Analysis of genetic diversity of Russian regional populations based on STR markers used in DNA identification

We conducted the first genetic analysis of a wide a range of rural Russian populations in European Russia with a panel of DNA markers commonly used in Human DNA identification. We examined a total of 647 samples from indigenous ethnic Russian populations in Arkhangelsk, Belgorod, Voronezh, Kursk, Rostov, Ryazan, and Orel regions. We employed a multiplex genotyping kit, COrDIS Plus, to genotype Short Tandem Repeat (STR) loci, which included markers officially recommended for DNA identification in the Russian Federation, the United States, and the European Union. In the course of our study, we created a database of allele frequencies, examined the distribution of alleles and genotypes in seven rural Russian populations, and analyzed the genetic relationships between these populations. We found that, although multidimensional analysis indicated a difference between the Northern Russian gene pool and the rest of the Russian European populations, a pairwise comparison using 19 STR markers among all populations did not reveal significant differences. This is in concordance with previous studies, which examined up to 12 STR markers in urban Russian populations. Therefore, the database of allele frequencies created in this study can be applied for forensic examinations and DNA identification among the ethnic Russian population over European Russia. We also noted a decrease in the levels of heterozygosity in the northern Russian population compared to southern and central Russian populations, which is consistent with trends identified previously using classical gene markers and analysis of mitochondrial DNA.     

Development and initial characterization of a HAPPY panel for mapping the X. tropicalis genome

HAPPY mapping was designed to pursue the analysis of approximately random HAPloid DNA breakage samples using the PolYmerase chain reaction for mapping genomes. In the present study, we improved the method and integrated two other molecular techniques into the process: whole genome amplification and the Sequenom SNP (single nucleotide polymorphism) genotyping assay in order to facilitate whole genome mapping of X. tropicalis. The former technique amplified enough DNA materials to genotype a large number of markers, while the latter allowed for relatively high throughput marker genotyping with multiplex assays on the HAPPY lines. A total of 58 X. tropicalis genes were genotyped on an initial panel of 383 HAPPY lines, which contributed to formation of a working panel of 146 lines. Further genotyping of 29 markers on the working panel led to construction of a HAPPY map for the X. tropicalis genome. We believe that our improved HAPPY method described in the present study has paved the way for the community to map different genomes with a simple, but powerful approach.     

Etiological heterogeneity in X-linked spastic paraplegia.

We describe a large family (K313) having 12 males affected with X chromosome-linked recessive hereditary spastic paraplegia (HSP). The disease phenotype in K313 is characterized by hyperreflexia and a spastic gait, but intelligence is normal. Carrier females have normal gait and unremarkable neurologic profiles. Eight widely spaced X-linked DNA markers were used to genotype 43 family members. In contrast to a published study of another family, in whom complete linkage of X-linked recessive HSP to distal chromosome Xq markers DXS15 and DXS52 was reported, we observed complete linkage with two DNA markers, pYNH3 and DXS17, located on the middle of the long arm of the X chromosome. These data have been combined with linkage data from a large reference panel of normal families to localize the new X-chromosome marker, pYNH3, and to provide evidence of significant locus heterogeneity between phenotypically distinct forms of X-linked recessive HSP.     

Variability and identification power of 60 X-chromosome in two native Siberian populations

Genetic diversity of 60 X-chromosome single nucleotide polymorphisms (XSNPid panel) in populations of Siberian Tatars and Tuvinians is described. A close spectrum of allele frequencies and a low level of their genetic differentiation (G_st = 0.021) is revealed. High discriminating power of the XSNPid panel in populations under study is demonstrated. The random matching probability (MP) of multilocus genotypes in males is 1.12 × 10^–18 in Siberian Tatars and 7.77 × 10^–16 in Tuvans. In females, MP is several orders of magnitude lower: 1.51 × 10^–25 in Siberian Tatars and 1.83 × 10^–23 in Tuvinians.     

X-linked dominant hypophosphatemia is closely linked to DNA markers DXS41 and DXS43 at Xp22

Summary Two families with X-linked dominant hypophosphatemia (McKusick No. ^*30780) were investigated for linkage of the disease locus with several marker genes defined by cloned, single-copy DNA sequences derived from defined regions of the X chromosome. Close linkage was found with DNA markers DXS41 (p99-6) and DXS43 (pD2) at Xp22, suggesting a location of the HPDR gene on the distal short arm of the X chromosome.     

Cross-amplification of ungulate microsatellite markers in the endemic Indian antelope or blackbuck (Antilope cervicapra) for population monitoring and conservation genetics studies in south Asia

The Indian antelope or blackbuck (Antilope cervicapra) is endemic to the Indian subcontinent, inhabiting scrublands and dry grasslands. Most of the blackbuck populations are small, isolated, and threatened by habitat fragmentation and degradation. Management of such disjunct populations requires genetic characterization, which is critical for assessing hazards of stochastic events and inbreeding. Addressing the scarcity of such information on the blackbuck, we describe a novel panel of microsatellite markers that could be used to monitor blackbuck demography and population genetic parameters using non-invasive faecal sampling. We screened microsatellites (n?=?40) that had been reported to amplify in bovid and cervid species using faecal samples of the blackbuck collected from Kaimoor Wildlife Sanctuary, Uttar Pradesh, India and its vicinities. We selected 12 markers for amplification using faecal DNA extracts (n?=?140) in three multiplex reactions. We observed a mean amplification success rate of 72.4% across loci (92.1–25.7%) with high allele diversity (mean number of alleles/locus?=?8.67?±?1.03). Mean genotyping error rates across the markers were low to moderate (allelic drop-out rate?=?0.09; false allele rate?=?0.11). The proportions of first- and second-order relatives in the study population were 0.69% and 6.21%, respectively. Based on amplification success, genotyping error rates and the probability of identity (P_ID), we suggest (i) a panel of five microsatellite markers (cumulative P_ID?=?1.24?×?10^–5) for individual identification and population monitoring and (ii) seven additional markers for conservation genetics studies. This study provides essential tools capable of augmenting blackbuck conservation strategies at the landscape level, integral to protecting the scrubland-grassland ecosystem.

     

Brazilian genetic database of chromosome X

The X chromosome is a singular source of information in population genetics, anthropological research and in forensic cases. Thus, many researchers have been interested in characterizing X chromosome markers in different populations. The Brazilian Genetic Database of Chromosome X (BGBX—Banco Genético Brasileiro do Cromossomo X) website is freely available in Portuguese and English versions and was developed with the main purpose of compiling all Brazilian population genetic data for X chromosome short tandem repeats (X-STRs) markers published in scientific journals searchable via PubMed. Furthermore, this database presents other relevant information concerning X-STRs, such as genetic and physical locations, allele structure, nomenclature, mutation rates, primers described in the literature and likelihood ratio calculation. The entire scientific community is now encouraged to submit their X-STR population genetic data to this website, available at http://www.bgbx.com.br. Regarding future prospects of BGBX, the authors intend to expand the website with data and information of X-linked insertion–deletion polymorphisms.     

Development of Multiplex Genotyping Method of Polymorphic Markers of Genes Associated with Cognitive Abilities

A developed method of multiplex genotyping of polymorphic markers of genes associated with cognitive abilities and neuropsychiatric diseases is based on multilocus PCR and MALDI-TOF mass spectrometry of DNA molecules. The frequencies of 32 single-nucleotide markers localized in 24 genes are analyzed in a sample of elderly people from the Russian population of Tomsk. The data obtained are compared with data for populations from the 1000 Genomes Project.     

X-Chromosome STR markers data in a Cabo Verde immigrant population of Lisboa

Population genetic data of 12 X chromosomal short tandem repeats markers (DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, DXS7132, DXS7423, DXS8378 and HPRTB) were analysed in 54 females and 95 males of an immigrant population from Cabo Verde living in Lisboa. The obtained results for forensic statistical parameters such as observed heterozigosity, polymorphism information content, power of discrimination and mean exclusion chance, based on single allele frequencies, reveal that this multiplex system is highly informative and can represent an important tool for genetic identification purposes in the immigrant population of Cabo Verde. Since the studied short tandem repeats genetic markers are distributed on four linkage groups, that can provide independent genotype information, we studied those groups as haploytes. The forensic efficiency parameters for the linked groups were all higher than 0.97, with linkage group I being the most polymorphic and linkage group III the less informative.     

一个先天性眼球震颤家系致病基因的初步定位

为确定一个X染色体显性遗传先天性眼球震颤家系的致病基因与X染色体的连锁关系, 选用X染色体上的DXS1214、DXS1068、DXS993、DXS8035、DXS1047、DXS8033、DXS1192和DXS1232共8个微卫星DNA标记对该家系进行基因扫描与基因分型,并利用LINKAGE等软件包对基因分型结果进行分析,探讨该家系致病基因与X染色体的连锁关系。 两点连锁分析时X染色体短臂4个基因座最大LOD值均小于-1,不支持与该家系致病基因连锁; X染色体长臂4个基因座中最大LOD值达到2,提示存在较大的连锁可能性。该家系的致病基因可初步定位于X染色体长臂,且提示Xq26-Xq28区间附近可能是先天性眼球震颤一个共同的致病基因座,但区间范围仍较大,仍须进一步选择合适的微卫星标记进行精确的定位以缩小候选基因的筛查范围。Abstract： To investigate the relationship between X chromosome and obligatory gene of a pedigree with congenital nystagmus,we used the following markers: DXS1214、DXS1068、DXS993、DXS8035、DXS1047、DXS8033、DXS1192 and DXS1232.Genome screening and genotyping were conducted in this pedigree of congenital nystagmus, and linkage analysis by LINKAGE package was used to determine the potential location. The linkage was not found on the Xp ( All LOD score <-1) but on Xq (the maximum LOD score=2). The related gene of this pedigree was located on the long arm of X chromosome. We demonstrate that Xq26-Xq28 is a common locus for CMN. It bring us closer to the identification of a gene responsible for X-linked CMN.     

Design of a multiplex PCR for genotyping 16 short tandem repeats in degraded DNA samples

The molecular genotyping of individuals and reconstruction of kinship through short and high polymorphic DNA markers, so-called short tandem repeats (STR), has become an important and efficient method in anthropology and forensic science. The here introduced experimental design describes a multiplex PCR capable of simultaneously amplifying 16 STRs and the sex determinant locus amelogenin in a short fragment lengths range from 84 bp to 275 bp. Thus, the design depends predominantly on the routines for DNA typing of historical samples with highly degraded ancient DNA. It is shown, that the newly designed multiplex PCR is suitable for successful typing of both forensic and historical material.     

Allelic Polymorphism of the Five X-Linked (CA) n Dinucleotide Repeats in Russia

A PCR-based survey of allelic polymorphism of three microsatellite markers, DXS998, DXS548, and FRAXAC1, mapped to chromosome region Xq27.3, and two microsatellite markers, DXS8091and DXS1691 located on Xq28 was carried out using a series of DNA samples obtained from 98 unrelated individuals from Russia. The number of alleles detected on electrophregrams for each marker tested was 4, 6, 4, 5, and 3, respectively. The values of heterozygosity index for the markers examined were 0.65, 0.27, 0.38, 0.70, and 0.29, respectively. The observed distribution of the allelic frequencies for each microsatellite marker examined fitted Hardy–Weinberg expectations. The values of individualization potential determined for each marker were 0.24, 0.53, 0.43, 0.12, and 0.52, respectively. In the sample tested the genotype distribution with regard to above loci was determined. The perspectives of using the analyzed allelic polymorphisms for indirect DNA diagnostics of the monogenic diseases located in this chromosome region (X-linked mental retardations, FRAXA and FRAXE) as well as for human population genetics and personal identification is discussed.     

Development of the first standardised panel of two new microsatellite multiplex PCRs for gilthead seabream (Sparus aurata L.)

The high number of multiplex PCRs developed for gilthead seabream (Sparus aurata L.) from many different microsatellite markers does not allow comparison among populations. This highlights the need for developing a reproducible panel of markers, which can be used with safety and reliability by all users. In this study, the first standardised panel of two new microsatellite multiplex PCRs was developed for this species. Primers of 138 specific microsatellites from the genetic linkage map were redesigned and evaluated according to their genetic variability, allele size range and genotyping reliability. A protocol to identify and classify genotyping errors or potential errors was proposed to assess the reliability of each marker. Two new multiplex PCRs from the best assessed markers were designed with 11 markers in each, named SMsa1 and SMsa2 (SuperMultiplex Sparus aurata). Three broodstocks (59, 47 and 98 breeders) from different Spanish companies, and a sample of 80 offspring from each one, were analysed to validate the usefulness of these multiplexes in the parental assignation. It was possible to assign each offspring to a single parent pair (100% success) using the exclusion method with SMsa1 and/or SMsa2. In each genotyped a reference sample (Ref‐sa) was used, and its DNA is available on request similar to the kits of bin set to genotype by genemapper (v.3.7) software (kit‐SMsa1 and kit‐SMsa2). This will be a robust and effective tool for pedigree analysis or characterisation of populations and will be proposed as an international panel for this species.     

Genetic variability and discriminating power of four microsatellite loci in Russian population

Allele and genotype frequencies of 4 STR loci (LPL, vWA, FES/FPS H F 13B), used in forensic medicine, were analyzed in Russian Siberian population. Genetic and molecular diversity of these polymorphic systems were characterized in comparison with US Caucasoid population. High discriminating power (PD = 0.99975) of the system of four studied STR loci was shown. Comparative analysis of genetic diversity in Russian population and Caucasoid US population revealed the significant differences between two populations and demonstrated that STR frequency data for US population should not be used for forensic expertise in Russia. The data obtained in the current investigation may be used as reference data for forensic medicine laboratories in Siberia.     

Highly Punctuated Patterns of Population Structure on the X Chromosome and Implications for African Evolutionary History

It is well known that average levels of population structure are higher on the X chromosome compared to autosomes in humans. However, there have been surprisingly few analyses on the spatial distribution of population structure along the X chromosome. With publicly available data from the HapMap Project and Perlegen Sciences, we show a strikingly punctuated pattern of X chromosome population structure. Specifically, 87% of X-linked HapMap SNPs within the top 1% of F_ST values cluster into five distinct loci. The largest of these regions spans 5.4 Mb and contains 66% of the most highly differentiated HapMap SNPs on the X chromosome. We demonstrate that the extreme clustering of highly differentiated SNPs on the X chromosome is not an artifact of ascertainment bias, nor is it specific to the populations genotyped in the HapMap Project. Rather, additional analyses and resequencing data suggest that these five regions have been substrates of recent and strong adaptive evolution. Finally, we discuss the implications that patterns of X-linked population structure have on the evolutionary history of African populations.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.     

A microarray system for genotyping 150 single nucleotide polymorphisms in the coding region of human mitochondrial DNA

We established a genotyping system for a panel of 150 SNPs in the coding regions of mitochondrial DNA based on multiplex tag-array minisequencing. We show the feasibility of this system for simultaneous identification of individuals and prediction of the geographical origin of the mitochondrial DNA population lineage of the sample donors by genotyping the panel of SNPs in 265 samples representing nine different populations from Africa, Europe, and Asia. Nearly 40,000 genotypes were produced in the study, with an overall genotyping success rate of 95% and accuracy close to 100%. The gene diversity value of the panel of 150 SNPs was 0.991, compared to 0.995 for sequencing 500 nucleotides of the hypervariable regions I and II of mtDNA. For 17 individuals with identical sequences in the hypervariable regions of mtDNA, our panel of SNPs increased the power of discrimination. We observed 144 haplotypes that correspond to previously determined mitochondrial "haplogroups," and they allowed prediction of the origin of the maternal population lineage of 97% of the analyzed samples.     

Application of Short Tandem Repeat markers in diagnosis of chromosomal aneuploidies and forensic DNA investigation in Pakistan

Purpose

Short Tandem Repeat (STR) genetic markers hold great potential in forensic investigations, molecular diagnostics and molecular genetics research. AmpFlSTR® Identifiler™ PCR amplification kit is a multiplex system for co-amplification of 15 STR markers used worldwide in forensic investigations. This study attempts to assess forensic validity of these STRs in Pakistani population and to investigate its applicability in quick and simultaneous diagnosis and tracing parental source of common chromosomal aneuploidies.

Methodology

Samples from 554 healthy Pakistani individuals from 5 different ethnicities were analyzed for forensic parameters using Identifiler STRs and 74 patients' samples with different aneuploidies were evaluated for diagnostic strengths of these markers.

Results

All STRs hold sufficient forensic applicability in Pakistani population with paternity index between 1.5 and 3.5, polymorphic information content from 0.63 to 0.87 and discrimination power ≥ 0.9 (except TPOX locus). Variation from Hardy–Weinberg equilibrium was observed at some loci reflecting selective breeding and intermarriages trend in Pakistan. Among aneuploidic samples, all trisomies were precisely detectable while aneuploidies involving sex chromosomes or missing chromosomes were not clearly detectable using Identifiler STRs. Parental origin of aneuploidy was traceable in 92.54% patients.

Conclusion

The studied STR markers are valuable tools for forensic application in Pakistan and utilizable for quick and simultaneous identification of some common trisomic conditions. Adding more sex chromosome specific STR markers can immensely increase the diagnostic and forensic potential of this system.     

High resolution genotyping of clinical Aspergillus flavus isolates from India using microsatellites

Background

Worldwide, Aspergillus flavus is the second leading cause of allergic, invasive and colonizing fungal diseases in humans. However, it is the most common species causing fungal rhinosinusitis and eye infections in tropical countries. Despite the growing challenges due to A. flavus, the molecular epidemiology of this fungus has not been well studied. We evaluated the use of microsatellites for high resolution genotyping of A. flavus from India and a possible connection between clinical presentation and genotype of the involved isolate.

Methodology/Principal Findings

A panel of nine microsatellite markers were selected from the genome of A. flavus NRRL 3357. These markers were used to type 162 clinical isolates of A. flavus. All nine markers proved to be polymorphic displaying up to 33 alleles per marker. Thirteen isolates proved to be a mixture of different genotypes. Among the 149 pure isolates, 124 different genotypes could be recognized. The discriminatory power (D) for the individual markers ranged from 0.657 to 0.954. The D value of the panel of nine markers combined was 0.997. The multiplex multicolor approach was instrumental in rapid typing of a large number of isolates. There was no correlation between genotype and the clinical presentation of the infection.

Conclusions/Significance

There is a large genotypic diversity in clinical A. flavus isolates from India. The presence of more than one genotype in clinical samples illustrates the possibility that persons may be colonized by multiple genotypes and that any isolate from a clinical specimen is not necessarily the one actually causing infection. Microsatellites are excellent typing targets for discriminating between A. flavus isolates from various origins.     

Identification of individuals by analysis of biallelic DNA markers, using PCR and solid-phase minisequencing.

We have developed a new method for forensic identification of individuals, in which a panel of biallelic DNA markers are amplified by the PCR, and the variable nucleotides are detected in the amplified DNA fragments by the solid-phase minisequencing method. A panel of 12 common polymorphic nucleotides located on different chromosomes with reported allele frequencies close to .5 were chosen for the test. The allele frequencies for most of the markers were found to be similar in the Finnish and other Caucasian populations. We also introduce a novel approach for rapid determination of the population frequencies of biallelic markers. By this approach we were able to determine the allele frequencies of the markers in the Finnish population, by quantitative analysis of three pooled DNA samples representing 3,000 individuals. The power of discrimination and exclusion of the solid-phase minisequencing typing test with 12 markers was similar to that of three VNTR markers that are routinely used in forensic analyses at our institute. The solid-phase minisequencing method was successfully applied to type paternity and forensic case samples. We also show that the quantitative nature of our method allows typing of mixed samples.