Stefania Ceccardi

Different informativeness of the three hypervariable mitochondrial DNA regions in the population of Bologna (Italy)

Mitochondrial DNA (mtDNA) sequence variations at hypervariable regions HVI, HVII and HVIII were analysed in 100 unrelated Italians from Bologna. The values of the statistical parameters are in agreement with the range of European populations. We suggest that the less informative HVIII region may be useful to distinguish HVI-HVII identical sequences in forensic analysis especially when nuclear DNA cannot be investigated.

Expanding X-chromosomal forensic haplotype frequencies database: Italian population data of four linkage groups

Requests for solving complex kinship casework involving at least one female are increasing and in these circumstances the analysis of X-chromosomal STR markers plays a relevant role. Actually, it is well known the superior statistical power of X-STRs compared to autosomal markers in solving relationship when two sisters or half-sisters are involved and none of parents is available, in maternity testing or in cases involving close relatives as alternative putative fathers. In addition, the possibility to amplify more loci simultaneously and the strategy based on the analysis of four linkage groups to obtain the X-haplotype provide a powerful and validated tool. Nevertheless, haplotypes frequency distribution in different populations is still needed for calculation of probabilities in relationship testing. Published haplotype frequencies from German population data are available, but in different caseworks we found unreported X-haplotypes. To enlarge the forensic X-chromosome database, we present haplotype frequencies and other parameter of forensic interest obtained from 200 anonymous DNA samples of unrelated Italian males for the four linkage groups included in the Investigator Argus X-12 kit. From the comparison of the Italian sample haplotype frequencies with other populations, significant genetic distances were found with Asian and African populations, but not with Europeans. Finally, casework examples of complex kinship analysis are presented.

Cancerous Tissues in Forensic Genetic Analysis

Microsatellites or short tandem repeats (STRs) markers are important tools for mapping disease-causing genes by linkage, for performing investigations in forensic medicine, for population genetic studies and for studying genetic modifications in tumors. In forensic applications neoplastic tissues can be used as a source of genetic information for personal identification or paternity testing when no other specimen is available. Cancer tissues can show microsatellite instability (MSI) and loss of heterozygosity (LOH) also for the STRs used in the forensic field. In this study, we screened 56 sporadic gastrointestinal carcinomas in order to provide further data for the evaluation of the incidence of allelic alterations for 15 STR loci and the suitability of using cancerous tissues in forensic applications. Sixty-six percent of the cancerous tissues were found to possess allelic alterations of the microsatellites analyzed with a high incidence of MSI-L (microsatellite instability low) when compared to the corresponding normal tissue. The most frequently altered loci were D18S51, VWA, and FGA. From a forensic perspective, great care must be taken in evaluating the DNA typing results obtained from cancerous tissue samples.

Minisequencing-based genotyping of Duffy and ABO blood groups for forensic purposes.

ABSTRACT: Duffy and ABO blood group genetic polymorphisms were studied by minisequencing analysis of single‐nucleotide polymorphisms (SNPs) at nucleotide positions—33, 125, 265, and 298 of the Duffy gene and at nucleotide positions—261, 297, 467, 646, and 703 of the ABO gene. In an Italian population sample, we found four alleles and seven genotypes for the Duffy and six alleles and 16 genotypes for the ABO systems. The lower limit for reproducible results was 200 pg DNA, with a range of up to 10 ng and an optimum at 1 ng. All of the 16 analyzed inclusive paternity tests were also consistent with parentage and two out of four inconsistencies with parentage cases were excluded by one or more SNPs. Although Duffy and ABO SNP typing show lower informativeness than most current forensic tests, their robustness, the limited population distribution of FY*Fy type, and the sensitivity of the minisequencing technology suggest that these markers can be useful in selected forensic applications.

CYP2D6 Genotyping in Natives and Immigrants from the Emilia-Romagna Region (Italy)

Pharmacogenetic testing of drug metabolizing enzyme polymorphisms provides an important tool to improve prescribing decisions, avoiding therapeutic failure and adverse drug reactions. Cytochrome P450 2D6 isoform plays an important role in the metabolism of about 20%-25% of widely used clinical drugs. Interethnic differences in allele frequency distribution of the CYP2D6 gene are well established, but interethnic admixture, introducing variations in population ancestry and resulting in distinct levels of population structure, should be acknowledged in pharmacogenomic studies to avoid inappropriate extrapolation of CYP2D6 data. The aim of the present research was to characterize CYP2D6 polymorphism in a random sample of 122 natives and 175 immigrants from Africa, Asia, and South America living in the Emilia-Romagna region (Italy), considering the present scenario of immigration and back migration events, which is a source of admixture. The results are today consistent with the known interethnic genetic variation, but the observed significant divergence between natives and Africans or South-East Asians predicts that admixture will reshape the population structure and the native metabolic ratio curve requiring, for drug prescription and pharmacogenetics studies, an interdisciplinary approach applied in an appropriate biogeographical and anthropological frame.

Development of a heptaplex PCR system to analyse X-chromosome STR loci from five Italian population samples. A collaborative study

Abstract A heptaplex PCR has been developed to amplify DXS6789, HUMARA, DXS10011, DXS7423, HPRTB, DXS6807 and DXS101 on Italian samples from Bologna, Modena, Padova, Ancona and Pisa. Statistical analyses were performed for all the loci.

Population data of mitochondrial DNA region HVIII in 150 individuals from Bologna (Italy)

Abstract Mitochondrial DNA (mtDNA) sequence variations at the HVIII region in 150 unrelated Italian individuals from Bologna were analysed. A comparison of our sequence data with the Anderson sequence showed that nucleotide substitutions predominate over insertions and deletions. Among nucleotide substitutions the most frequent was a T–C transition.

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.

Sex chromosome analysis in Turner Syndrome by a pentaplex PCR assay.

In this study, we describe a pentaplex PCR to determine the parental origin of the X chromosome and the presence of mosaicism, via amplification of four polymorphic markers located along the X chromosome (DXS10011, DXS6807, HUMARA, DXS101) and the X-Y amelogenin marker, in 41 families having a daughter with Turner Syndrome. Our results confirmed the cytogenetic findings and we found that the parental origin of the single X chromosome to be maternal in 84% of cases.

Myotonic dystrophy CTG repeats in an Italian population sample: evaluation of the polymorphism for forensic applications.

The myotonic dystrophy (DM) CTG repeat polymorphism has been studied in an Italian population sample. Polymerase chain reaction (PCR) amplification, manual polyacrylamide gel electrophoresis (PAGE), and silver staining were employed. Alleles were typed by comparison with a sequenced allelic ladder. A total of 25 different alleles, spanning the range from 5 to 31 CTG triplets, was observed. The heterozygosity was 79%, and no significant deviation from Hardy-Weinberg equilibrium was found. Eighty-one meioses from parentage testing were also analyzed, and a Mendelian pattern of inheritance was observed in all cases. In addition, we could successfully type the DM locus in 20 laboratory-prepared bloodstains, with 1 ng of DNA allowing clear definition of alleles. We conclude that the CTG repeats at the DM locus may be useful for forensic applications.