I. Fernández-Fernández

The Basques according to polymorphic Alu insertions

Abstract. Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000–2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups.

Mitochondrial DNA haplogroup diversity in Basques: a reassessment based on HVI and HVII polymorphisms.

This study provides a more complete characterization of the mitochondrial genome variability of the Basques, including data on the hypervariable segment HVII of the D‐loop region, which remains relatively unknown. To that end, genomic DNA from 55 healthy men living in the Arratia Valley (Biscay province) and the Goiherri region (Guipúzcoa province) was examined by direct sequencing. Three‐generation pedigree charts were compiled to ensure the collection from autochthonous individuals. The most notable findings emerging from the analysis of haplogroup composition are: (i) lack of U8a mitochondrial lineage, a rare subhaplogroup recently identified in Basques and proposed as a Paleolithic marker, (ii) low frequency of haplogroup V, which conflicts with results of earlier analyses describing high frequencies in southwestern Europe, and (iii) high frequency of haplogroup J, especially subhaplogroups J1c1 and J2a. The frequency of haplogroup J does not coincide with previous mtDNA studies in present‐day Basques, but is congruent with frequencies found in prehistoric and historic Basque populations. In explaining divergence in haplogroup composition between modern Basque samples, we hypothesized spatial heterogeneity promoted by population fragmentation due to extreme limitation of dispersal opportunities during the Pleistocene glaciations. Similarities between extinct and extant Basque populations as for the high frequency of lineage J, as well as the abundance of this haplogroup in northern Spain endorse a shift in the focus of attention of mtDNA analysts. A refined dissection of haplogroup J might provide more solid evidence about the process of postglacial recolonization of Europe, and thus about the shaping of the European gene pool. Am. J. Hum. Biol., 2008.

Sequence polymorphisms of the mtDNA control region in a human isolate: the Georgians from Swanetia

AbstractIn this work, we analyzed the sequence diversity of the mtDNA control region (HVI and HVII) in a sample of 48 individuals from Swanetia (Georgia), using direct fluorescent-based sequencing methods. We identified 43 different mtDNA haplotypes resulting from 78 polymorphic sites (46 in HVI and 32 in HVII). Most of the variable positions identified in both HVI and HVII were transitions (82.6 and 71.9%, respectively). The frequency of length heteroplasmy in the homopolymeric C-stretch regions was the same for both segments (10.4%). The sequence diversity increased markedly when both hypervariable regions were analyzed jointly (HVI: 0.985, HVII: 0.975, HVI+HVII: 0.994). Accordingly, the probability of two randomly selected sequences matching (random match probability, RMP) decreased from 3.4% (HVI) to 2.6% (HVI+HVII), despite which the RMP values in Georgians remained higher than estimated in most Europeans. This suggests that the variability of maternal lineages tends to be lower in traditional human isolates and, therefore, the potential of discrimination of mtDNA in forensic analysis is more limited in this type of population. The incorporation of HVII data also contributed to the refinement of results regarding the genetic relationships among the samples included in the analyses, which stress the importance of considering HVII in both population and forensic genetics.

Population Genetics and Forensic Applications Using Multiplex PCR (CSF1PO, TPOX, and TH01) Loci in the Basque Country

A population study in a sample of 200 unrelated individuals from the Basque Country (Northern Spain) was carried out using the GenePrint STR Multiplex System. The PCR products were electrophorized on a denaturing polyacrylamide gel and visualized by silver staining. The loci are TH01, TPOX, and CSF1PO. All loci meet Hardy-Weinberg expectations, and independence of alelles at these STR loci was found. A comparison with other population groups appeared to indicate that frequencies are well conserved in Caucasians, but differ from those of other racial groups. We have also calculated Fst as a measure of population subdivision. No appreciable genetic subdivision in the Caucasian populations studied here was found. Some statistical parameters of forensic interest (Pex, PM and PD) were also calculated. No exclusions were found in 100 mother-child and father-child meiosis. To evaluate the applicability of these systems to forensic casework, we studied the minimum quantity of DNA which can be used applying the multiplex methodology, and the minimum quantity that can be typed in a mixed sample. We also examined several samples such as hair roots, semen stains, vaginal swabs, blood stains and temporary teeth, each of these of varying ages.

D1S80 Locus Typing by Micro Thermal Cycler. Application to Genetic Identity Testing

Based on a micro thermal cycler apparatus, a new protocol for amplification of the D1S80 locus has been developed. The advantages of this system consist of a reduction in time and costs of the amplification process, which greatly facilitates the analysis of the D1S80 locus at the population level and considerably increases its applicability in forensic samples. Using this protocol, an allele distribution study of the D1S80 locus has been carried out in a sample of 257 individuals residing in the Basque Country. In this study, up to 22 different alleles, ranging from 17 to 40 repetitions have been observed; moreover, the 35 and 38-repetition alleles, not reported in the european populations analyzed to date, have been detected. The sample studied fits Hardy-Weinberg equilibrium, the observed heterozygosity being 0.74, and the expected heterozygosity 0.804 +/- 0.012. The Chance of Exclusion and Index of Discrimination are 0.638 and 0.072 respectively. Moreover, the gene frequency distribution from the Basque resident population does not show significant differences when compared to other European and U.S. Caucasian populations.

Cytochrome b and HVI sequences of mitochondrial DNA to identify domestic animal hair in forensic casework

Biological traces that appear at the scene of a crime or on the body of the victim may be of human, animal and/or vegetable origin. Among those of animal origin, household pets are a common source, with pet hair being one of the most frequent traces found. Consequently, it is necessary to have laboratory methods capable of identifying traces from pets and domesticated animals in general. Savolainen et al. [1] have developed a basic method of sequencing the HVI region of mitochondrial DNA from Canis familiaris using single hairs as template, with a discrimination capacity of 1 in 10 individuals. The species from which a biological trace has come can be identified by analyzing a short fragment of the cytochrome b (cytb) gene sequence of the mitochondrial genome. This gene contains species-specific information and has been used in phylogenetic as well as in forensic investigations in a number of studies. Parson et al. [2] have confirmed the usefulness of cytb analysis in identifying the biological origin of casework specimens.

Newborn genetic identification: a protocol using microsatellite DNA as an alternative to footprinting

Newborn identification by foot- or finger-printing presents serious drawbacks. This study proposes an alternative method based on DNA analysis of blood-spots taken from the newborn child. CSF1PO, TPOX and TH01 microsatellite loci were chosen to develop a fast and reliable protocol to be applied in cases where it is suspected that newborn children have been exchanged. The advantage of these loci is that one can simultaneously amplify them by PCR multiplex reaction and determine their alleles, thereby reducing the time needed for identification tests. Moreover, the amplification products of these loci are very small (< 350 bp) and so can be analyzed in samples with degraded DNA. We have been able to prove that it is possible to obtain results in blood-spots taken from newborns up to 13 years before and kept at room temperature. Thus the protocol proposed here can be applied in long-term post-natal identification cases.

Genetic structure of autochthonous Basques through analysis of the HVI and HVII regions of mitochondrial DNA

Mitochondrial DNA (mtDNA) analysis has proven to be of great interest in human population studies to study phylogenetic relationships and differentiation among individuals [1]. MtDNA differs from nuclear DNA in the absence of recombination, maternal inheritance, large number of copies per cell and a mutation rate that is 5–10 times higher than that of nuclear DNA [2,3]. The non-coding region of mtDNA, known as the D-loop, accumulates more mutations than the rest of the molecule, making it a useful tool for studying short-term evolutionary phenomena [4], and the tool of choice for diagnosing matrilineal relationships. A number of scientists have studied the mtDNA of Basque populations. Bertranpetit [5] and Corte-Real [6] have analyzed the base sequences of the hypervariable segment I (HVI). All of these studies were based on an underlying assumption of homogeneous population structure in the autochthonous Basque group. The D-loop regions analyzed in this study were HVI and HVII in two autochthonous population samples Arratia and Goierri, linguistically and geographically differentiated. Since the HVII segment has not been previously studied in samples from the Basque Country, the analysis in these populations will provide better information on the variability of mtDNA in the autochthonous Basque population and its usefulness in forensic casework. Objectives: (a) to determine the homogeneity or heterogeneity of mtDNA in the autochthonous population of the Basque Country and to use the information contained in the sequence variations of the mtDNA of these fragments to analyze their phylogenetic

Multilocus Profiles of the Basque Country Population with Dig-Labelled Probes 33.15 and 33.6

Probes 33.15 and 33.6 are frequently used due to their high discrimination power. Usually methods for labeling these multilocus probes are based on radioactive isotopes. The main disadvantages of this labeling-technique are the short half-life of the isotopes, the requirement of safety facilities, and the manipulation risk. For these reasons, probes labeled with molecules such as biotin or digoxigenin (DIG) have emerged as practical alternatives to radiolabeled probes (1).

Y chromosome STRs in the north of Portugal

The interest in Y chromosome polymorphisms has continuously increased, not only in evolutionary/genealogical studies, but also in forensic genetics. The objective of this study is to analyse the loci DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393 and DYS385I/II in a population sample from the North of Portugal. One hundred one unique haplotypes were found and the haplotypic diversity was 0.998F0.001 which reinforces the forensic utility of these markers. Phylogenetic analysis shows that our population can be included in the European Y chromosome lineages and distinct from the Asian ones. D 2003 Published by Elsevier B.V.