Probabilistic expert systems for forensic inference from DNA markers in horses: applications to confirm genealogies with lack of genetic data.

Microsatellites have been used for parentage testing and individual identification in forensic science because they are highly polymorphic and show abundant sequences dispersed throughout most eukaryotic nuclear genomes. At present, genetic testing based on DNA technology is used for most domesticated animals, including horses, to confirm identity, to determine parentage, and to validate registration certificates. But if genetic data of one of the putative parents are missing, verifying a genealogy could be questionable. The aim of this paper is to illustrate a new approach to analyze complex cases of disputed relationship with microsatellites markers. These cases were solved by analyzing the genotypes of the offspring and other horses' genotypes in the pedigrees of the putative dam/sire with probabilistic expert systems (PESs). PES was especially efficient in supplying reliable, error-free Bayesian probabilities in complex cases with missing pedigree data. One of these systems was developed for forensic purposes (FINEX program) and is particularly valuable in human analyses. We applied this program to parentage analysis in horses, and we will illustrate how different cases have been successfully worked out.

A 9-loci Y chromosome haplotype in three Italian populations.

Three geographic areas of Italy have been sampled and genotyped for 9 Y chromosome STRs: DYS19, DYS385, DYS388, DYS389 I and II, DYS390, DYS391, DYS392, DYS393. Sampling was focused on residents of small areas, well distant from major urban centres. Only individuals whose grandfather would live in the same area were included. A total of 210 unrelated individuals were collected. Distribution of genetic variation across the three samples and comparison with previously published Italian database indicated that so far Y chromosome diversity has been only partially explored in the Italian Peninsula.

STR analysis of artificially degraded DNA-results of a collaborative European exercise.

Degradation of human DNA extracted from forensic stains is, in most cases, the result of a natural process due to the exposure of the stain samples to the environment. Experiences with degraded DNA from casework samples show that every sample may exhibit different properties in this respect, and that it is difficult to systematically assess the performance of routinely used typing systems for the analysis of degraded DNA samples. Using a batch of artificially degraded DNA with an average fragment size of approx. 200 bp a collaborative exercise was carried out among 38 forensic laboratories from 17 European countries. The results were assessed according to correct allele detection, peak height and balance as well as the occurrence of artefacts. A number of common problems were identified based on these results such as strong peak imbalance in heterozygous genotypes for the larger short tandem repeat (STR) fragments after increased PCR cycle numbers, artefact signals and allelic drop-out. Based on the observations, strategies are discussed to overcome these problems. The strategies include careful balancing of the amount of template DNA and the PCR cycle numbers, the reaction volume and the amount of Taq polymerase. Furthermore, a careful evaluation of the results of the fragment analysis and of automated allele calling is necessary to identify the correct alleles and avoid artefacts.

Expansion to full mutation of a FMR1 intermediate allele over two generations.

Fragile X syndrome is due to an expanded CGG repeat in the 5' UTR of the FMR1 gene. According to repeat size, we distinguish four allele categories: normal (<40 CGG), intermediate (46-60 CGG), premutated (55-200 CGG) and full mutated (>200 CGG). However, the boundaries among these categories are unclear, making it difficult to classify unstable alleles and to estimate the risk of expansion. We report a family with a proband, carrying a methylated full mutation with an amplification of 1.2 kb. PCR analysis demonstrated two alleles of 29 and 61 CGGs in the mother. Sequencing of the 61 CGG allele showed no AGG interruptions. Both mother's sisters had two alleles of 31 and 44 CGGs, and the daughter of one of these had two alleles of 22 and 44 repeats, demonstrating stable transmission of the 44 CGG allele. The maternal grandfather was deceased, but haplotype reconstruction using markers DXS548 and FRAXAC1 demonstrated that he was carrier of the premutated allele. Furthermore, molecular analysis confirmed the same paternity with a probability of 99.79% for all the three sisters. According to these findings, it is likely that the maternal grandfather carried the 44 CGG allele, showing unstable transmission, given that it expanded first to 61 CGGs in one daughter, and then to full mutation in her child. Although we cannot exclude paternal mosaicism, it is likely that a rare event of progression from an intermediate to a premutated and on to a full mutated allele occurred in this family over two generations.

Allele sharing in first-degree and unrelated pairs of individuals in the Ge F I AmpFlSTR Profiler Plus database.

Eleven Italian forensic laboratories participated in a population study based on the AB Profiler Plus loci with proficiency testing. The validated database, including 1340 individuals, is available on-line. Tests for Hardy-Weinberg equilibrium, gametic unbalance, and heterogeneity of gene frequency were generally not significant. Gene frequencies at each locus were consistent with those of two previously published Italian studies, but different from a third. Individuals of each subsample were paired, and the total number of alleles shared across the nine loci was determined in each pair. The analysis was replicated over the total sample. In addition, two samples of mother-child pairs (N=315) and full-sib pairs (N=91) were subjected to allele sharing analysis. The resulting distributions were sufficiently distinct from the sample of unrelated pairs as to be of practical usefulness.