Phylogeographic review of Y chromosome haplogroups in Europe.

The Y chromosome has been widely explored for the study of human migrations. Due to its paternal inheritance, the Y chromosome polymorphisms are helpful tools for understanding the geographical distribution of populations all over the world and for inferring their origin, which is really useful in forensics. The remarkable historical context of Europe, with numerous migrations and invasions, has turned this continent into a melting pot. For this reason, it is interesting to study the Y chromosome variability and how it has contributed to improving our knowledge of the distribution and development of European male genetic pool as it is today. The analysis of Y lineages in Europe shows the predominance of four haplogroups, R1b-M269, I1-M253, I2-M438 and R1a-M420. However, other haplogroups have been identified which, although less frequent, provide significant evidence about the paternal origin of the populations. In addition, the study of the Y chromosome in Europe is a valuable tool for revealing the genetic trace of the different European colonizations, mainly in several American countries, where the European ancestry is mostly detected by the presence of the R1b-M269 haplogroup. Therefore, the objective of this review is to compile the studies of the Y chromosome haplogroups in current European populations, in order to provide an outline of these haplogroups which facilitate their use in forensic studies.

Two ovine mitochondrial DNAs harboring a fifth 75/76 bp repeat motif without altered gene expression in Northern Spain.

The Basque Country is home to the Latxa sheep breed, which is divided in several varieties such as Latxa Black Face (LBKF) and Latxa Blonde Face (LBLF). Mitochondrial DNA control region analysis of 174 male sheep (97 LBKF and 77 LBLF) was performed with the objective of characterizing the maternal lineages of these two varieties that are the basis to produce the cheese with Idiazabal quality label. The percentage of unique haplotypes was 77.32% in LBKF and 67.53% in LBLF. Most of the individuals were classified into B haplogroup (98.85%), while A haplogroup was much less frequent. Two Latxa individuals (one LBKF and one LBLF), both belonging to B haplogroup, displayed an additional 75/76 bp tandem repeat motif. Only 33 other sequences with this repeat motif were found among 11 061 sheep sequences included in the GenBank database. Gene expression was analyzed in peripheral blood leukocytes since the additional 75/76 bp repeat motif falls within ETAS1, a domain with a possible function in regulation of replication and transcription. The mRNA expression from four mitochondrial genes (COI, cyt b, ND1, and ND2) was analyzed in the two individuals of this study with a fifth repeat motif and in four without it. Although lower transcription was observed when the additional 75/76 bp repeat motif was present, no statistically significant differences were observed. Therefore, the variation in the number of the 75/76 repeat motif does not seem to modify the gene expression rate in mitochondrial genes.

Genomewide miRNA profiling of oral lichenoid disorders and oral squamous cell carcinoma.

OBJECTIVE: To dissect the aberrant microRNA profile of oral lichenoid disorders (OLD) by analyzing the larger set of OLD samples tested so far. MATERIALS AND METHODS: MicroRNA expression profiles were assessed using TLDA card in 32 samples (16 OLD, 8 OSCC, and 8 control). The findings were validated using RT-qPCR in an independent cohort of 91 samples. RESULTS: We identified 20 differentially expressed microRNAs in OLD, of which several are functionally related to cell proliferation, response to organic substances, or immune processes. Further validation of the top-ranked microRNAs revealed that they were all aberrantly expressed in OLD. CONCLUSION: We have identified a new microRNA signature associated with OLD that may provide a meaningful basis for better understanding the physiopathology of the disease. In addition, we validated seven microRNAs whose expression was shown to be higher in OLD tissue in comparison with the control and OSCC tissues.

Global DNA methylation: uncommon event in oral lichenoid disease.

OBJECTIVES: Accumulating evidence indicates that aberrant DNA methylation is closely related to oral carcinogenesis, and it has been shown that methylation changes might be used as prognostic biomarker in oral squamous cell carcinoma. Oral lichenoid disease (OLD) is the most common oral potentially malignant disorder in our region. The aim of this study was to perform the first wide DNA methylation study in OLD in order to investigate the relevance of DNA methylation changes in this premalignant disorder. MATERIALS AND METHODS: Two different Illumina microarray platforms, namely the GoldenGate Cancer Panel I and the HumanMethylation27 DNA Analysis BeadChip, were utilized in the discovery phase to interrogate the methylation profile of 59 OLD cases and 9 healthy individuals. Top-ranked genes were further validated by pyrosequencing in a second sample set consisting of 160 OLD and 65 controls. RESULTS: Our results show that the frequency of aberrant DNA methylation is rare in OLD, and this finding was further corroborated by pyrosequencing in the biological validation. CONCLUSIONS: These findings reinforce the notion that molecular alterations associated with oral carcinogenesis do not seem to be common events in OLD, which in turn might explain the low rate of malignization of this disorder.

Genetic uniqueness of the Waorani tribe from the Ecuadorian Amazon.

South America and especially the Amazon basin is known to be home to some of the most isolated human groups in the world. Here, we report on a study of mitochondrial DNA (mtDNA) in the Waorani from Ecuador, probably the most warlike human population known to date. Seeking to look in more depth at the characterization of the genetic diversity of this Native American tribe, molecular markers from the X and Y chromosomes were also analyzed. Only three different mtDNA haplotypes were detected among the Waorani sample. One of them, assigned to Native American haplogroup A2, accounted for more than 94% of the total diversity of the maternal gene pool. Our results for sex chromosome molecular markers failed to find close genetic kinship between individuals, further emphasizing the low genetic diversity of the mtDNA. Bearing in mind the results obtained for both the analysis of the mtDNA control region and complete mitochondrial genomes, we suggest the existence of a 'Waorani-specific' mtDNA lineage. According to current knowledge on the phylogeny of haplogroup A2, we propose that this lineage could be designated as subhaplogroup A2s. Its wide predominance among the Waorani people might have been conditioned by severe genetic drift episodes resulting from founding events, long-term isolation and a traditionally small population size most likely associated with the striking ethnography of this Amazonian community. In all, the Waorani constitute a fine example of how genetic imprint may mirror ethnopsychology and sociocultural features in human populations.

Progression from amnesic mild cognitive impairment to Alzheimer's disease: ESR1 and ESR2 polymorphisms and APOE gene.

BACKGROUND: Many genes have been studied to determine how they might be involved in Alzheimer's disease (AD). Estrogens have a protective effect in the central nervous system. The mechanisms of action of estrogens are mediated by two estrogen receptors (ERs), ERα and ERß. Thus, these genes could also play a role in the progression of amnesic mild cognitive impairment (MCIa) to AD. The aim of this study was to examine the role of ER single nucleotide polymorphisms (SNPs) as a risk factor for MCIa, as well as the interaction with apolipoprotein E (APOE) ε4 in the progression to AD. METHODS: 79 MCIa patients and 138 healthy controls were analyzed. SNPs were genotyped via restriction fragment length polymorphisms and real-time PCR, RT-PCR or RT-PCR (TaqMan) assays. RESULTS: There is a lack of association between MCIa patients who converted to AD and ER SNPs. APOE ε4 allele is an independent risk factor of MCIa (OR=1.86; 95% CI=1.02-3.38, p=0.042) with a high prevalence in converted subjects. APOE ε4 is able to predict the progression from MCIa patients to AD (OR=2.55; 95% CI=1.20-5.42, p=0.015). CONCLUSIONS: The presence of the APOE ε4 allele, and not the alleles of ER SNPs, is a risk factor for MCIa. Furthermore, APOE genotype seems to predict the conversion from MCIa to AD.

Microsatellites and Alu elements from the human MHC in Valencia (Spain): analysis of genetic relationships and linkage disequilibrium.

Two different sets of noncoding markers (microsatellites and Alu elements) from the human chromosome six were analysed in 106 individuals from Valencia (Spain), with the aim of exploring the effect of evolutionary forces on the genetic variability of the major histocompatibility complex (MHC) and assessing the potential usefulness of these genetic loci in phylogenetic studies. Linkage disequilibrium (LD) analyses revealed statistically significant associations among markers located in the MHC class I region, and also between the microsatellite D6S2792 and several genetic loci from MHC class I, II and III regions. Results of the Ewens-Watterson test indicated that only D6S2792 showed significant departure from selective neutrality. Despite the paucity of haplotype data in the literature, results of the phylogenetic analyses at world scale (Alu elements) showed that the genetic relationships of Valencia were mainly determined by the ethnic ancestry of the populations considered, whereas at European scale (microsatellites) population affinities were strongly influenced by geography. Our findings suggest that noncoding markers from the MHC such as Alu and microsatellite loci might have a potential value as lineage (ancestry) markers in investigations into evolutionary, medical and forensic perspectives.

Genetic variability in autochthonous Basques from Guipuzcoa: a view from MHC microsatellites.

Five short tandem repeats (STRs) located at human chromosome 6 were analysed in 97 autochthonous Basques from Guipuzcoa (northern Spain), with the aim of assessing the genetic relationships of Basques at a European scale, based on the variability of the major histocompatibility complex (MHC) region, and comparing the phylogenetic information obtained from STRs, and from HLA class I genes (HLA-A and HLA-B) for the same set of European populations. The integrative approach was focused on D6S265 and D6S2792, according to availability of population databases. F(ST) genetic distances obtained from STRs and from HLA loci were very similar, thereby describing a comparable pattern of genetic structuring among the European populations. These findings were supported by results of the Mantel test of matrix correspondence (r = 0.796, P = 0.0022) and by significant correlations between the first two F(ST) eigenvectors of STRs and HLA genes. Coinciding with previous phylogenetic studies, Basques showed substantial genetic differentiation within the European context, probably as a result of the impact of random genetic drift and high inbreeding levels for extended periods of isolation even from adjacent populations. Analysis of the geographical distribution of the allele frequencies revealed a great number of latitudinal frequency clines in both the MHC STRs and the HLA class I genes, which supports the notion of the post-glacial resettlement of Europe being a crucial factor in the genetic make-up of Europeans. Our results indicate that analysing the genetic variability of MHC microsatellites could be a suitable strategy in evaluating the role of evolutionary forces such as natural selection (because of genetic hitchhiking effect), genetic drift and gene flow in the maintenance of polymorphism at the MHC region, because STRs can efficiently complement the genetic information obtained from HLA genes.

Genetic characterization and founder effect analysis of recently introduced Salers cattle breed population.

Salers are a native French breed used for beef and dairy production that has expanded to all the continents. The Salers breed was introduced to the north of Spain in 1985 with only 15 individuals from France and has successfully increased to over 20 000 animals. Although over time new animals have been imported from France for breeding, it is possible that the limiting number of founder animals could have resulted in a reduction of the genetic diversity found in Spanish Salers. Thus, the purpose of the present study has been to characterize the genetic diversity of Salers breed in Spain and evaluate a possible founder effect due to reduced number of the first reproducers. A total of 403 individuals from 12 Salers herds were analyzed using 12 microsatellite markers and compared with phylogenetically and geographically close related Blonde d'Aquitaine, Limousin and Charolais French breeds but also other 16 European breeds. Microsatellites in Salers were polymorphic, with a mean allelic richness of 5.129 and an expected heterozygosity of 0.621 across loci (0.576 to 0.736 among all breeds). Average observed heterozygosity was 0.618. All the loci fit the Hardy-Weinberg (HW) equilibrium except TGLA227 locus due to a significant deficit of heterozygotes in only one of the herds, probably attributable to a sampling effect. When all loci were combined, Salers inbreeding coefficient did not differ statistically from 0 (F IS=0.005), indicating not significant excess or deficit of heterozygotes (P=0.309). Based in allelic distribution, Salers revealed a frequency of 0.488 in BM2113-131 and 0.064 in BM2113-143 diagnostic alleles, which are specific to the African zebu. These zebu alleles are also found in some French breeds, supported by STR data previously postulated hypothesis of a migration route through Mediterranean route by which North African cattle may have left a genetic signature in southern Europe. Phylogenetic tree and population structure analyses could unambiguously differentiate Salers cattle from the other populations and 10% of the total genetic variability could be attributed to differences among breeds (mean R ST=0.105; P<0.01). Mutation-drift equilibrium tests (sign test and Wilcoxon's sign rank test) were in correspondence to the absence of founder effect when Bonferroni was applied. Gene diversity previously reported in French Salers was comparable with the observed in our population. Thus, high genetic diversity in Spanish Salers highlights the resources of this population, which looks toward future breeding and selection programs.

Species identification in meat products: A new screening method based on high resolution melting analysis of cyt b gene.

Meat adulteration by substitution with lower value products and/or mislabeling involves economic, health, quality and socio-religious issues. Therefore, identification and traceability of meat species has become an important subject to detect possible fraudulent practices. In the present study the development of a high resolution melt (HRM) screening method for the identification of eight common meat species is reported. Samples from Bos taurus, Ovis aries, Sus scrofa domestica, Equus caballus, Oryctolagus cuniculus, Gallus gallus domesticus, Meleagris gallopavo and Coturnix coturnix were analyzed through the amplification of a 148 bp fragment from the cyt b gene with a universal primer pair in HRM analyses. Melting profiles from each species, as well as from several DNA mixtures of these species and blind samples, allowed a successful species differentiation. The results demonstrated that the HRM method here proposed is a fast, reliable, and low-cost screening technique.

New cyt b gene universal primer set for forensic analysis.

Analysis of mitochondrial DNA, and in particular the cytochrome b gene (cyt b), has become an essential tool for species identification in routine forensic practice. In cases of degraded samples, where the DNA is fractionated, universal primers that are highly efficient for the amplification of the target region are necessary. Therefore, in the present study a new universal cyt b primer set with high species identification capabilities, even in samples with highly degraded DNA, has been developed. In order to achieve this objective, the primers were designed following the alignment of complete sequences of the cyt b from 751 species from the Class of Mammalia listed in GenBank. A highly variable region of 148bp flanked by highly conserved sequences was chosen for placing the primers. The effectiveness of the new pair of primers was examined in 63 animal species belonging to 38 Families from 14 Orders and 5 Classes (Mammalia, Aves, Reptilia, Actinopterygii, and Malacostraca). Species determination was possible in all cases, which shows that the fragment analyzed provided a high capability for species identification. Furthermore, to ensure the efficiency of the 148bp fragment, the intraspecific variability was analyzed by calculating the concordance between individuals with the BLAST tool from the NCBI (National Center for Biotechnological Information). The intraspecific concordance levels were superior to 97% in all species. Likewise, the phylogenetic information from the selected fragment was confirmed by obtaining the phylogenetic tree from the sequences of the species analyzed. Evidence of the high power of phylogenetic discrimination of the analyzed fragment of the cyt b was obtained, as 93.75% of the species were grouped within their corresponding Orders. Finally, the analysis of 40 degraded samples with small-size DNA fragments showed that the new pair of primers permits identifying the species, even when the DNA is highly degraded as it is very common in forensic samples.

Associations between STR autosomal markers and longevity.

Life span is a complex and multifactorial trait, which is shaped by genetic, epigenetic, environmental, and stochastic factors. The possibility that highly hypervariable short tandem repeats (STRs) associated with longevity has been largely explored by comparing the genotypic pools of long lived and younger individuals, but results so far have been contradictory. In view of these contradictory findings, the present study aims to investigate whether HUMTHO1 and HUMCSF1PO STRs, previously associated with longevity, exert a role as a modulator of life expectancy, as well as to assess the extent to which other autosomal STR markers are associated with human longevity in population from northern Spain. To that end, 21 autosomal microsatellite markers have been studied in 304 nonagenarian individuals (more than 90 years old) and 516 younger controls of European descent. Our results do not confirm the association found in previous studies between longevity and THO1 and CSF1PO loci. However, significant association between longevity and autosomal STR markers D12S391, D22S1045, and DS441 was observed. Even more, when we compared allelic frequency distribution of the 21 STR markers between cases and controls, we found that 6 out of the 21 STRs studied showed different allelic frequencies, thus suggesting that the genomic portrait of the human longevity is far complex and probably shaped by a high number of genomic loci.

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.

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.

Population frequency distribution of HumF13A01, HumFXIIIB, and HumLIPOL loci in the Basque Country (Northern Spain).

A population study of unrelated individuals from the Basque Country (Northern Spain) was carried out using the GenePrint STR System. The PCR products were separated on denaturing polyacrylamide gels and visualized by silver staining. Three tetrameric loci were evaluated: HumF13A01, HumFXIIIB, and HumLIPOL. All loci fit Hardy-Weinberg expectations, and independence of alleles was found between these STR loci. A comparison with other population groups indicated allele frequencies are well conserved in Caucasians, but differ from other racial groups. The calculated parameters a priori probability of exclusion (Pex) and "power of discrimination" (PD) show how informative these loci are for the determination of identity and relatedness of individuals.

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.

[Polymorphism of six Alu-insertions in residents of Morocco: comparative study in Arab and Berber populations and residents of Casablanca]. / Polimorfism shesti Alu-insertsii u zhitelei Marokko: sravnitel'noe izuchenie v populiatsiiakh Arabov, Berberov i zhitelei Kasablanki.

Alu elements are the largest family of short tandem interspersed elements (SINEs) in human who have arisen to a copy number with an excess of 500,000 copies per haploid human genome and mobilize through an RNAse polymerase III derived transcript in a process termed "retroposition." Several features make Alu insertions a powerful tool used in population genetic studies: the polymorphic nature of many Alu insertions, the stability of an Alu insertion event and, furthermore, the ancestral state of an Alu insertion is known to be the absence (complete and exact) of the Alu element at a particular locus and the presence of an Alu insertion at the site that forward mutational change. Here we report on the distribution of six polymorphic Alu insertions in a general Moroccan population and in the Arab and Berber populations from Morocco and their relationships with other populations previously studied. Our results show that there is a small difference between Arabs and Berbers and that the Arab population was closer to African populations than Berber population which is closest to Europeans.

Recent advances and considerations for the future in forensic analysis of degraded DNA by autosomic miniSTR multiplex genotyping.

STR genotyping from degraded DNA samples requires genetic profiles to be obtained from DNA fragments no bigger than 200-300 bp. It requires the use of miniSTRs, which are smaller than the STRs used in standard typing. This paper reviews recent advances in miniSTR genotyping, beginning with a brief introduction to the processes involved in DNA fragmentation and how it hinders standard STR genotyping before proceeding further to the loci included in the main DNA databases and finishing with the International Workgroups' recommended design strategies for developing miniSTR reactions. The results of the efforts of many laboratories achieving different STR multiplexes and patents are also described and compared. Finally, a consideration of the perspectives for the future in this area is presented.

The GHEP-EMPOP collaboration on mtDNA population data--A new resource for forensic casework.

Mitochondrial DNA (mtDNA) population data for forensic purposes are still scarce for some populations, which may limit the evaluation of forensic evidence especially when the rarity of a haplotype needs to be determined in a database search. In order to improve the collection of mtDNA lineages from the Iberian and South American subcontinents, we here report the results of a collaborative study involving nine laboratories from the Spanish and Portuguese Speaking Working Group of the International Society for Forensic Genetics (GHEP-ISFG) and EMPOP. The individual laboratories contributed population data that were generated throughout the past 10 years, but in the majority of cases have not been made available to the scientific community. A total of 1019 haplotypes from Iberia (Basque Country, 2 general Spanish populations, 2 North and 1 Central Portugal populations), and Latin America (3 populations from São Paulo) were collected, reviewed and harmonized according to defined EMPOP criteria. The majority of data ambiguities that were found during the reviewing process (41 in total) were transcription errors confirming that the documentation process is still the most error-prone stage in reporting mtDNA population data, especially when performed manually. This GHEP-EMPOP collaboration has significantly improved the quality of the individual mtDNA datasets and adds mtDNA population data as valuable resource to the EMPOP database (www.empop.org).

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.