Chiara Turchi

Polymorphism of the mitochondrial DNA control region in Italians

Abstract Mitochondrial DNA sequences of the hypervariable regions HVI and HVII were analysed in 83 Caucasians living in central Italy to expand the database for forensic identification purposes, and 75 different haplotypes resulting from 62 polymorphic positions in HVI and 44 in HVII were observed. The most frequent haplotype (263G, 309.1C, 315.1C) was shared by 7 individuals, 2 haplotypes were shared by 2 individuals, and 72 were unique. The genetic diversity was found to be 0.99 and the random match probability 1.9%. A condition of sequence heteroplasmy was found in only one case at nt 16311, whereas a length heteroplasmy was found in the homopolymeric stretch of cytosines 303–315. Our results indicate that in direct sequencing beyond the poly-cytosine stretch, the overlap is due to length heteroplasmy, whereas the blurred signal occurs when the stretch is composed of more than 10 cytosines.

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.

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/¬7025 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%).

Subtyping mtDNA haplogroup H by SNaPshot minisequencing and its application in forensic individual identification

Sequence variation of the hypervariable segments (HVS) I/II of mitochondrial DNA (mtDNA) and the haplogroup affiliation were determined in a sample of 271 Italian subjects. This analysis showed that 42% of the individuals could be ascribed to H, the most frequent haplogroup in European Caucasian populations. This fraction was then screened for specific single nucleotide polymorphisms located in the coding region to identify H subclades H1–H15. We set up two multiplex polymerase chain reactions and specific SNaPshot assays to investigate the frequency distribution of these subgroups in our population sample and to examine their usefulness in discriminating among commonly shared HVS I/II sequences. This allowed the assignment of a large portion of the mtDNAs (∼70%) to specific subhaplogroups, with H1 and H5 being the most represented. About two-thirds of the individuals sharing common HVS I/II sequences were subdivided and ascribed to specific H subhaplogroups with a significant reduction of the frequencies of the most common mtDNA haplotypes. Haplogroup H subtyping could thus be extremely useful in forensic identification when many samples have to be analysed and compared, avoiding excessive time-consuming and labor-intensive sequencing analysis.

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 alcoholisn 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.

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).

Multiplex mtDNA coding region SNP assays for molecular dissection of haplogroups U/K and J/T.

Mitochondrial DNA (mtDNA) U/K and J/T are sister haplogroups within the superhaplogroup R. They are both common in Europe, with a combined overall frequency similar to the one reported for H, the most common European haplogroup (40-50%). In this study, we selected 159 Italian subjects, already ascribed to U/K and J/T by RFLP typing, and assigned each mtDNA to specific clades/subclades by investigating at least one diagnostic coding region SNP. For each sister haplogroup, one multiplex PCR and one SNaPshot minisequencing reaction were set up targeting 16 U/K and 7 J/T coding region SNPs. Each mtDNA sample was clearly assigned to a specific subclade, which could be further subdivided into several minor sub-branches according to peculiar HVS I/II motifs. Such a molecular dissection of haplogroups U/K and J/T could be extremely useful to reduce the overall analysis time and labor intensive sequencing procedures in high volume forensic casework, for example when it is important to rapidly exclude samples in order to restrict the number of suspects.

Multiplex PCR Development of Y-chromosomal Biallelic Polymorphisms for Forensic Application

Single-nucleotide polymorphisms of Y chromosome (Y-SNPs) are a class of markers of interest in forensic investigations, because many of them show regional specificity, providing useful information about the geographic origin of a subject or evidence under investigation. A first multiplex with 7 SNPs (M35, M89, M9, M170, M172, M45, M173), which occur in the basal branches of the phylogenetic tree and are able to assign a subject to known most frequent European haplogroups, was designed. SNP genotyping was accomplished by hot-start PCR with primers amplifying fragments between 96 and 136 nucleotides, minisequencing, and capillary electrophoresis of extension products. Ninety seven subjects of known geographic provenance were studied, of which 68 from Europe. Of these, 57 had mutations found more frequently in European haplogroups and 11 more frequent in Asian populations. Subjects from non-European countries were also examined and had haplogroups common in their regions of provenance. Experiments with low molecular weight DNA gave positive amplification from 1 ng of DNA for all seven SNPs.

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 (r2 = 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.

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.