteria, referred to our Center for genetic counselling. The proband, and some of her relatives, have been investigated for microsatellite instability (MSI), immunohistochemical MMR protein staining and by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: All patients carried the same novel MSH2 germline missense mutation (R359S) in exon 7, which determines the substitution of an Arginine, which is a basic amino acid, with a polar Serine residue (R359S). The mutation was associated with lack of expression of MSH2 protein and high microsatellite instability in tumour tissues. The same mutation has been detected in one healthy relative. CONCLUSIONS: The mutation here reported shows a high correlation with phenotype. The mutation is located in an evolutionary conserved domain. Taken together, our findings suggest evidence that the amino acid substitution can be interpreted as pathogenetic.