Alejandro Blanco-Verea

Hierarchical analysis of 30 Y-chromosome SNPs in European populations

Analysis of Y-chromosome haplogroups defined by binary polymorphisms, has became a standard approach for studying the origin of modern human populations and for measuring the variability between them. Furthermore, the simplicity and population specificity of binary polymorphisms allows inferences to be drawn about the population origin of any male sample of interest for forensic purposes. From the 245 binary polymorphisms that can be analysed by PCR described in the Y Chromosome Consortium tree, we have selected 30 markers. The set of 30 has been grouped into 4 multiplexes in order to determine the most frequent haplogroups in Europe, using only 1 or 2 multiplexes. In this way, we avoid typing unnecessary SNPs to define the final haplogroup saving effort and cost, since we only need to type 9 SNPs in the best case and in the worst case, no more than 17 SNPs to define the haplogroup. The selected method for allele discrimination was a single base extension reaction using the SNaPshot multiplex kit. A total of 292 samples from 8 different districts of Galicia (northwest Spain) were analysed with this strategy. No significant differences were detected among the different districts, except for the population from Mariña Lucense, which showed a distant haplogroup frequency but not higher Φst values.

Ancestry analysis in the 11-M Madrid bomb attack investigation

The 11-M Madrid commuter train bombings of 2004 constituted the second biggest terrorist attack to occur in Europe after Lockerbie, while the subsequent investigation became the most complex and wide-ranging forensic case in Spain. Standard short tandem repeat (STR) profiling of 600 exhibits left certain key incriminatory samples unmatched to any of the apprehended suspects. A judicial order to perform analyses of unmatched samples to differentiate European and North African ancestry became a critical part of the investigation and was instigated to help refine the search for further suspects. Although mitochondrial DNA (mtDNA) and Y-chromosome markers routinely demonstrate informative geographic differentiation, the populations compared in this analysis were known to show a proportion of shared mtDNA and Y haplotypes as a result of recent gene-flow across the western Mediterranean, while any two loci can be unrepresentative of the ancestry of an individual as a whole. We based our principal analysis on a validated 34plex autosomal ancestry-informative-marker single nucleotide polymorphism (AIM-SNP) assay to make an assignment of ancestry for DNA from seven unmatched case samples including a handprint from a bag containing undetonated explosives together with personal items recovered from various locations in Madrid associated with the suspects. To assess marker informativeness before genotyping, we predicted the probable classification success for the 34plex assay with standard error estimators for a naïve Bayesian classifier using Moroccan and Spanish training sets (each n = 48). Once misclassification error was found to be sufficiently low, genotyping yielded seven near-complete profiles (33 of 34 AIM-SNPs) that in four cases gave probabilities providing a clear assignment of ancestry. One of the suspects predicted to be North African by AIM-SNP analysis of DNA from a toothbrush was identified late in the investigation as Algerian in origin. The results achieved illustrate the benefit of adding specialized marker sets to provide enhanced scope and power to an already highly effective system of DNA analysis for forensic identification.

Uniparental markers of contemporary Italian population reveals details on its pre-Roman heritage.

Background According to archaeological records and historical documentation, Italy has been a melting point for populations of different geographical and ethnic matrices. Although Italy has been a favorite subject for numerous population genetic studies, genetic patterns have never been analyzed comprehensively, including uniparental and autosomal markers throughout the country. Methods/Principal Findings A total of 583 individuals were sampled from across the Italian Peninsula, from ten distant (if homogeneous by language) ethnic communities — and from two linguistic isolates (Ladins, Grecani Salentini). All samples were first typed for the mitochondrial DNA (mtDNA) control region and selected coding region SNPs (mtSNPs). This data was pooled for analysis with 3,778 mtDNA control-region profiles collected from the literature. Secondly, a set of Y-chromosome SNPs and STRs were also analyzed in 479 individuals together with a panel of autosomal ancestry informative markers (AIMs) from 441 samples. The resulting genetic record reveals clines of genetic frequencies laid according to the latitude slant along continental Italy – probably generated by demographical events dating back to the Neolithic. The Ladins showed distinctive, if more recent structure. The Neolithic contribution was estimated for the Y-chromosome as 14.5% and for mtDNA as 10.5%. Y-chromosome data showed larger differentiation between North, Center and South than mtDNA. AIMs detected a minor sub-Saharan component; this is however higher than for other European non-Mediterranean populations. The same signal of sub-Saharan heritage was also evident in uniparental markers. Conclusions/Significance Italy shows patterns of molecular variation mirroring other European countries, although some heterogeneity exists based on different analysis and molecular markers. From North to South, Italy shows clinal patterns that were most likely modulated during Neolithic times.

Sarcomeric gene mutations in sudden infant death syndrome (SIDS).

In developed countries, sudden infant death syndrome (SIDS) represents the most prevalent cause of death in children between 1 month and 1 year of age. SIDS is a diagnosis of exclusion, a negative autopsy which requires the absence of structural organ disease. Although investigators have confirmed that a significant percentage of SIDS cases are actually channelopathies, no data have been made available as to whether other sudden cardiac death-associated diseases, such as hypertrophic cardiomyopathy (HCM), could be responsible for some cases of SIDS. The presence of a genetic mutation in the sarcomeric protein usually affects the force of contraction of the myocyte, whose weakness is compensated with progressive hypertrophy and disarray. However, it is unclear whether in the most incipient forms, that is, first years of life, the lack of these phenotypes still confers a risk of arrhythmogenesis. The main goal of the present study is to wonder whether genetic defects in the sarcomeric proteins, previously associated with HCM, could be responsible for SIDS. We have analysed 286 SIDS cases for the most common genes implicated in HCM in adults. A total of 680 mutations localised in 16 genes were analysed by semi-automated matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDITOF-MS) using the Sequenom MassARRAY(®) System. Ten subjects with completely normal hearts showed mutated alleles at nine of the genetic variants analysed, and one additional novel mutation was detected by conventional sequencing. Therefore, a genetic mutation associated with HCM may cause sudden cardiac death in the absence of an identifiable phenotype.

Y-chromosome lineages in native South American population

The present work tries to investigate the population structure and variation of the Amerindian indigenous populations living in Argentina. A total of 134 individuals from three ethnic groups (Kolla, Mapuche and Diaguitas) living in four different regions were collected and analysed for 26 Y-SNPs and 11 Y-STRs. Intra-population variability was analysed, looking for population substructure and neighbour populations were considered for genetic comparative analysis, in order to estimate the contribution of the Amerindian and the European pool, to the current population. We observe a high frequency of R1b1 and Q1a3a* Y-chromosome haplogroups, in the ethnic groups Mapuche, Diaguita and Kolla, characteristic of European and Native American populations, respectively. When we compare our native Argentinean population with other from the South America we also observe that frequency values for Amerindian lineages are relatively lower in our population. These results show a clear Amerindian genetic component but we observe a predominant European influence too, suggesting that typically European male lineages have given rise to the displacement of genuinely Amerindian male lineages in our South American population.

Prevalence of HCM and long QT syndrome mutations in young sudden cardiac death-related cases

Cardiomyopathies and channelopathies are major causes of sudden cardiac death. The genetic study of these diseases is difficult because of their heterogenic nature not only in their genetic traits but also in their phenotypic expression. The purpose of the present study is the analysis of a wide spectrum of previously known genetic mutations in key genes related to hypertrophic cardiomyopathy (HCM), long QT syndrome (LQTS), and Brugada syndrome (BrS) development. The samples studied include cases of sudden cardiac death (SCD) in young adults and their relatives in order to identify the real impact of genetic screening of SCD in forensic cases. Genetic screening of described variation in 16 genes implicated in the development of HCM and three more genes implicated in LQTS and BrS was performed by using MassARRAY™ technology. In addition, direct sequencing of the two most prevalent genes implicated in the development of SQTL type 1 and 2 was also carried out. Genetic screening allowed us to unmask four possibly pathogenic mutation carriers in the 49 SCD cases considered; carriers of mutation represent 9% (2/23) of the probands with structural anomalies found after autopsy and 7% (1/14) of the probands with structurally normal hearts after in depth autopsy protocol. One mutation was found among 12 of the recovered SCD cases considered. In people with direct family history of sudden cardiac death, but not themselves, 11 additional mutation carriers were found. Three different mutations were found in six of the 19 LQTS patients, representing three families and two different mutations were found among six patients with previous syncope. Genetic analysis in sudden cardiac death cases could help to elucidate the cause of death, but it also can help in the prevention of future deaths in families at risk. The study presented here shows the importance and relevance of genetic screening in patients with signs of cardiac hypertrophy and in family cases with more than one relative affected.

Massive parallel sequencing applied to the molecular autopsy in sudden cardiac death in the young

Sudden cardiac death in the young is a very traumatic event that occurs often in apparently healthy individuals without an explainable cause of death after a comprehensive medico-legal investigation. Knowledge about the pathologies with a risk of sudden death is increasingly showing a greater underlying genetic heterogeneity, which provides one of the main handicaps for molecular autopsy. On the other hand the enormous technological advances in sequencing technologies, allow us to analyse as many genes as we want at a cost increasingly reduced. The sum of these two factors (increased knowledge of genetics and available technologies) allow us to make an individualized study of the causes of sudden cardiac death in young adults, through massive sequencing of all potential genes involved in the process. We define this approach as massive genomic autopsy, and with this review we will try to explain the possible scenarios and methods available for its implementation.

Patterns of Y-STR variation in Italy

The 17 Y-chromosomal short tandem repeats (STRs) included in the AmpFlSTR Yfiler Amplification Kit (AB Applied Biosystems) (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635 and GATA H4.1) were typed in 292 samples from seven Italian regions. Population comparisons with other European samples were undertaken; for this purpose, two databases were collated from the literature: (a) 19 population samples including >2900 Yfiler profiles, and (b) 67 population samples including >15,000 minimum haplotype profiles. A total of 276 different Yfiler haplotypes were observed in Italy, and only one of them was shared among our seven population samples. The overall haplotype diversity (0.9996) was comparable to other European samples. AMOVA indicates that among population variance depends on the amount of Y-STRs used, being higher when using minimal haplotypes. This is probably due to the fact that Yfiler profiles are represented by singleton haplotypes in all the population samples raising the diversity values to the maximum theoretical value. AMOVA results seems to depend even more strongly on the amount of population samples used, the among population variance in Italy ranging from 2.82% to 11.03% (using 15 and 32 Italian populations samples, respectively). Variance is not as strongly stratified geographically within Italy, although it is notorious that latitude is more important than longitude in the distribution of variance. The results also indicated that Italy is less stratified than other European samples. The present study contributes to enrich the Y-chromosome databases regarding high-resolution Y-chromosome data sets and demonstrates that extended Y-STR profiles substantially increases the discriminatory capacity in individual identification for forensic purposes.

Broad-based molecular autopsy: a potential tool to investigate the involvement of subtle cardiac conditions in sudden unexpected death in infancy and early childhood

Objectives Sudden unexplained death in children is a tragic and traumatic event, often worsened when the cause of death cannot be determined. This work aimed to investigate the presence of putative pathogenic genetic variants in a broad spectrum of cardiomyopathy, channelopathy and aortic disease associated genes that may have increased these childrens vulnerability to sudden cardiac death. Design We performed molecular autopsy of 41 cases of sudden unexplained death in infants and children through massive parallel sequencing of up to 86 sudden cardiac death-related genes. Multiple in silico analyses were conducted together with a thorough review of the literature in order to prioritise the putative pathogenic variants. Results A total of 63 variants in 35 cases were validated. The largest proportion of these variants is located within cardiomyopathy genes although this would have been more expected of channelopathy gene variants. Subtle microscopic features of heart tissue may indicate the presence of an early onset cardiomyopathy as a predisposing condition to sudden unexpected death in some individuals. Conclusions Next-generation sequencing technologies reveal the existence of a wide spectrum of rare and novel genetic variants in sarcomere genes, compared with that of cardiac ion channels, in sudden unexplained death in infants and children. Our findings encourage further investigation of the role of early onset inherited cardiomyopathies and other diseases involving myocardial dysfunction in these deaths. Early detection of variants in these individuals could help to unmask subtle forms of disease within their relatives, who would eventually benefit from better counselling about their genetic history.

Next generation sequencing challenges in the analysis of cardiac sudden death due to arrhythmogenic disorders.

Inherited arrhythmogenic disorders is a relatively common cause of cardiac sudden death in young people. Diagnosis has been difficult so far due to the genetic heterogeneity of the disease. Next generation sequencing (NGS) is offering a new scenario for diagnosis. The purpose of our study was to validate NGS for the analysis of twenty‐eight genes known to be associated with inherited arrhythmogenic disorders and therefore with sudden cardiac death. SureSelect hybridization was used to enrich DNA from 53 samples, prior to be sequenced with the SOLID™ System of Life Technologies. Depth of coverage, consistency of coverage across samples, and location of variants identified were assessed. All the samples showed a depth of coverage over 200×, except one of them discarded because of its coverage below 30×. Average percentage of target bp covered at least 20× was 96.45%. In the remaining samples, following a prioritization process 46 possible variants in 31 samples were found, of which 45 were confirmed by Sanger sequencing. After filtering variants according to their minor allele frequency in the Exome Sequencing Project 27 putative pathogenic variants in 20 samples remained. With the use of in silico tools, 13 variants in 11 samples were classified as likely pathogenic. In conclusion, NGS allowed us to accurately detect arrhythmogenic disease causing mutations in a fast and cost‐efficient manner that is suitable for daily clinical and forensic practice of genetic testing of this type of disorders.