Valutazione e validazione di protocolli sperimentali ad uso forense

During the last 20 years, a revolution has taken place within the field of forensic genetics. DNA profiling is now considered an indispensable tool in criminal investigations: serious crimes, as rapes and murders, cases concerning paternity, relationship, disaster victim identification are often solved by DNA investigations in which, DNA profiles obtained from biological traces match the DNA profile of a suspect or a person in a criminal offender DNA database. At present, DNA investigations for forensic purposes are based on autosomal STRs analysis and can solve most of the cases, providing a high power of discrimination. Nevertheless, this technique shows some limitations in particular situations, such as in cases of degraded DNA or LT-DNA (low template) and in complex or deficiency relationship studies. Y-STRs and mitochondrial DNA analysis may support forensic activity, but it may be not sufficient to solve all the cases. Single nucleotide polymorphisms are a class of genetic markers that may be considered for a potentially useful role in forensic human identification, especially in highly degraded samples. In this work a panel of 36 SNPs was selected for forensic purposes from a number of markers already investigated and validated for forensic purposes in previous studies. All the SNPs have been selected on the basis of very stringent criteria and validated in a large number (n= 1040) of unrelated individuals originating from three different populations (Italian, Benin Gulf and Mongolian). Analisys of genetic data confirmed for all markers the high informativity (heterozigosity), the absence of departure from the Hardy–Weinberg equilibrium and the constant frequency across the populations, as demonstrated by an average value of Fst of 0,041 ± 0,06. The Random Match Probability (RMP) for the 36 SNPs panel was calculated to be the order of 1 x 10-15 in all the tested populations. The knowledge of population specific allele frequencies is essential for using the selected markers for forensic identification purposes. Allele frequencies of the 36 SNPs were assessed in 250 unrelated individuals originating from five different countries of Europe (Spain, Croatia, Bulgaria, Turkey and Serbia). All the SNPs showed high eterozigosity and generated extremely low Fst values. As a consequence of such Fst values, similar values of random match probability were observed across the populations: 6,21 × 10-15 in the Spanish population, 5,40 × 10-15 in the Croatian population, 1,13 × 10-14 in the Bulgarian population, 6,45 × 10-15 in the Serbian population and 3,57 × 10-15 in the Turkish population. Moreover, the sensitivity of the method was tested genotyping some artificially degraded DNA and DNA extracted from real forensic evidences, comparing results with those obtained with classical technologies. Partial information often associated with some kinship cases make challenging their resolution with autosomal markers. X-chromosomal markers could represent an efficient supplementation of autosomal STR, mitochondrial DNA and Y-chromosomal STR analysis in complicated or deficiency kinship questions. In particular, the analysis of X-cromosomal markers can clarify deficiency paternity testing and other complicated kinship situations in which only remote relatives are available. The need of additional methods in forensic practice is due to the increase in requests for family rejoin in the context of the world-wide migration and in the necessity of an efficient identification of wars and/or mass disasters victims. In this work, the commercial kit Elucigene XY-QST, designed and validated for prenatal diagnosis of aneuploidies, was investigated for potential use for forensic purposes. The Elucigene XY-QST protocol provides a multiplex PCR of 10 XY-STRs, with subsequent detection of genetic profiles by capillary electrophoresis. The kit includes 7 X-chromosomal markers (DXS6803, DXS981, DXS6807, DXS1187, XHPRT, DXS7423 and DXS6809), many of which have been previously studied for possible use in forensics. Allele frequencies of the 10 XY-STRs were assessed on 595 unrelated individuals originating from Italy (311 females and 284 males). Moreover, structures of all markers were investigated by direct sequencing of a large number of omo- and emi-zygote individuals for each alleles. In particular, an extensive sequencing analysis was performed for DXS1187 and DXYS218 markers, no previously studied. Linkage and linkage disequilibrium analysis led to the subdivision of the markers in four linkage groups, which may be considered as independent factors. The Content in Polymorphism (PIC), Mean Exclusion Chance (MEC) and Power of Discrimination (PD) for females and males were estimated from frequency data. Analysis of the genetic data showed absence of departure from Hardy–Weinberg equilibrium and high heterozigosity for all the X-STRs. The overall Mean Exclusion Chance of the X-markers is 0,99988, while the combined Power of Discrimination for females and males reaches the 0,99999995 and 0,9999197, respectively.