Characterization of ancestry informative markers in the Tigray population of Ethiopia: A contribution to the identification process of dead migrants in the Mediterranean Sea.

Determination of bio-geographical ancestry by means of DNA ancestry informative markers (AIMs) can contribute to the identification of human remains in missing person cases and mass disasters. While the presence of Eastern Africans among the migrant victims of trafficking accidents in the Mediterranean Sea is often suspected, few studies have addressed the ability of autosomal AIM panels in current use in forensic laboratories to provide differentiation of populations within the African continent. In this study, two assays consisting of 46 AIM-Indels and 31 AIM-SNPs were typed in a Tigray population sample from Northern Ethiopia. STRUCTURE analysis showed that the Tigray population is characterized by a strong (∼50 %) non-African genetic component shared with European and Middle Eastern populations. The intermediate position of the Tigray sample between sub-Saharan African and European / Middle Eastern reference population samples was confirmed by principal component analysis. Both AIM panels provided effective differentiation between Tigray and sub-Saharan African populations. Classification accuracy of other populations involved in the current Mediterranean migrant crisis, like South Asians, was superior with the AIM-SNP panel compared to the AIM-Indel panel. Misclassification of Middle Eastern samples as Tigray was frequent with both AIM-indel (∼30 % misclassified) and AIM-SNPs (∼20 %). However, with AIM-SNPs, error rates were reduced to acceptable levels by applying cautionary minimum thresholds to assignment likelihoods. Establishment of an Eastern African reference database of AIMs that can be genotyped by means of low cost, small-scale assays compatible with capillary electrophoresis, sets a balance between the need for ancestry inference tools and the budget limitations faced by Italian laboratories engaged in the humanitarian identification of dead migrants recovered from the Mediterranean Sea.

Phylogeographic and genome-wide investigations of Vietnam ethnic groups reveal signatures of complex historical demographic movements.

The territory of present-day Vietnam was the cradle of one of the world's earliest civilizations, and one of the first world regions to develop agriculture. We analyzed the mitochondrial DNA (mtDNA) complete control region of six ethnic groups and the mitogenomes from Vietnamese in The 1000 Genomes Project (1000G). Genome-wide data from 1000G (~55k SNPs) were also investigated to explore different demographic scenarios. All Vietnamese carry South East Asian (SEA) haplotypes, which show a moderate geographic and ethnic stratification, with the Mong constituting the most distinctive group. Two new mtDNA clades (M7b1a1f1 and F1f1) point to historical gene flow between the Vietnamese and other neighboring countries. Bayesian-based inferences indicate a time-deep and continuous population growth of Vietnamese, although with some exceptions. The dramatic population decrease experienced by the Cham 700 years ago (ya) fits well with the Nam tien ("southern expansion") southwards from their original heartland in the Red River Delta. Autosomal SNPs consistently point to important historical gene flow within mainland SEA, and add support to a main admixture event occurring between Chinese and a southern Asian ancestral composite (mainly represented by the Malay). This admixture event occurred ~800 ya, again coinciding with the Nam tien.

FAS system deregulation in T-cell lymphoblastic lymphoma.

The acquisition of resistance towards FAS-mediated apoptosis may be required for tumor formation. Tumors from various histological origins exhibit FAS mutations, the most frequent being hematological malignancies. However, data regarding FAS mutations or FAS signaling alterations are still lacking in precursor T-cell lymphoblastic lymphomas (T-LBLs). The available data on acute lymphoblastic leukemia, of precursor origin as well, indicate a low frequency of FAS mutations but often report a serious reduction in FAS-mediated apoptosis as well as chemoresistance, thus suggesting the occurrence of mechanisms able to deregulate the FAS signaling pathway, different from FAS mutation. Our aim at this study was to determine whether FAS-mediated apoptotic signaling is compromised in human T-LBL samples and the mechanisms involved. This study on 26 T-LBL samples confirms that the FAS system is impaired to a wide extent in these tumors, with 57.7% of the cases presenting any alteration of the pathway. A variety of mechanisms seems to be involved in such alteration, in order of frequency the downregulation of FAS, the deregulation of other members of the pathway and the occurrence of mutations at FAS. Considering these results together, it seems plausible to think of a cumulative effect of several alterations in each T-LBL, which in turn may result in FAS/FASLG system deregulation. Since defective FAS signaling may render the T-LBL tumor cells resistant to apoptotic cell death, the correct prognosis, diagnosis and thus the success of anticancer therapy may require such an in-depth knowledge of the complete scenario of FAS-signaling alterations.

A novel mutation in the OFD1 (Cxorf5) gene may contribute to oral phenotype in patients with oral-facial-digital syndrome type 1.

BACKGROUND: Oral-facial-digital syndrome (OFDS) type 1 (OFD1) is an X-linked dominant condition associated with embryonic male lethality. It almost always affects the oral cavity, face, and digits. It is considered to be a ciliopathy caused by mutations in the OFD1 gene. A variety of mutations have been described, and a genotype-phenotype correlation has been suggested. OBJECTIVE AND METHODS: The proband was an 8-year-old Spanish girl with suspected OFD1. We extended the pedigree to three proband's generations, performing a thorough physical examination and screening for OFD1 mutations in nine individuals. RESULTS: The proband, her mother, and her sister showed oral findings consistent with OFD1. Ultrasound evaluation revealed the existence of renal cysts only in the proband's mother. The rest of the family (all male) had no relevant morphological abnormalities. A single-base deletion in exon 16 of OFD1 (c.2183delG) leading to a frameshift was detected in the proband, her mother, and her sister. CONCLUSION: Because all three women had a similar oral phenotype, this new mutation might be involved in the development of the OFD1 oral manifestations. In cases of OFDS, physical examination (including the oral cavity and renal function) and genetic screening of the probands and their relatives are mandatory.

Testing the performance of mtSNP minisequencing in forensic samples.

There is a growing interest among forensic geneticists in developing efficient protocols for genotyping coding region mitochondrial DNA (mtDNA) SNPs (mtSNPs). Minisequencing is becoming a popular method for SNP genotyping, but it is still used by few forensic laboratories. In part, this is due to the lack of studies testing its efficiency and reproducibility when applied to real and complex forensic samples. Here we tested a minisequencing design that consists of 71 mtSNPs (in three multiplexes) that are diagnostic of known branches of the R0 phylogeny, in real forensic samples, including degraded bones and teeth, hair shafts, and serial dilutions. The fact that amplicons are short coupled with the natural efficiency of the minisequencing technique allow these assays to perform well with all the samples tested either degraded and/or those containing low DNA amount. We did not observe phylogenetic inconsistencies in the 71 mtSNP haplotypes generated, indicating that the technique is robust against potential artefacts that could arise from unintended contamination and/or spurious amplification of nuclear mtDNA pseudogenes (NUMTs).

Gender bias in the multiethnic genetic composition of central Argentina.

A sample of central Argentina (Córdoba) was genotyped for the first hypervariable region (HVS-I) plus a set of coding region mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) (N = 102) and compared with a data set of Y-chromosome short tandem repeats (Y-STRs; N = 100) previously genotyped in the same individuals. We additionally compiled a database containing more than 4,000, 6,800, and 12,000 HVS-I sequences of Native American, sub-Saharan African, and European origin, respectively. The Y-Chromosome Haplotype Reference Database (YHRD) was used as a reference for the Y-STR profiles from Córdoba. The Native American component is highly prevalent on the maternal side (approximately 41%) in contrast to the Y-chromosome paternal contribution (approximately 2%), indicating a strong gender bias in the colonization and admixture processes that occurred in the recent history of Argentina, in agreement with historical records. The demographic input of African slaves in Córdoba was very high in the eighteenth century (approximately 40% of the total population) but decreased dramatically after a few decades; therefore, the minor traces of sub-Saharan Y-chromosome and mtDNA lineages observed in our sample fit well with these historical records. The European Y-chromosome component of Córdoba (approximately 97%; in contrast to the 57% observed in the mtDNA side) also mirrors the substantial immigration experienced by Argentina during the beginning of the last century, predominantly from Italy and Spain.

The mtDNA ancestry of admixed Colombian populations.

A total of 185 individuals from Colombia were sequenced for the first hypervariable region (HVS-I) of the mitochondrial DNA (mtDNA) genome, and a subset of these individuals were additionally genotyped for the second hypervariable segment (HVS-II). These individuals were collected according to their "self-reported ethnicity" in Colombia, comprising "Mestizos," "Mulatos," and "Afro-Colombians." We used databases containing more than 4,300 Native American lineages, 6,800 Africans, and 15,600 Europeans for population comparisons and phylogeographic inferences. We observe that Mulatos and Afro-Colombians have a dominant African mtDNA component, whereas Mestizos carry predominantly Native American haplotypes. All the populations analyzed have high diversity indices and there are no signatures of dramatic genetic drift episodes. Central and South America are the main candidate source populations of the Colombian Native American lineages, whereas west-central, southwest, and southeast Africa are the main original mtDNA sources for the African Colombian mtDNAs. We found that our results differ from those obtained in other studies for the same "population groups" in terms of haplogroup frequencies. This observation leads us to conclude that (i) self-reported ancestry is not a reliable proxy to indicate an individual's "ethnicity" in Colombia, (ii) our results do not support the use of outmoded race descriptions (Mestizos, Mulatos, etc.) mainly because these labels do not correspond to any genetically homogeneous population group, and (iii) studies relying on these terms to describe the population group of the individual, which then treat them as genetically homogeneous, carry a high risk of type I error (false positives) in medical studies in this country and of misinterpretation of the frequency of observed variation in forensic casework.

Analysis of body fluid mixtures by mtDNA sequencing: An inter-laboratory study of the GEP-ISFG working group.

The mitochondrial DNA (mtDNA) working group of the GEP-ISFG (Spanish and Portuguese Group of the International Society for Forensic Genetics) carried out an inter-laboratory exercise consisting of the analysis of mtDNA sequencing patterns in mixed stains (saliva/semen and blood/semen). Mixtures were prepared with saliva or blood from a female donor and three different semen dilutions (pure, 1:10 and 1:20) in order to simulate forensic casework. All labs extracted the DNA by preferential lysis and amplified and sequenced the first mtDNA hypervariable region (HVS-I). Autosomal and Y-STR markers were also analysed in order to compare nuclear and mitochondrial results from the same DNA extracts. A mixed stain prepared using semen from a vasectomized individual was also analysed. The results were reasonably consistent among labs for the first fractions but not for the second ones, for which some laboratories reported contamination problems. In the first fractions, both the female and male haplotypes were generally detected in those samples prepared with undiluted semen. In contrast, most of the mixtures prepared with diluted semen only yielded the female haplotype, suggesting that the mtDNA copy number per cell is smaller in semen than in saliva or blood. Although the detection level of the male component decreased in accordance with the degree of semen dilution, it was found that the loss of signal was not consistently uniform throughout each electropherogram. Moreover, differences between mixtures prepared from different donors and different body fluids were also observed. We conclude that the particular characteristics of each mixed stain can deeply influence the interpretation of the mtDNA evidence in forensic mixtures (leading in some cases to false exclusions). In this sense, the implementation of preliminary tests with the aim of identifying the fluids involved in the mixture is an essential tool. In addition, in order to prevent incorrect conclusions in the interpretation of electropherograms we strongly recommend: (i) the use of additional sequencing primers to confirm the sequencing results and (ii) interpreting the results to the light of the phylogenetic perspective.

Coding region mitochondrial DNA SNPs: targeting East Asian and Native American haplogroups.

We have developed a single PCR multiplex SNaPshot reaction that consists of 32 coding region SNPs that allows (i) increasing the discrimination power of the mitochondrial DNA (mtDNA) typing in forensic casework, and (ii) haplogroup assignments of mtDNA profiles in both human population studies (e.g. anthropological) and medical research. The selected SNPs target the East Asian phylogeny, including its Native American derived branches. We have validated this multiplex assay by genotyping a sample of East Asians (Taiwanese) and Native Americans (Argentineans). In addition to the coding SNP typing, we have sequenced the complete control region for the same samples. The genotyping results (control region plus SNaPshot profiles) are in good agreement with previous human population genetic studies (based on e.g. complete sequencing) and the known mtDNA phylogeny. We observe that the SNaPshot method is reliable, rapid, and cost effective in comparison with other techniques of multiplex SNP genotyping. We discuss the advantages of our SNP genotyping selection with respect to previous attempts, and we highlight the importance of using the known mtDNA phylogeny as a framework for SNP profile interpretation and as a tool to minimize genotyping errors.

Results of the 2003-2004 GEP-ISFG collaborative study on mitochondrial DNA: focus on the mtDNA profile of a mixed semen-saliva stain.

We report here a review of the seventh mitochondrial DNA (mtDNA) exercise undertaken by the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) corresponding to the period 2003-2004. Five reference bloodstains from five donors (M1-M5), a mixed stain of saliva and semen (M6), and a hair sample (M7) were submitted to each participating laboratory for nuclear DNA (nDNA; autosomal STR and Y-STR) and mtDNA analysis. Laboratories were asked to investigate the contributors of samples M6 and M7 among the reference donors (M1-M5). A total of 34 laboratories reported total or partial mtDNA sequence data from both, the reference bloodstains (M1-M5) and the hair sample (M7) concluding a match between mtDNA profiles of M5 and M7. Autosomal STR and Y-STR profiling was the preferred strategy to investigate the contributors of the semen/saliva mixture (M6). Nuclear DNA profiles were consistent with a mixture of saliva from the donor (female) of M4 and semen from donor M5, being the semen (XY) profile the dominant component of the mixture. Strikingly, and in contradiction to the nuclear DNA analysis, mtDNA sequencing results yield a more simple result: only the saliva contribution (M4) was detected, either after preferential lysis or after complete DNA digestion. Some labs provided with several explanations for this finding and carried out additional experiments to explain this apparent contradictory result. The results pointed to the existence of different relative amounts of nuclear and mtDNAs in saliva and semen. We conclude that this circumstance could strongly influence the interpretation of the mtDNA evidence in unbalanced mixtures and in consequence lead to false exclusions. During the GEP-ISFG annual conference a validation study was planned to progress in the interpretation of mtDNA from different mixtures.

Mitochondrial DNA error prophylaxis: assessing the causes of errors in the GEP'02-03 proficiency testing trial.

We report the results of the Spanish and Portuguese working group (GEP) of the International Society for Forensic Genetics (ISFG) Collaborative Exercise 2002-2003 on mitochondrial DNA (mtDNA) analysis. Six different samples were submitted to the participating laboratories: four blood stains (M1-M2-M3-M4), one mixture blood sample (M5), and two hair shaft fragments (M6). Most of the labs reported consensus results for the blood stains, slightly improving the results of previous collaborative exercises. Although hair shaft analysis is still carried out by a small number of laboratories, this analysis yielded a high rate of success. On the contrary, the analysis of the mixture blood stain (M5) yielded a lower rate of success; in spite of this, the whole results on M5 typing demonstrated the suitability of mtDNA analysis in mixture samples. We have found that edition errors are among the most common mistakes reported by the different labs. In addition, we have detected contamination events as well as other minor problems, i.e. lack of standarization in nomenclature for punctual and length heteroplasmies, and indels. In the present edition of the GEP-ISFG exercise we have paid special attention to the visual phylogenetic inspection for detecting common sequencing errors.

Typing of mitochondrial DNA coding region SNPs of forensic and anthropological interest using SNaPshot minisequencing.

The development of new methodologies for high-throughput SNP analysis is one of the most stimulating areas in genetic research. Here, we describe a rapid and robust assay to simultaneously genotype 17 mitochondrial DNA (mtDNA) coding region SNPs by minisequencing using SNaPshot. SNaPshot is a methodology based on a single base extension of an unlabeled oligonucleotide with labeled dideoxy terminators. The set of SNPs implemented in this multiplexed SNaPshot reaction allow us to allocate common mitochondrial West Eurasian haplotypes into their corresponding branch in the mtDNA skeleton, with special focus on those haplogroups lacking unambiguous diagnostic positions in the first and second hypervariable regions (HVS-I/II; by far, the most common segments analyzed by sequencing). Particularly interesting is the set of SNPs that subdivide haplogroup H; the most frequent haplogroup in Europe (40-50%) and one of the most poorly characterized phylogenetically in the HVS-I/II region. In addition, the polymorphic positions selected for this multiplex reaction increase considerably the discrimination power of current mitochondrial analysis in the forensic field and can also be used as a rapid screening tool prior to full sequencing analysis. The method has been validated in a sample of 266 individuals and shows high accuracy and robustness avoiding both the use of alternative time-consuming classical strategies (i.e. RFLP typing) and the need for high quantities of DNA template.