Kinship analysis and allelic dropout: a forensic approach on an archaeological case.

BACKGROUND: This study relies on the discovery of two pit burials (LTA and LTB) of the Bronze Age Cogotas I archaeological culture (circa 3600-2950 BP) in Spain. LTA was a single burial and LTB contained three skeletal remains of two adults and a newborn or foetus at term. AIM: The central question posed by this find was whether the LTB tomb constituted a traditional nuclear family (father, mother and son or daughter). METHODS: Ancient and forensic DNA protocols were employed to obtain reliable results. Autosomal, X-STR markers and mitochondrial DNA were amplified. Subsequently, different kinship probabilities were estimated by means of LR values calculated using the Familias 3 software. Furthermore, an allelic dropout sensitivity test was developed in order to evaluate the influence of allelic dropout phenomena on the results. RESULTS: It was possible to determine the molecular sex of all individuals and to establish a maternal relationship between the perinatal individual and one of the adults. CONCLUSION: The remains in the LTB tomb were not a traditional nuclear family (father, mother and son/daughter) and it was probably a tomb where two women, one of them pregnant, were buried.

Ancient DNA analysis of 8000 B.C. near eastern farmers supports an early neolithic pioneer maritime colonization of Mainland Europe through Cyprus and the Aegean Islands.

The genetic impact associated to the Neolithic spread in Europe has been widely debated over the last 20 years. Within this context, ancient DNA studies have provided a more reliable picture by directly analyzing the protagonist populations at different regions in Europe. However, the lack of available data from the original Near Eastern farmers has limited the achieved conclusions, preventing the formulation of continental models of Neolithic expansion. Here we address this issue by presenting mitochondrial DNA data of the original Near-Eastern Neolithic communities with the aim of providing the adequate background for the interpretation of Neolithic genetic data from European samples. Sixty-three skeletons from the Pre Pottery Neolithic B (PPNB) sites of Tell Halula, Tell Ramad and Dja'de El Mughara dating between 8,700-6,600 cal. B.C. were analyzed, and 15 validated mitochondrial DNA profiles were recovered. In order to estimate the demographic contribution of the first farmers to both Central European and Western Mediterranean Neolithic cultures, haplotype and haplogroup diversities in the PPNB sample were compared using phylogeographic and population genetic analyses to available ancient DNA data from human remains belonging to the Linearbandkeramik-Alföldi Vonaldiszes Kerámia and Cardial/Epicardial cultures. We also searched for possible signatures of the original Neolithic expansion over the modern Near Eastern and South European genetic pools, and tried to infer possible routes of expansion by comparing the obtained results to a database of 60 modern populations from both regions. Comparisons performed among the 3 ancient datasets allowed us to identify K and N-derived mitochondrial DNA haplogroups as potential markers of the Neolithic expansion, whose genetic signature would have reached both the Iberian coasts and the Central European plain. Moreover, the observed genetic affinities between the PPNB samples and the modern populations of Cyprus and Crete seem to suggest that the Neolithic was first introduced into Europe through pioneer seafaring colonization.

The Y-chromosome tree bursts into leaf: 13,000 high-confidence SNPs covering the majority of known clades.

Many studies of human populations have used the male-specific region of the Y chromosome (MSY) as a marker, but MSY sequence variants have traditionally been subject to ascertainment bias. Also, dating of haplogroups has relied on Y-specific short tandem repeats (STRs), involving problems of mutation rate choice, and possible long-term mutation saturation. Next-generation sequencing can ascertain single nucleotide polymorphisms (SNPs) in an unbiased way, leading to phylogenies in which branch-lengths are proportional to time, and allowing the times-to-most-recent-common-ancestor (TMRCAs) of nodes to be estimated directly. Here we describe the sequencing of 3.7 Mb of MSY in each of 448 human males at a mean coverage of 51×, yielding 13,261 high-confidence SNPs, 65.9% of which are previously unreported. The resulting phylogeny covers the majority of the known clades, provides date estimates of nodes, and constitutes a robust evolutionary framework for analyzing the history of other classes of mutation. Different clades within the tree show subtle but significant differences in branch lengths to the root. We also apply a set of 23 Y-STRs to the same samples, allowing SNP- and STR-based diversity and TMRCA estimates to be systematically compared. Ongoing purifying selection is suggested by our analysis of the phylogenetic distribution of nonsynonymous variants in 15 MSY single-copy genes.

Continent-wide decoupling of Y-chromosomal genetic variation from language and geography in native South Americans.

Numerous studies of human populations in Europe and Asia have revealed a concordance between their extant genetic structure and the prevailing regional pattern of geography and language. For native South Americans, however, such evidence has been lacking so far. Therefore, we examined the relationship between Y-chromosomal genotype on the one hand, and male geographic origin and linguistic affiliation on the other, in the largest study of South American natives to date in terms of sampled individuals and populations. A total of 1,011 individuals, representing 50 tribal populations from 81 settlements, were genotyped for up to 17 short tandem repeat (STR) markers and 16 single nucleotide polymorphisms (Y-SNPs), the latter resolving phylogenetic lineages Q and C. Virtually no structure became apparent for the extant Y-chromosomal genetic variation of South American males that could sensibly be related to their inter-tribal geographic and linguistic relationships. This continent-wide decoupling is consistent with a rapid peopling of the continent followed by long periods of isolation in small groups. Furthermore, for the first time, we identified a distinct geographical cluster of Y-SNP lineages C-M217 (C3*) in South America. Such haplotypes are virtually absent from North and Central America, but occur at high frequency in Asia. Together with the locally confined Y-STR autocorrelation observed in our study as a whole, the available data therefore suggest a late introduction of C3* into South America no more than 6,000 years ago, perhaps via coastal or trans-Pacific routes. Extensive simulations revealed that the observed lack of haplogroup C3* among extant North and Central American natives is only compatible with low levels of migration between the ancestor populations of C3* carriers and non-carriers. In summary, our data highlight the fact that a pronounced correlation between genetic and geographic/cultural structure can only be expected under very specific conditions, most of which are likely not to have been met by the ancestors of native South Americans.

Genetic contribution to iron status: SNPs related to iron deficiency anaemia and fine mapping of CACNA2D3 calcium channel subunit.

Numerous studies associate genetic markers with iron- and erythrocyte-related parameters, but few relate them to iron-clinical phenotypes. Novel SNP rs1375515, located in a subunit of the calcium channel gene CACNA2D3, is associated with a higher risk of anaemia. The aim of this study is to further investigate the association of this SNP with iron-related parameters and iron-clinical phenotypes, and to explore the potential role of calcium channel subunit region in iron regulation. Furthermore, we aim to replicate the association of other SNPs reported previously in our population. We tested 45 SNPs selected via systematic review and fine mapping of CACNA2D3 region, with haematological and biochemical traits in 358 women of reproductive age. Multivariate analyses include back-step logistic regression and decision trees. The results replicate the association of SNPs with iron-related traits, and also confirm the protective effect of both A allele of rs1800562 (HFE) and G allele of rs4895441 (HBS1L-MYB). The risk of developing anaemia is increased in reproductive age women carriers of A allele of rs1868505 (CACNA2D3) and/or T allele of rs13194491 (HIST1H2BJ). Association of SNPs from fine mapping with ferritin and serum iron suggests that calcium channels could be a potential pathway for iron uptake in physiological conditions.

Exploring the relationship between lifestyles, diets and genetic adaptations in humans.

BACKGROUND: One of the most important dietary shifts underwent by human populations began to occur in the Neolithic, during which new modes of subsistence emerged and new nutrients were introduced in diets. This change might have worked as a selective pressure over the metabolic pathways involved in the breakdown of substances extracted from food. Here we applied a candidate gene approach to investigate whether in populations with different modes of subsistence, diet-related genetic adaptations could be identified in the genes AGXT, PLRP2, MTRR, NAT2 and CYP3A5. RESULTS: At CYP3A5, strong signatures of positive selection were detected, though not connected to any dietary variable, but instead to an environmental factor associated with the Tropic of Cancer. Suggestive signals of adaptions that could indeed be connected with differences in dietary habits of populations were only found for PLRP2 and NAT2. Contrarily, the demographic history of human populations seemed enough to explain patterns of diversity at AGXT and MTRR, once both conformed the evolutionary expectations under selective neutrality. CONCLUSIONS: Accumulated evidence indicates that CYP3A5 has been under adaptive evolution during the history of human populations. PLRP2 and NAT2 also appear to have been modelled by some selective constrains, although clear support for that did not resist to a genome wide perspective. It is still necessary to clarify which were the biological mechanisms and the environmental factors involved as well as their interactions, to understand the nature and strength of the selective pressures that contributed to shape current patterns of genetic diversity at those loci.

Genetic polymorphism of 15 STR loci in El Salvador.

The aim of this study was to estimate the allelic frequencies of the 15 short tandem repeat (STR) loci included in AmpFlSTRIdentifiler PCR Amplification Kit. Biological samples were obtained from 109 unrelated individuals from El Salvador. Allelic frequencies and forensic parameters were calculated. All loci showed no departure from Hardy-Weinberg equilibrium after Bonferroni correction. The obtained frequencies were compared with other previously reported population data. The multidimensional scaling plot and the neighbor-joining phylogeny supported a high native Mesoamerican contribution.

Influence of diet, menstruation and genetic factors on iron status: a cross-sectional study in Spanish women of childbearing age.

The aim of this study was to investigate the combined influence of diet, menstruation and genetic factors on iron status in Spanish menstruating women (n = 142). Dietary intake was assessed by a 72-h detailed dietary report and menstrual blood loss by a questionnaire, to determine a Menstrual Blood Loss Coefficient (MBLC). Five selected SNPs were genotyped: rs3811647, rs1799852 (Tf gene); rs1375515 (CACNA2D3 gene); and rs1800562 and rs1799945 (HFE gene, mutations C282Y and H63D, respectively). Iron biomarkers were determined and cluster analysis was performed. Differences among clusters in dietary intake, menstrual blood loss parameters and genotype frequencies distribution were studied. A categorical regression was performed to identify factors associated with cluster belonging. Three clusters were identified: women with poor iron status close to developing iron deficiency anemia (Cluster 1, n = 26); women with mild iron deficiency (Cluster 2, n = 59) and women with normal iron status (Cluster 3, n = 57). Three independent factors, red meat consumption, MBLC and mutation C282Y, were included in the model that better explained cluster belonging (R2 = 0.142, p < 0.001). In conclusion, the combination of high red meat consumption, low menstrual blood loss and the HFE C282Y mutation may protect from iron deficiency in women of childbearing age. These findings could be useful to implement adequate strategies to prevent iron deficiency anemia.

Loneliness, Depression, and Genetics in the Elderly: Prognostic Factors of a Worse Health Condition?

Loneliness is considered a prognostic factor for poorer health status in the elderly. It is proposed to analyze the role of loneliness in health status in terms of various factors. A total of 1747 individuals from the pilot survey of the Aging in Spain Longitudinal Study (ELES-PS) were reviewed. ELES is a cross-sectional study for collecting health variables, food habits, socioeconomic data, and cognitive and functional capacities, which was carried out on a Spanish representative sample of noninstitutionalized persons of 50 years of age or older. Moreover, since telomere shortening is associated with cellular senescence, 35 telomere-related SNPs and cognitive impairments were analyzed. The results characterize the "solos" as males of 50-60 years, who were overweight and had lower levels of hemoglobin and neutrophils. There is also an association between five SNPs related to telomere length and BDNF. A group of people with loneliness and depression was identified with poorer health and cognitive status, poorer perception of their quality of life, poorer quality of sleep, and lower physical activity. Therefore, it follows that telomeres and BDNF play a role as intermediaries between loneliness and depression and their relationship with a worse state of health.

Brief communication: Ancient nuclear DNA and kinship analysis: the case of a medieval burial in San Esteban Church in Cuellar (Segovia, Central Spain).

The aim of this work was to investigate a very common situation in the archaeological and anthropological context: the study of a burial site containing several individuals, probably related genetically, using ancient DNA techniques. We used available ancient DNA and forensic protocols to obtain reliable results on archaeological material. The results also enabled molecular sex determination to be compared with osteological data. Specifically, a modified ancient DNA extraction method combined with the amplification of nuclear markers with the AmpFlSTR®MiniFiler™ kit(Applied Biosystems) was used. Seven medieval individuals buried in four niches dated in the 15th Century at San Esteban Church in Cuellar (Segovia, Central Spain) were analyzed by the proposed method, and four of seven provided complete autosomal short tandem repeat (STRs) profiles. Kinship analyses comprising paternity and sibship relations were carried out with pedigree-specific software used in forensic casework. A 99.98% paternity probability was established between two individuals, although lower percentages (68%) were obtained in other cases, and some hypothetical kinship relations were excluded. The overall results could eventually provide evidence for reconstructing the historical record.

Acenocoumarol Pharmacogenetic Dosing Algorithm versus Usual Care in Patients with Venous Thromboembolism: A Randomised Clinical Trial.

Patients with venous thromboembolism (VTE) require immediate treatment with anticoagulants such as acenocoumarol. This multicentre randomised clinical trial evaluated the effectiveness of a dosing pharmacogenetic algorithm versus a standard-of-care dose adjustment at the beginning of acenocoumarol treatment. We included 144 patients with VTE. On the day of recruitment, a blood sample was obtained for genotyping (CYP2C9*2, CYP2C9*3, VKORC1, CYP4F2, APOE). Dose adjustment was performed on day 3 or 4 after the start of treatment according to the assigned group and the follow-up was at 12 weeks. The principal variable was the percentage of patients with an international normalised ratio (INR) within the therapeutic range on day 7. Thirty-four (47.2%) patients had an INR within the therapeutic range at day 7 after the start of treatment in the genotype-guided group compared with 14 (21.9%) in the control group (p = 0.0023). There were no significant differences in the time to achieve a stable INR, the number of INRs within the range in the first 6 weeks and at the end of study. Our results suggest the use of a pharmacogenetic algorithm for patients with VTE could be useful in achieving target INR control in the first days of treatment.

Spanish allele and haplotype database for 32 X-chromosome Insertion-Deletion polymorphisms.

X-chromosome markers have been proved to be decisive both complementing and solving kinship analysis, particularly when autosomal markers are not able to produce adequate likelihood ratios between different hypothesis. On the other hand, Pereira et al., (2012) have demonstrated that 32 Insertion/Deletion (InDel) markers located on the X-Chromosome have a very important power of discrimination in human populations, being a novel tool in the forensic and population fields. So, the aim of the present work was testing the forensic and population genetic efficiency of the 32 X-InDel polymorphisms in the Spanish population, and subsequently build an allele/haplotype frequencies database. To accomplish this objective, a total of 555 samples comprising male individuals from 13 Spanish regions were analysed for the above mentioned 32 X-InDels in two independent laboratories. A pairwise FST analysis was performed in order to understand if the studied Spanish sub-populations present significant differences among them, detecting possible population substructure. Also, linkage disequilibrium analyses were computed to investigate the presence of association between markers in the Spanish population. After Bonferroni correction, the absence of significant differences among the studied regions supports a global Spanish population database. Concerning LD, besides previously reported linked markers MID356-MID357 and MID3690-MID3719-MID2089, we also detected significant association between MID3703-MID3774, even after Bonferroni correction. Finally, after computing allele and haplotype frequencies, forensic efficiency parameters were calculated (PDmales = 99.999976 %; PDfemales = 99.99999999998 %). Mean exclusion chance values for duos were 0.999 and trios 0.99999. These results reinforce the suitability of the 32 X-InDels marker set both in identification and kinship studies.

Genetic variability of 16 Y-chromosome STRs in a sample from Equatorial Guinea (Central Africa).

Nine Y-STR loci from the "minimal haplotype" included in Y-STR Haplotype Reference Databases (YHRD) together with eight additional Y-STRs (DYS437, DYS438, DYS439, DYS460, DYS461, GATA C4, GATA H4 and GATA A10) were analyzed in a sample of 101 males from Equatorial Guinea living in Madrid. Haplotype and allelic frequencies were calculated and genetic diversities were estimated for each genetic system as well as for the whole haplotype. An unexpected high frequency (6%) of intermediate alleles (13.2 and 14.2) was found in DYS385. For DYS19, two alleles were found in one sample. Another sample failed to amplify with DYS393 primers using either PowerPlex Y System (Promega Corporation) or the Y-PLEXtrade mark 12 (Reliagene, New Orleans, LA) commercial kits. Comparison between Equatorial Guinea and another African population (Mozambique; South East Coast) revealed a significant pairwise Phi(st) value between them (Phi(st)=0.03309; P=0.00000).

STR allelic frequencies for an African population sample (Equatorial Guinea) using AmpFlSTR Identifiler and Powerplex 16 kits.

We present allele frequencies and forensic parameters for 17 STRs included in the AmpFlSTR Identifiler (CSF1PO, D2S1338, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D19S433, D21S11, FGA, TH01, TPO and VWA) and Powerplex 16 System (CSF1PO, D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, FGA, Penta D, Penta E, TH01, TPO and VWA) in a sample of 134 unrelated individuals from Equatorial Guinea located in Western Africa, between Cameroon and Gabon. Hardy-Weinberg equilibrium was tested for each locus and the sample was compared with five African databases: Promega's and AB Applied Biosystems African-Americans and samples from Mozambique, from Cabinda (Angola) and Guinea-Bissau.

Large-scale recent expansion of European patrilineages shown by population resequencing.

The proportion of Europeans descending from Neolithic farmers ∼ 10 thousand years ago (KYA) or Palaeolithic hunter-gatherers has been much debated. The male-specific region of the Y chromosome (MSY) has been widely applied to this question, but unbiased estimates of diversity and time depth have been lacking. Here we show that European patrilineages underwent a recent continent-wide expansion. Resequencing of 3.7 Mb of MSY DNA in 334 males, comprising 17 European and Middle Eastern populations, defines a phylogeny containing 5,996 single-nucleotide polymorphisms. Dating indicates that three major lineages (I1, R1a and R1b), accounting for 64% of our sample, have very recent coalescent times, ranging between 3.5 and 7.3 KYA. A continuous swathe of 13/17 populations share similar histories featuring a demographic expansion starting ∼ 2.1-4.2 KYA. Our results are compatible with ancient MSY DNA data, and contrast with data on mitochondrial DNA, indicating a widespread male-specific phenomenon that focuses interest on the social structure of Bronze Age Europe.

STR data for nine Y-chromosomal loci in Guinea Equatorial (central Africa).

Allele and haplotype frequencies for nine Y-specific STR loci (DYS19, DYS389-I, DYS389-II, DYS390, DYS391, DYS393, DYS434, DYS437, and DYS439) were obtained from a sample of 57 males from Guinea Equatorial.

Population data for 16 Y-chromosome STRs in four populations from Pyrenees (Spain).

Population frequencies for the eight Y-STR loci included in the "minimal haplotype" from Y-STR Haplotype Reference Database (YHRD) plus other eight Y-STRs (DYS434, DYS435, DYS436, DYS437, DYS438, DYS439, GATA H4 and GATA A10) were obtained for a sample of 133 males from four main geographical areas in the Pyrenees (Spain): Vall D'Aran (Lérida), Cerdanya (Gerona), Alt Urgell (Lérida) and Jacetania (Huesca). Haplotype diversities were estimated in the four populations.

Genetic polymorphism of 15 STR loci in Chinese Han population from Shanghai municipality in East China.

The aim of this study was to estimate the allelic frequencies of the fifteen STRs included in AmpFlSTR(®) Identifiler(®) PCR Amplification Kit (Applied Biosystems, USA) in a sample of 186 unrelated individuals resident in Shanghai. Allelic frequencies and forensic parameters were calculated. All loci showed no departure from Hardy-Weinberg equilibrium. The obtained frequencies were compared with other previously reported population data.

The genetic landscape of Equatorial Guinea and the origin and migration routes of the Y chromosome haplogroup R-V88.

Human Y chromosomes belonging to the haplogroup R1b1-P25, although very common in Europe, are usually rare in Africa. However, recently published studies have reported high frequencies of this haplogroup in the central-western region of the African continent and proposed that this represents a 'back-to-Africa' migration during prehistoric times. To obtain a deeper insight into the history of these lineages, we characterised the paternal genetic background of a population in Equatorial Guinea, a Central-West African country located near the region in which the highest frequencies of the R1b1 haplogroup in Africa have been found to date. In our sample, the large majority (78.6%) of the sequences belong to subclades in haplogroup E, which are the most frequent in Bantu groups. However, the frequency of the R1b1 haplogroup in our sample (17.0%) was higher than that previously observed for the majority of the African continent. Of these R1b1 samples, nine are defined by the V88 marker, which was recently discovered in Africa. As high microsatellite variance was found inside this haplogroup in Central-West Africa and a decrease in this variance was observed towards Northeast Africa, our findings do not support the previously hypothesised movement of Chadic-speaking people from the North across the Sahara as the explanation for these R1b1 lineages in Central-West Africa. The present findings are also compatible with an origin of the V88-derived allele in the Central-West Africa, and its presence in North Africa may be better explained as the result of a migration from the south during the mid-Holocene.

Identification of a novel quantitative trait nucleotype related to iron status in a calcium channel gene.

Several iron-related parameters have been reported to show significant heritability, and thus, seemed to be genetically regulated. A genome wide family-based study revealed two regions that showed a linkage signal with transferrin receptor levels. The aim of the study was to identify genetic markers associated with iron status biomarkers. Ten SNPs selected from the literature were tested, and parameters related to iron metabolism were analysed, in a group (n=284) of Spanish women. Data were analyzed using Bayesian Model Averaging (BMA) test and decision trees. The rs1375515, located in an intronic region of the calcium channel gene CACNA2D3, showed strong associations with levels of mean corpuscular volume according to BMA test, and with levels of haemoglobin and ferritin according to decision trees. The allele G was associated to low levels of these parameters which suggests higher iron deficiency anaemia risk. This SNP along with the C282Y mutation explained significant differences in the distribution of individuals in three iron-related clinical phenotypes (normal, iron deficient and iron deficiency anaemic). In conclusion, the rs1375515, or other genetic polymorphisms in linkage, may play important roles in iron status, probably by affecting the function of a calcium channel. These findings may be useful for further investigation in the etiology of iron diseases.