Identification of Wolf-Dog Hybrids in Europe – An Overview of Genetic Studies

Significant development of genetic tools during the last decades provided opportunities for more detailed analyses and deeper understanding of species hybridization. New genetic markers allowed for reliable identification of admixed individuals deriving from recent hybridization events (a few generations) and those originating from crossings up to 19 generations back. Implementation of microsatellites (STRs) together with Bayesian clustering provided abundant knowledge regarding presence of admixed individuals in numerous populations and helped understand the problematic nature of studying hybridization (i.a., defining a reliable thresholds for recognizing individuals as admixed or obtaining well-grounded results representing actual proportion of hybrids in a population). Nevertheless, their utilization is limited to recent crossbreeding events. Single Nucleotide Polymorphisms (SNPs) proved to be more sensible tools for admixture analyses furnishing more reliable knowledge, especially for older generation backcrosses. Small sets of Ancestry Informative Markers (AIMs) of both types of markers were effective enough to implement in monitoring programs, however, SNPs seem to be more appropriate because of their ability to identify admixed individuals up to 3rd generations. The main aim of this review is to summarize abundant knowledge regarding identification of wolf-dog hybrids in Europe and discuss the most relevant problems relating to the issue, together with advantages and disadvantages of implemented markers and approaches.

Evaluation of a custom QIAseq targeted DNA panel with 164 ancestry informative markers sequenced with the Illumina MiSeq

Introduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations.

Reliable wolf-dog hybrid detection in Europe using a reduced SNP panel developed for non-invasively collected samples

Background Understanding the processes that lead to hybridization of wolves and dogs is of scientific and management importance, particularly over large geographical scales, as wolves can disperse great distances. However, a method to efficiently detect hybrids in routine wolf monitoring is lacking. Microsatellites offer only limited resolution due to the low number of markers showing distinctive allele frequencies between wolves and dogs. Moreover, calibration across laboratories is time-consuming and costly. In this study, we selected a panel of 96 ancestry informative markers for wolves and dogs, derived from the Illumina CanineHD Whole-Genome BeadChip (174 K). We designed very short amplicons for genotyping on a microfluidic array, thus making the method suitable also for non-invasively collected samples. Results Genotypes based on 93 SNPs from wolves sampled throughout Europe, purebred and non-pedigree dogs, and suspected hybrids showed that the new panel accurately identifies parental individuals, first-generation hybrids and first-generation backcrosses to wolves, while second- and third-generation backcrosses to wolves were identified as advanced hybrids in almost all cases. Our results support the hybrid identity of suspect individuals and the non-hybrid status of individuals regarded as wolves. We also show the adequacy of these markers to assess hybridization at a European-wide scale and the importance of including samples from reference populations. Conclusions We showed that the proposed SNP panel is an efficient tool for detecting hybrids up to the third-generation backcrosses to wolves across Europe. Notably, the proposed genotyping method is suitable for a variety of samples, including non-invasive and museum samples, making this panel useful for wolf-dog hybrid assessments and wolf monitoring at both continental and different temporal scales.

Genetic ancestry plays a central role in population pharmacogenomics

Recent studies have pointed out the essential role of genetic ancestry in population pharmacogenetics. In this study, we analyzed the whole-genome sequencing data from The 1000 Genomes Project (Phase 3) and the pharmacogenetic information from Drug Bank, PharmGKB, PharmaADME, and Biotransformation. Here we show that ancestry-informative markers are enriched in pharmacogenetic loci, suggesting that trans-ancestry differentiation must be carefully considered in population pharmacogenetics studies. Ancestry-informative pharmacogenetic loci are located in both protein-coding and non-protein-coding regions, illustrating that a whole-genome analysis is necessary for an unbiased examination over pharmacogenetic loci. Finally, those ancestry-informative pharmacogenetic loci that target multiple drugs are often a functional variant, which reflects their importance in biological functions and pathways. In summary, we develop an efficient algorithm for an ultrahigh-dimensional principal component analysis. We create genetic catalogs of ancestry-informative markers and genes. We explore pharmacogenetic patterns and establish a high-accuracy prediction panel of genetic ancestry. Moreover, we construct a genetic ancestry pharmacogenomic database Genetic Ancestry PhD (http://hcyang.stat.sinica.edu.tw/databases/genetic_ancestry_phd/).

Peptide Ancestry Informative Markers in Uterine Neoplasms from Women of European, African and Asian Ancestry

Deep proteogenomic analyses of diverse populations will improve our understanding of molecular drivers of disease, such as those underlying uterine neoplasms which exhibit marked racial disparities. The characterization of ancestry-linked peptide variants in disease-relevant patient tissues represents a foundational step towards investigating relationships linking patient ancestry with the proteome, genome and disease pathogenesis. We selected 1,037 nonsynonymous single nucleotide polymorphisms (nsSNPs) encoding missense amino acid substitutions occurring within putative tryptic peptides exhibiting high allele frequencies (≥ 50%) in European, African, and East Asian populations, termed peptide ancestry informative markers (pAIMs) and investigated these in cell line models and tissues of uterine neoplasms collected from women representing diverse ancestries. We quantified 62 pAIMs in total across >100 patient-derived samples by multiplexed, quantitative proteomic analyses. Our analyses of endometrial cancer cell lines established from European (n=7), African (n=3), and East Asian (n=3) women resulted in the quantitation of 43 pAIMs that significantly correlated with population-level allele frequencies consistent with global ancestry estimates defined by genotype analyses. We next investigated uterine leiomyoma (ULM) tissues from self-described African-American (n=31) and European-American (n=35) women and quantified 33 pAIMs that correlated with patient race. We validated 10 of these in an independent cohort of ULM tissues collected from women determined to be of European (n=16) and African (n=11) ancestry by genotype analyses. This validated subset was predominated by candidates of undetermined clinical significance (ClinVar) and included a substitution in GC vitamin D-binding protein, i.e. (D451E, rs7041), that exhibits a higher allele frequency in European (58%) versus African (7.3%) populations and correlates with altered GC protein and vitamin D levels. Comparison of cell line and tissue analyses revealed co-quantitation of pAIMs encoded within Protein-L-isoaspartate(D-aspartate) O-methyltransferase (V178I, >80% AF in African and East Asians), Serine/threonine-protein phosphatase CPPED1 (A19D, >75% AF in Africans) and AH receptor-interacting protein (Q228K, >99% AF in European and East Asians). We further assessed the performance of pAIMs to determine patient ancestry in silico relative to standard genotype estimates by random sampling analyses and find that as few as 20 population-specific pAIMs can determine ancestry proportions similarly to >260K SNPs (R2=0.9905). These efforts have quantified and validated pAIMs in diverse uterine neoplasms that represent a foundational set of ancestry-linked variant peptides observable by multiplexed proteomics and provides proof of concept that estimates of European, African and East Asian ancestry can be determined from routine proteomic analysis of disease-relevant patient cell lines and tissues. This work will support ongoing efforts focused on linking the proteome and genome with patient ancestry to better understand relationships with disease mechanisms driving racial disparities in the pathogenesis of uterine neoplasms.

Ancestry and TPMT-VNTR Polymorphism: Relationship with Hematological Toxicity in Uruguayan Patients with Acute Lymphoblastic Leukemia

6-Mercaptopurine (6-MP) is a thiopurine drug widely used in childhood acute lymphoblastic leukemia (ALL) therapy. Genes such as TPMT and NUDT15 have an outstanding role in 6-MP metabolism. Mutations in both genes explain a significant portion of hematological toxicities suffered by ALL Uruguayan pediatric patients. A variable number tandem repeat in the TPMT promoter (TPMT-VNTR) has been associated with TPMT expression. This VNTR has a conservative architecture (AnBmC). To explore new causes of hematological toxicities related to ALL therapy, we genotyped the TPMT-VNTR of 130 Uruguayan pediatric patients. Additionally, individual genetic ancestry was estimated by 45 ancestry-informative markers (AIMs). Hematological toxicity was measured as the number of leukopenia events and 6-MP dose along the maintenance phase. As previously reported, we found TPMT*2 and TPMT*3C alleles were associated to TPMT-VNTR A2BC and AB2C, respectively. However, contrasting with other reports, TPMT*3A allele was found in a heterogeneous genetic background in linkage equilibrium. Patients carrying more than 5 A repeats present a significant higher number of leukopenia events among patients without TPMT and/or NUDT15 variants. Native American ancestry and the number of A repeats were significantly correlated with the number of leukopenia events. However, the correlation between Native American ancestry and the number of leukopenia events was lost when the number of A repeats was considered as covariate. This suggests that TPMT-VNTR alleles are more relevant than Native American ancestry in the hematological toxicity. Our results emphasize that TPMT-VNTR may be used as a pharmacogenetic biomarker to predict 6-MP-related hematological toxicity in ALL childhood therapy.

Characterization of Ancestral Origin of Cystic Fibrosis of Patients with New Reported Mutations in CFTR

The incidence of cystic fibrosis (CF) and the frequency of the variants reported for CFTR depend on the population; furthermore, CF symptomatology is characterized by obstructive lung disease and pancreatic insufficiency among other symptoms, which are reliant on the individual's genotype. The Ecuadorian population is a mixture of Native Americans, Europeans, and Africans. That population admixture could be the reason for the new mutations reported in a previous study by Ruiz et al. (2019). A panel of 46 Ancestry Informative Markers was used to estimate the ancestral proportions of each available sample (12 samples in total). As a result, the Native American ancestry proportion was the most prevalent in almost all individuals, except for three patients from Guayaquil with the mutation [c.757G>A:p.Gly253Arg; c.1352G>T:p.Gly451Val] who had the highest European composition.

Characterization Of Ancestral Origin Of Cystic Fibrosis Of Patients With New Reported Mutations In CFTR

The incidence of Cystic fibrosis (CF) and the frequency of the variants for CFTR depend on the population; furthermore, CF symptomatology is characterized by obstructive lung disease, pancreatic insufficiency among others, reliant on the individual genotype. Ecuadorian population is a mixture of Native Americans, Europeans, and Africans. That population admixture could be the reason for the new mutations reported in a previous study by Ruiz et al. (2019). A panel of 46 Ancestry Informative Markers was used to estimate the ancestral proportions of each available sample (12 samples in total). As a result, the Native American ancestry proportion was the most prevalent in almost all individuals, except for three patients from Guayaquil with the mutation [c.757G>A:p.Gly253Arg; c.1352G>T:p.Gly451Val] who had the highest European composition.