STR Markers Research Articles

Algorithm aligen: Simple method of encryption and matching with genetic profiles in a system of STR identification database

During the DNA identification process of 15000 missing persons in Peru between 1980 and 2000, we observed many cases of random matches due to the population genetic structure (founder effect, low gene flow between communities and inbreeding). In this genetic context, since 2002, we have been developing an algorithm named ALIGEN with the aim of improving the match and identification. This algorithm performs a meiosis simulation in two DNA databases (relatives and missing persons). In each DNA database ALIGEN generated the haploid profiles (hap-file) for each genetic profile divided in five groups of four STR markers (match group) with a total of twenty STR markers. Simultaneously, we performs a kinship analysis using the model of allele Identity by descendent (IBD) using a threshold of 90% posterior probability in the case of fullsibs with the aim to obtain only the significative relationship and avoiding the random matches. To support the first analysis, the algorithm generated a genetic distances between two genetic profile using hap-file and this form the matrix of likeness that correspond to the genetic distance among all genetic profiles (relatives and missing persons). This matrix is used in MEGA with the aim to obtain a relationship tree. In this way we can confirm the matches, the random matches and evidence of unknown relationships. Other characteristic of the algorithm, it can able to ensure the DNA information privacy through the encryption of each genetic profile using a Hash encryption model with de hap-file generated and it is a criteria very important in the future of populations DNA databasing. Finally, ALIGEN have been validated in the last 13 years solving the identification of missing persons in many cases at national and international level. For this raison, we wish share this algorithm as an easy tool that it can be implementing for any laboratory using the formulas described in this article.

Quantifiler® Trio DNA validation and usefulness in casework samples

DNA profiling with sets of highly polymorphic STR markers is applied in various aspects of human identification. During the past few years significant progress has been made in solving technical challenges associated with STR profiling, including the ability to analyze degraded DNA, low amounts of DNA and/or the presence of PCR inhibitors. Quantifiler® Trio DNA method enables forensic laboratories to simultaneously obtain a quantitative and qualitative assessment of total human and human male DNA. Moreover, this kit has a set of primers to amplify and detected two autosomal multiple-copy target loci, known as the small autosomal (SA) and large autosomal (LA), which provide a quality index (DI) to detect the presence of degraded DNA along with PCR inhibitors. The experiments focus on kit performance parameters relevant to the intended use of the kits as DNA quantification assays and also application in casework studies. The workflow combining the DNA quantification with Quantifiler® Trio DNA quantification kit and STR amplification has the potencial to improve forensic laboratories efficiency.

Colombian results of the interlaboratory quality control exercise 2013–2014

Currently, all agencies of conformity assessment testing laboratory favor the participation in proficiency testing as a mechanism for quality assurance. In Colombia, the Genes Laboratory has been designated, since 2008, to perform, design and implement the proficiency testing for all the interested laboratories. In this report, we show the results of Colombian exercises Inter-laboratory Quality Control for the years 2013 and 2014. In both years the exercise consisted of one practical component, one theoretical mandatory component and a theoretical optional component. The participants were 23 and 20 laboratories in 2013 and 2014, respectively, representing seven different countries of Latin America and the Caribbean. For the practical component each participant laboratory receive; (1) samples of blood, saliva and/or semen stains, in this part they should report the routine own laboratory markers for each sample, (2) three optional theoretical cases of varying complexity, (3) a simple theoretical approach consistent of paternity case (father, mother and son) (2013) and a complex paternity case with a deceased father (2014). In the last two components of the exercise they had to submit only the calculations. In both years, for the practical component the consensus of 70 STR markers, distributed between autosomal and linked to the sex chromosomes, was achieved with an error rate of 1.75% and 3.07% for 2013 and 2014, respectively. On the other hand, for mandatory theoretical exercise, error rates of 21.74% (2013) and 10.63% (2014) were detected. This inter-laboratory exercise has become an important mechanism for quality assurance in the region.

Increasing relevance of non-human genetics in Hungarian forensic practice

In step with the evolution of molecular genetics, the application of non-human biological traces is an intensively researched field in forensic sciences [1]. Recently it has been widely acknowledged that this extended possibility – with associated abilities and pitfalls – for forensic DNA examination can provide innovative results in criminal and civil casework as well as in connection with associative evidence.Depending on the specific species or types of traces, the final results can have very different criminalistics value (e.g., plants, dogs or microbiome of soil traces), but it is unquestionable that non-human DNA has an increasing role in forensic biological evidence [2–4].However, in contrast to the investigation of human DNA-traces, there currently exist no restrictions concerning DNA loci and privacy of genetic information, and a wide spectrum of genetic information and technologies may be applied. The field is developing with animal rights, international (e.g. CITES) conventions, national regulations and professional recommendations [5–7].The Hungarian forensic practice on animal DNA examination has been developing since 2000 [8]. The range of cases is sufficiently broad, and includes fatal accidents and animal attacks (case #1) as well as illegal trade cases (case #2). In case of degraded animal hair samples we used two sets of Canine STR markers with shortened amplicons, and species specific primers (Ursus sp.) for mitochondrial DNA analysis of mixed samples (case #2), respectively. The results supported the evaluation of individualization (case #1), confirming the species identification (case #2).

Non-invasive prenatal paternity testing with STRs: A pilot study

Non-invasive prenatal paternity testing using foetal cell-free DNA (cfDNA) in the mother's blood eliminates the risks of miscarriage associated with invasive methods such as chorionic villus sampling and amniocentesis. However, the low abundance of cfDNA as well as the large excess of maternal cfDNA makes foetal DNA typing very challenging. As illustrated by our case study, SNP typing at 317,000 positions using cfDNA in the mother’s blood was shown to successfully identify the father. However, for forensic routine work, STR analysis would be preferable over SNP analysis, as for STR markers the frequencies and population data are available and the statistics are well established. We therefore initiated a pilot study to set up a next-generation sequencing (NGS) assay using an early access version of the Ion AmpliSeq HID STR 25-plex on the Ion PGM™. We successfully confirmed paternity from blood samples from two pregnant women with 10 and 16 weeks of gestation (GW). Drop-outs were not observed but further testing is necessary for a full evaluation. Overall, our data suggest that paternity testing with STRs and NGS is a promising approach for non-invasive prenatal paternity testing.

Establishing traceability to NIST SRM 2391c: PCR-Based DNA Profiling Standard

The NIST Standard Reference Material (SRM) 2391c: PCR-Based DNA Profiling Standard was updated in April 2015 to contain new information relevant to the forensic community. Previously, there were certified genotypes for 24 autosomal STR markers plus Amelogenin and 17 Y-STR markers. Due to the increase in markers present in larger commercial autosomal STR and Y-STR multiplex kits recently released, there is a need to add certified types for these new markers for each component of SRM 2391c (Components A–F). The updated Certificate of Analysis has certified values for 1 additional autosomal STR marker (D6S1043) and 12 additional Y-STR markers (29 total). Also, the number of STR multiplex assays tested increased from 24 to 43. Sanger sequencing was performed on Components A–C, E and F (Component D is a mixture of Components A and C) for all the certified markers to determine the STR repeat motifs and to characterize adjacent flanking regions and underlying polymorphisms (sequence, insertion-deletion, variation in complex motifs) typically not detected by fragment-based typing. SRM 2391c Components A-F can be used to establish traceability in a laboratory based on the updated certified and reference genotypes/haplotypes (information values cannot be used for NIST traceable materials). The simple steps to achieve NIST traceability with SRM 2391c will be explained.

Allele frequency of 19 autosomal STR loci in the Bai population from the southwestern region of mainland China.

The aim of this study was to investigate a 19 STR loci database using the Bai population from China. This multiplex amplification kit included 13 CODIS STR markers and six plus STR markers (D19S433, Penta E, D2S1338, Penta D, D6S1043, and D12S391) that were successfully analyzed by using 1158 DNA samples from the Bai population from the southwestern part of mainland China. These results indicate that this multiplex amplification kit may provide significant polymorphic information for kinship testing and relationship investigations.

Successful myeloid repopulation with a second hematopoietic stem cell transplant donor for relapse – AML

A 38 year old female was treated for AML M2 (FLT-3+, 3:10 translocation) with Sorafenib, AraC and Idarubcin. She subsequently received an unrelated HLA matched hematopoietic stem cell transplant (HSC TX). Post transplant monitoring (STR) indicated 100% donor in unseparated peripheral blood cells (PBL) and 100% donor in both lymphoid (CD3+) and myeloid (CD14+, 15+) compartments. At day 651 PBL unseparated was 77% donor, 100% CD3+ and 90% CD14+, 15+. Bone marrow showed unseparated 63% donor, CD3+ 98% donor and CD14+, 15+ 74% donor. Further studies indicated relapse – AML, 40% blasts in the bone marrow. The patient was given a second HSC TX from an unrelated HLA matched donor. At 60 days after the second HSC TX unsorted PBL was 100% donor, whereas the lymphoid compartment was 95% the first donor, 5% the second donor and the myeloid compartment was 100% second donor STR marker. Conclusion The second transplant was successful in repopulating the myeloid compartment after therapy for relapse – AML.

OR40 Site of HLA-B/C recombination identified by STR analysis and sequencing

Recombination events have been reported between HLA-A/C, HLA-B/DRB1 and HLA-DQB1/DPB1. To our knowledge no proven recombination event between HLA-B and -C has ever been found. In a sibling of a stem cell transplant candidate, the candidate and 3 siblings were typed and a pedigree with 4 different haplotypes could be established, but with very rare B-C segregation patterns (B*35∼C*05, B*44∼C*04). We performed STR analysis (Identifiler, ABI) to confirm sib relationship amongst the 4 individuals. If common HLA-B ∼C associations (B*35∼C*04, B*44∼C*05) were considered, 3 haplotypes could be identified and segregation patterns revealed one common haplotype for all individuals and the occurrence of a C/B recombination in one of the individuals. To investigate this possibility STR analysis was performed with informative markers D6S2838, D6S2851 (telomeric of HLA-A), D6S510, D6S265, D6S2931 (between HLA-A and -C), D6S2811, D6S2928 (between HLA-C and -B), MIB, D6S2792, D6S2787, D6S2920, D6S2894, D6S2883 (between HLA-B and -DRB1), D6S1666 and G51152 (centromeric of HLA-DQB1). The individual with the possible recombination event showed STR markers identical to one sibling with the same HLA-A and -C typing up to marker D6S2928, whereas the markers between HLA-B and -DRB1 and telomeric of DQB1 were identical to the other two siblings with identical HLA-B, -DRB1 and -DQB1 typing results. The region of the recombination was further narrowed by sequencing the area between HLA-B and D6S2928. This showed the recombination event to have taken place in the highly conserved sequence between 847 and 1228 bases after the HLA-B STOP codon. In conclusion, this is the first report confirming a recombination event between HLA-B and -C identified in a family of a stem cell transplant candidate.

Haplotype analysis of the polymorphic 40 Y-STR markers in Chinese populations

Forty Y-STR loci were analyzed in 1128 males from the following six Chinese ethnic populations: Han (n=300), Hui (n=244), Korean (n=100), Mongolian (n=100), Uighur (n=284) and Tibetan (n=100), utilizing two new generation multiplex Y-STR systems, AGCU Y24 STR and GFS Y24 STR genotyping kits, which allow for the genotyping of 24 loci from a single amplification reaction in each system. The lowest estimates of genetic diversity (below 0.5) correspond to markers DYS391 (0.441658) and DYS437 (0.496977), and the greatest diversity corresponds to markers DYS385a/b (0.969919) and DYS527a/b (0.94676). A considerable number of duplicate and off-ladder alleles were also revealed. Additionally, there were 1111 different haplotypes identified from the total 1128 samples, of which 1095 were unique. Notably, no shared haplotypes between populations were observed. The estimated overall haplotype diversity (HD) was 0.999085, and its discrimination capacity (DC) was 0.970745. An MDS plot based on the genetic distances between populations showed the genetic similarity of the southern Han population to the Northern populations of Hui, Korean, Mongolian and Uighur and a clear genetic departure of the Tibetan population from other populations. For the Y STR markers, population substructure correction was considered when calculating the rarity of the Y STR profile. However, because the haplotype based Fst values are extremely small within the present data (0.000153 with 40 Y-STRs), no substructure correction is required to estimate the rarity of a haplotype comprising 40 markers. In summary, the results of our study indicate that the 40 Y-STRs have a high level of polymorphism in Chinese ethnic groups and could therefore be a powerful tool for forensic applications and population genetic studies.

A comparative study of insertion/deletion polymorphisms applied among Southwest, South and Northwest Chinese populations using Investigator® DIPplex

The low mutation rates, short amplicon sizes and length variation characteristics of biallelic insertion/deletion polymorphisms (INDELs) are features that allow combining advantages of both STR and SNP markers for genetic analysis. The Qiagen Investigator DIPplex kit contains 30 biallelic autosomal INDELs plus Amelogenin for forensic personal identification and parentage testing. This study compares general performances, forensic and population data collected in 7 Chinese ethnic populations (Han, Tibetan, Yi, Zhuang, Dong, Miao and Uyghur) from three areas (Southwest, South and Northwest) of China by DIPplex kit. There were no significant departures from Hardy–Weinberg equilibrium (HWE) of the 30 markers in all the tested populations after Bonferroni correction. The combined matching probabilities ranged from 1.4273×10−10 for the Dong minority to 1.2817×10−12 for the Uyghur minority. The combined power of paternity exclusion was from 0.9854 to 0.9968. All pairwise genetic differences (FST) between the seven ethnic groups were less than 0.05, and the results of STRUCTURE analysis did not indicate the presence of substructures suggesting genetic similarity among the populations in these three areas. The DIPplex kit can be used as a good additional tool for forensic personal identification in these three areas of China.

Strong founder effect of p.P240L in CDH23 in Koreans and its significant contribution to severe-to-profound nonsyndromic hearing loss in a Korean pediatric population.

BackgroundDespite the prevalence of CDH23 mutations in East Asians, its large size hinders investigation. The pathologic mutation p.P240L in CDH23 is common in East Asians. However, whether this mutation represents a common founder or a mutational hot spot is unclear. The prevalence of CDH23 mutations with prelingual severe-to-profound sporadic or autosomal recessive sensorineural hearing loss (arSNHL) is unknown in Koreans.MethodsFrom September 2010 to October 2014, children with severe-to-profound sporadic or arSNHL without phenotypic markers, and their families, were tested for mutations in connexins GJB2, GJB6 and GJB3. Sanger sequencing of CDH23 p.P240L was performed on connexin-negative samples without enlarged vestibular aqueducts (EVA), followed by targeted resequencing of 129 deafness genes, including CDH23, unless p.P240L homozygotes were detected in the first screening. Four p.P240L-allele-linked STR markers were genotyped in 40 normal-hearing control subjects, and the p.P240L carriers in the hearing-impaired cohort, to identify the haplotypes.ResultsFour (3.1 %) of 128 children carried two CDH23 mutant alleles, and SLC26A4 and GJB2 accounted for 18.0 and 17.2 %, respectively. All four children showed profound nonsyndromic SNHL with minimal residual hearing. Interestingly, all had at least one p.P240L mutant allele. Analysis of p.P240L-linked STR markers in these children and other postlingual hearing-impaired adults carrying p.P240L revealed that p.P240L was mainly carried on a single haplotype.Conclusionsp.P240L contributed significantly to Korean pediatric severe arSNHL with a strong founder effect, with implications for future phylogenetic studies. Screening for p.P240L as a first step in GJB2-negative arSNHL Koreans without EVA is recommended.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0624-8) contains supplementary material, which is available to authorized users.

La identificación genética como herramienta en la investigación de adopciones irregulares y sustracción de recién nacidos en España: experiencia del Instituto Nacional de Toxicología y Ciencias Forenses (Departamento de Barcelona)

IntroductionCases of irregular adoptions and abduction of newborns in Spain have had got a great social impact which has led to regulatory reforms and the implementation of scientific guidelines and recommendations to help their investigation. The objective of the study was the genetic identification of the samples submitted by kinship studies. Materials and methodsIn the period between the year 2011 and 2014, the Barcelona Department of the National Institute of Toxicology and Forensic Sciences,received a total of 58 cases on which it was judicially required to carry out a study of genetic identification. Exhumed bones or clinical samples were analyzed. After previous study (anthropological, anatomo-pathological), the extraction of genetic material and its subsequent analysis were carried out. ResultsThe quality and the quantity of recovered DNA allowed carrying out genetic analysis in 67.2% of cases, compared to 32.8%, where it was not possible. The data show that for the majority ofthe cases studied (87.2%) was established a positive relationship, being minority the cases of exclusion (12.8%). The likelihood ratio (LR) obtained, ranged from 1.75×104-5.95×1017, and the STR markers studied from 8 to 21. ConclusionsExperience and data show the importance of gathering information, the history related to the caseand the importance of the anthropological study prior to genetic analysis. This information allows orienting the genetic studies and, in some cases, it helps to interpret the results. Also, the characteristics of this type of samples require establishing a quality protocol that ensures the authenticity of the final results.

Targeted gene capture and massively parallel sequencing identify TMC1 as the causative gene in a six-generation Chinese family with autosomal dominant hearing loss.

Hereditary nonsyndromic hearing loss is extremely heterogeneous. Mutations in the transmembrane channel-like gene1 (TMC1) are known to cause autosomal dominant and recessive forms of nonsyndromic hearing loss linked to the loci of DFNA36 and DFNB7/11, respectively. We characterized a six-generation Chinese family (5315) with progressive, postlingual autosomal dominant nonsyndromic hearing loss (ADNSHL). By combining targeted capture of 82 known deafness genes, next-generation sequencing and bioinformatic analysis, we identified TMC1 c.1714G>A (p. D572N) as the disease-causing mutation. This mutation co-segregated with hearing loss in other family members and was not detected in 308 normal controls. In order to determine the prevalence of TMC1 c.1714G>A in Chinese ADNSHL families, we used DNA samples from 67 ADNSHL families with sloping audiogram and identified two families carry this mutation. To determine whether it arose from a common ancestor, we analyzed nine STR markers. Our results indicated that TMC1 c.1714G>A (p.D572N) account for about 4.4% (3/68) of ADNSHL in the Chinese population.

Autosomal SNPs in different forensic applications

The aim of this work was to test a 52 SNP-plex in our laboratory in order to apply it in casework cases, namely in kinship involving relatives of the true father leading to low Parentage Index (PI) values, and in unusual cases such as paraffin-embedded tissue samples or in other cases involving severely degraded samples related to some identification cases. For this, a standard DNA was used, artificially degraded by digestion with DNase I and by different UV exposure times. STR previously-analysed cases were revisited and studied. All the samples were typed for the SNP for ID 52plex using the SNaPshotTM assay. In all the cases, a SNP profile was achieved. In two cases involving paraffin-embedded tissue samples, it was only possible to obtain results for 48% (11 out of 23) and 61% (14 out of 23) STR and mini-STR markers, respectively. Concerning SNPs, results were possible for all the SNPs (52) in one of the cases and for 50 SNPs (96%) in the other. The PI or LR calculated values suffered a minimum of a fourfold increment. Autosomal SNPs can be used as additional markers to the profiling of ‘difficult’ cases that emerge in the routine of many laboratories.

A GENETIC ANALYSIS OF HUMAN REMAINS FROM AK-ALAKHA-3 BURIAL MOUND 1, GORNY ALTAI

A genetic analysis of human remains from burial 1 in mound 1 at Ak-Alakha-3, Gorny Altai, focused on mitochondrial DNA, sex markers, and autosomal hypervariable STR markers. Variants of mtDNA extracted from the remains of an adult individual and a child fall into Eastern Eurasian haplogroups A4 and C, respectively, which are common in modern and prehistoric populations of Gorny Altai and the adjacent regions of southern Siberia and Central Asia. These variants must be considered autochthonous in the gene pool of the Early Iron Age Altai, and were shared by otherwise dissimilar populations of that region in the Scythian Age. The adult individual was shown to be male, and the child was a girl. The results corroborate the efficiency of aDNA testing using the well preserved cancellous bone samples.

Homozygosity mapping reveals novel and known mutations in Pakistani families with inherited retinal dystrophies.

Inherited retinal dystrophies are phenotypically and genetically heterogeneous. This extensive heterogeneity poses a challenge when performing molecular diagnosis of patients, especially in developing countries. In this study, we applied homozygosity mapping as a tool to reduce the complexity given by genetic heterogeneity and identify disease-causing variants in consanguineous Pakistani pedigrees. DNA samples from eight families with autosomal recessive retinal dystrophies were subjected to genome wide homozygosity mapping (seven by SNP arrays and one by STR markers) and genes comprised within the detected homozygous regions were analyzed by Sanger sequencing. All families displayed consistent autozygous genomic regions. Sequence analysis of candidate genes identified four previously-reported mutations in CNGB3, CNGA3, RHO, and PDE6A, as well as three novel mutations: c.2656C > T (p.L886F) in RPGRIP1, c.991G > C (p.G331R) in CNGA3, and c.413-1G > A (IVS6-1G > A) in CNGB1. This latter mutation impacted pre-mRNA splicing of CNGB1 by creating a -1 frameshift leading to a premature termination codon. In addition to better delineating the genetic landscape of inherited retinal dystrophies in Pakistan, our data confirm that combining homozygosity mapping and candidate gene sequencing is a powerful approach for mutation identification in populations where consanguineous unions are common.

Analysis of 16 autosomal STRs and 17 Y-STRs in an indigenous Maya population from Guatemala.

The aim of this study was to contribute new data on autosomal STR and Y-STR markers of the Mayas from Guatemala in order to improve available databases of forensic interest. We analyzed 16 autosomal STR markers in a population sample of 155 indigenous Maya and 17 Y-chromosomal STR markers in the 100 males of the sample. Deviations from Hardy-Weinberg equilibrium and linkage disequilibrium between autosomal STR markers were not observed at any loci. The combined power of exclusion was estimated as 99.9991% and the combined power of discrimination was >99.999999999999%. Haplotype diversity of Y-STRs was calculated as 0.9984 ± 0.0018 and analysis of pairwise genetic distances (Rst) supported the Native American background of the population.

Genetic variability of X-linked STR markers in Siberian populations

X-chromosome microsatellite markers are a convenient tool for studying genetic variability in human populations and DNA identification, especially during the decreased informativeness of autosomal markers. The results of genetic analysis of Siberian populations by ten X-linked microsatellite markers (DXS8378, GATA172D05, DXS7132, DXS9898, DXS7423, DXS8377, DXS101, DXS6809, DXS6789, and HPRTB) are presented. The allele frequencies, criminalistics parameters, and genetic relations between populations were calculated. The average level of expected heterozygosity (He) was 0.73 in populations. The total level of genetic differentiation in ten populations was relatively low (Fst = 0.031) compared to the level determined by autosomal and Y chromosome markers. The high probability of the establishment of differences (PD) between two unrelated individuals using a ten X-STR marker system was demonstrated. The average PD value in the panel of ten X chromosome microsatellite markers was 0.9999999997 in women and 0.999998 in men. The total level of genetic differentiation in a pool of ten populations is 0.03186.

Variation of X-chromosomal microsatellites in Belarus within the context of their genetic diversity in Europe

More and more X-STR data are becoming available for worldwide human populations for forensic and anthropological investigations, but the European datasets analysed so far represent mainly the central, northern, western and southern part of the continent with populations of Eastern Europe being practically uninvestigated. In the present study, we assessed genetic variation and linkage disequilibrium of 19 X-chromosomal STR markers (DXS7132, DXS7133, DXS7423, DXS7424, DXS8377, DXS8378, DXS9895, DXS10074, DXS10075, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10147, DXS10148, GATA172D05, HPRTB) in four regional populations of an Eastern European state of Belarus, including 12 loci incorporated in the Argus X-12 kit. Our results revealed cumulative power of discrimination of the tested X-STR loci to amount to 0.999999999999996 and 0.999999997 in females and males, respectively. Analysis of molecular variance demonstrated regional stratification within the country, excluding the use of a common X-STR database for Belarus in forensic casework. However, development of a separate X-STR database for the northwestern part of the country or exclusion of four loci displaying regional differences from the dataset were shown to eliminate the observed geographic substructure among Belarusians. Comparison of the Belarusian genotypes with X-STR data from other European populations disclosed a geography-driven northeast-southwest gradient extending from Belarus and Finland to Iberia and Italy. This study is the first extensive report on variation of X-STR markers in populations from Eastern Europe and the first comprehensive analysis of diversity of X-chromosomal microsatellites in Europe.