Adriano Tagliabracci

Correlation between Genetic and Geographic Structure in Europe

Understanding the genetic structure of the European population is important, not only from a historical perspective, but also for the appropriate design and interpretation of genetic epidemiological studies. Previous population genetic analyses with autosomal markers in Europe either had a wide geographic but narrow genomic coverage [1, 2], or vice versa [3-6]. We therefore investigated Affymetrix GeneChip 500K genotype data from 2,514 individuals belonging to 23 different subpopulations, widely spread over Europe. Although we found only a low level of genetic differentiation between subpopulations, the existing differences were characterized by a strong continent-wide correlation between geographic and genetic distance. Furthermore, mean heterozygosity was larger, and mean linkage disequilibrium smaller, in southern as compared to northern Europe. Both parameters clearly showed a clinal distribution that provided evidence for a spatial continuity of genetic diversity in Europe. Our comprehensive genetic data are thus compatible with expectations based upon European population history, including the hypotheses of a south-north expansion and/or a larger effective population size in southern than in northern Europe. By including the widely used CEPH from Utah (CEU) samples into our analysis, we could show that these individuals represent northern and western Europeans reasonably well, thereby confirming their assumed regional ancestry.

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.

DNA degradation and genetic analysis of empty puparia: genetic identification limits in forensic entomology.

Puparial cases are common remnants of necrophagous flies in crime investigations. They usually represent the longest developmental time and, therefore, they can be very useful for the estimation of the post-mortem interval (PMI). However, before any PMI estimate, it is crucial to identify the species of fly eclosed from each puparium associated with the corpse. Morphological characteristics of the puparium are often distinctive enough to permit a species identification. But, even an accurate morphological analysis of empty puparia cannot discriminate among different species of closely related flies. Furthermore, morphological identification may be impossible if the fly puparia are poorly preserved or in fragments. This study explores the applicability of biomolecular techniques on empty puparia and their fragments for identification purposes. A total of 63 empty puparia of necrophagous Diptera resulting from forensic casework were examined. Samples were divided into three groups according to size, type and time of eclosion in order to verify whether the physical characteristics and puparia weathering can influence the amount of DNA extraction. The results suggest that a reliable genetic identification of forensically important flies may also be performed from empty puparia and/or their fragments. However, DNA degradation can deeply compromise the genetic analysis since the older the fly puparia, the smaller are the amplified fragments.

Moors and Saracens in Europe : estimating the medieval North African male legacy in southern Europe

To investigate the male genetic legacy of the Arab rule in southern Europe during medieval times, we focused on specific Northwest African haplogroups and identified evolutionary close STR-defined haplotypes in Iberia, Sicily and the Italian peninsula. Our results point to a higher recent Northwest African contribution in Iberia and Sicily in agreement with historical data. southern Italian regions known to have experienced long-term Arab presence also show an enrichment of Northwest African types. The forensic and genomic implications of these findings are discussed.

A cautionary note on switching mitochondrial DNA reference sequences in forensic genetics

The first human mitochondrial DNA (mtDNA) sequence was produced in 1981 from an individual of European descent [1]. Since then, this sequence has been known as the Cambridge Reference Sequence (CRS) with a total length of 16,569 base pairs. As is common practice in other fields of genome research, this first mitochondrial genome (mtGenome) served as reference for the scientific community, relative to which other mtDNA haplotypes were reported. Eighteen years later the CRS was re-sequenced and corrected at 10 positions (3423T, 4985A, 9559C, 11335C, 13702C, 14199T, 14272C, 14365C, 14368C, and 14766C) to form the revised Cambridge Reference Sequence (rCRS) [2]. The new analysis also revealed that this mtGenome consists of only 16,568 nucleotides, as a base at position 3107 was mistakenly reported in the CRS. Instead of redefining all nucleotide positions downstream of 3107, this position is indicated in the rCRS as a deletion (unfortunately often indicated as ‘‘N’’, which is reserved for any base in the IUPcode [3]). Thus, the numbering system employed for the CRS and the body of established data can continuously be used with the rCRS. More than 15,500 mtGenomes and well over 150,000 (partial) control region sequences (including databases) have been published to date (http://www.phylotree.org/, Phylotree Build 14 [4]), in which the CRS and the rCRS have been cited 5603 and 968 times, respectively (http://apps.isiknowledge.com/; queried on May 2012). In a recent study, the switch to a new reference sequence, the so-called Reconstructed Sapiens Reference Sequence (RSRS), has been proposed [5]. This ancestral reconstructed sequence represents the deepest root in the known human mtDNA phylogeny at the base of the split of haplogroups L0 and L10203040506 after combining sequence information from all available mtGenomes from Homo neanderthalensis and novel human mtGenomes. The authors believe that the switch would solve misunderstandings and problems associated with the existing nomenclature relative to the rCRS, which belongs to the recently coalescing European haplogroup H2a2a1. In the following we briefly review developments in forensic mitochondrial genetics and discuss possible implications of the proposed switch. Mitochondrial DNA is highly abundant in cells compared to nuclear DNA (nDNA) with increased typing success rates for analysis of highly degraded samples and also hair shafts that often do not harbor detectable amounts of nDNA. To assess the significance of a match between two mtDNA haplotypes (e.g. from the crime scene and from a suspect), mtDNA databases have been developed. Earlier, the difference-coded haplotypes (with respect to the rCRS) were directly compared to the haplotypes in the mtDNA databases with the risk that multiple different alignments of the same sequence led to biased results [6]. The

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).

Determination of lamotrigine in whole blood with on line solid phase extraction

A simple, sensitive and reproducible method was developed for the determination of lamotrigine in whole blood with on-line solid phase extraction followed by HPLC separation with UV detection. Whole blood samples were diluted 1:1 with water and then injected directly on a clean-up column dry-packed with 40microm C8 silica and separated on a C18 reversed-phase column (150x4.6mm) at room temperature. The extraction column was activated with methanol and conditioned with phosphate buffer of pH 4.5. Mobile phases consisted of phosphate buffer of pH 4.5 for the extraction column and of phosphate buffer of pH 4.5 - acetonitrile (60:40, v/v) for the analytical column. At a flow rate of 1.0ml/min and a connection time of 1.0min, the complete cycle time was 10.0min. Detection was carried out at 260nm. No internal standard was necessary. The method was linear over concentration range 0.2-20.0microg/ml for lamotrigine. Recovery was 98%. Within-day and between-day coefficients of variation ranged from 1.8 to 6.7%.