Linkage and linkage disequilibrium analysis of X-STRs in Italian families

Twenty X-chromosomal short tandem repeat (STR) loci were typed in 80 families of Italian descent, composed by mother and two or more sons, for a total of 93 meiosis. The analyzed X-STR panel included six clusters of closely linked markers (each spanning<3cM): DXS10135-DXS10148-DXS8378 (Xp22); DXS7132-DXS10074-DXS10079 (Xq12); DXS6801-DXS6809-DXS6789 (Xq21); DXS7424-DXS101 (Xq22); DXS10103-HPRTB-DXS10101 (Xq26); DXS8377-DXS10134-DXS7423-DXS10146 (Xq28). Recombination fractions between pairs of markers calculated by pedigree analysis were compared with those obtained from the second-generation Rutgers combined linkage-physical map of the human genome. The observed differences confirm that recombination is not homogeneous along the X chromosome and that the conventional subdivision of X-STRs in four groups of completely unlinked markers cannot be regarded as true. Significant linkage disequilibrium was found between markers DXS6801 and DXS6809 (p=0.017). The effect on likelihood calculations of inferring haplotype frequencies from allele distributions rather than haplotype count in the relevant population was evaluated.     

Genetic diversity study on 12 X-STR loci of investigator® Argus X STR kit in Bangladeshi population

The X-chromosome short tandem repeat (STR) loci are of particular interest for solving complex kinship and paternity cases. Here, we report the genetic data from 209 unrelated Bangladeshi individuals (102 males and 107 females) that were genotyped using the 12 X-chromosomal STR markers included in the Investigator® Argus X-12 kit (Qiagen). The 12 X-STR markers are located in four linkage groups (linkage group I: DXS10135, DXS10148, and DXS8378; linkage group II: DXS7132, DXS10079, and DXS10074; linkage group III: DXS10103, HPRTB, and DXS10101; and linkage group IV: DXS10146, DXS10134, and DXS7423). Allelic frequencies of the 12 X-STR loci and haplotype frequencies of the four linkage groups were investigated. No significant difference was observed in the allele frequencies of males and females. Distributions of heterozygosity were observed from 64.5 to 92.5% among the studied 12 X STR loci. DXS10135 and DXS10101 loci were found to be most polymorphic. For all the four linkage groups, the haplotype diversity was found to be greater than 0.986. A total of 95, 73, 66, and 74 haplotypes were observed in linkage groups I, II, III, and IV, respectively. Hardy–Weinberg equilibrium tests showed no significant deviation from expected values for all 12 loci (p > 0.05). The exact test for pairwise linkage disequilibrium for the 12 loci in the male samples did not show any significant linkage disequilibrium except the DXS10103 and DXS10101 loci after the p values were corrected by Bonferroni’s correction for multiple testing (p > 0.05/66). A combined power of discrimination in male and female individuals were 0.999999998159791 and 0.999999999999993, respectively. The combined mean exclusion chance were 0.999997635 in deficiency cases, 0.999999996 in normal trio cases, and 0.999999178 in duo cases. The currently investigated Bangladeshi population showed significant differences when compared with previously reported X-STR data from other 12 populations. The results of the data analysis indicated that all the loci in the Investigator® Argus X 12 kit were fairly informative and might be useful in forensic application and kinship analysis in Bangladeshi population.

     

Population genetic study of six closely linked groups of X-STRs in a Japanese population

X chromosome STR (X-STR) polymorphisms are a useful tool in the fields of human population genetics and personal identification and are quite informative in the investigation of complex kinship or deficiency cases, especially where it is necessary to determine relationships with second-generation offspring in which the same X chromosome may have been inherited. We investigated eight X-STR systems using the Mentype Argus X-8 kit and further developed decaplex PCR for the DXS10148, DXS10161, DXS10160, DXS10159, DXS10079, DXS10075, DXS6799, DXS10102, DXS10106, and DXS10146 loci with the aim of constructing closely linked groups on the X chromosome. The studied population comprised 569 Japanese individuals (390 males and 179 females). Heterozygosity among the present 18 X-STRs showed a distribution of from 54.2% to 90.5%. We constructed six closely linked groups, each comprising three to five X-STRs: DXS10148- DXS10135-DXS8378, DXS10161- DXS10160-DXS10159, DXS7132-DXS10079-DXS10074-DXS10075-DXS981, DXS6809-DXS6789-DXS6799, DXS10102-HPRTB-DXS10101-DXS10106, and DXS8377-DXS10146-DXS10134-DXS7423. The forensic utility of these groups as haplotypes was then evaluated. Haplotype diversity values showed a distribution of from 0.9699 to 0.9959. Analysis of the present closely linked haplotypes will contribute to solving complex kinship cases involving X chromosome inheritance.     

Characterisation of the STR markers DXS10146, DXS10134 and DXS10147 located within a 79.1 kb region at Xq28

Three polymorphic X-chromosomal STR markers within a 79 kb region at Xq28 were studied and registered in the GDB as DXS10146, DXS10134 and DXS10147. These markers were molecular characterised and evaluated for their forensic usage. As a result DXS10134 was recently integrated in the commercial available test kit Mentype Argus X-8. At locus DXS10146 we found 23 alleles with PIC and HET values of 0.878 and 0.887. Locus DXS10134 showed 17 alleles with PIC and HET values of 0.844 and 0.858. At locus DXS10147 only 5 alleles with some lower PIC and HET values of 0.636 and 0.692 were found. Additionally, the already known and closely linked STR DXS7423 was included into the haplotyping and recombination studies. Testing this cluster a German population of 404 males revealed the presence of 311 haplotypes. Recombination analysis was performed in 109 father–daughter–grandson trios in which two crossing over events were observed located in the 65.8 kb region between DXS10146 and DXS10134. By using this STR complex for haplotyping in kinship testing further genetic analyses are required to establish an exact recombination rate.     

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.     

Allele and haplotype diversity of X-chromosomal STRs in Ivory Coast

Twenty-one X-chromosomal short tandem repeat (STR) loci, including the six clusters of linked markers DXS10148–DXS10135–DXS8378 (Xp22), DXS7132–DXS10079–DXS10074 (Xq12), DXS6801–DXS6809–DXS6789 (Xq21), DXS7424–DXS101 (Xq22), DXS10103–HPRTB–DXS10101 (Xq26), DXS8377–DXS10146–DXS10134–DXS7423 (Xq28) and the loci DXS6800, GATA172D05 and DXS10011 were typed in a popula3tion sample from Ivory Coast (n = 125; 51 men and 74 women). Allele and haplotype frequencies as well as linkage disequilibrium data for kinship calculations are provided. On the whole, no significant differences in the genetic variability of X-STR markers were observed between Ivorians and other sub-Saharan African populations belonging to the Niger–Kordofanian linguistic group.     

Diversity of 15 human X chromosome microsatellite loci in Polish population

X-STR analysis is a powerful tool in both phylogeny reconstruction and forensic investigation. Hereby, we provide new population data concerning 15 X-STR loci (included in commercially available typing kit Mentype Argus X-8 (Biotype AG, Dresden, Germany) (DXS10135, DXS8378, DXS7132, DXS10074, HPRTB, DXS10101, DXS10134 and DXS7423) and another seven (DXS6807, DXS9898, DXS101, DXS7424, DXS7133, DXS8377 and DXS10011) that were previously described by Poetsch et al. [1] obtained from a sample of 311 individuals from Poland and compared to the results previously obtained from other populations of European, Asian and African origin [2-4]. Numerous experiments seem to prove that X-STRs are valuable markers for human identification, kinship testing and even phylogenetic research - thus serving as a complement for autosomal microsatellites, Y-STRs and mtDNA [5-7].     

Validation and forensic application of a new 19 X-STR loci multiplex system

The Microreader™ 19X Direct ID System was a newly developed multiplex PCR kit, which could detect 19 X-chromosomal STR loci (DXS6795, DXS9907, DXS6803, GATA172D05, DXS6807, GATA31E08, DXS7423, DXS6810, DXS101, DXS9902, DXS7133, DXS6800, DXS981, DXS10162, DXS6809, DXS10135, HPRTB, GATA165B12, DXS10079) and the sex determination locus of AMEL simultaneously. Different from other X-STR multiplex PCR kits, no linkage groups are included in this system, so the likelihood ratios could be calculated without the consideration of linkage groups. In this study, PCR conditions, sensitivity, species specificity, stability, DNA mixtures, concordance, stutter, sizing precision and population studies were conducted according to the SWGDAM developmental validation guidelines. The results indicated that this new X-STRs multiplex system was an efficient and reliable detection system, which could facilitate human kinship analysis and identification testing, as a powerful supplementary to autosomal STR kits.     

Characteristics of eight X-STR loci for forensic purposes in the Chinese population

X-chromosomal short tandem repeats (ChrX STRs) loci are used for forensic practice in recent years. Considering the unique heredity characteristics of ChrX, recombination and linkage disequilibrium (LD) among ChrX STR loci vary between male and female and different populations as well. However, there is a lack of data for analysis of recombination and linkage disequilibrium on ChrX STR loci in the Chinese population. In this work, a total of 303 unrelated individuals (203 males and 100 females) in the Chinese Han population were analyzed with Mentype Argus X-8 PCR amplification kit (DXS10135-DXS8378, DXS7132-DXS10074, HPRTB-DXS10101, and DXS10134-DXS7423). The recombination and linkage disequilibrium of the eight ChrX STR loci were investigated with HapMap LD plots and software ARLEQUIN 3.1. Allele frequencies of the eight loci and further population forensic genetic parameters were obtained. Our results revealed hotspots for recombination, and there was no obvious evidence for LD among the eight loci in the Chinese population. Our work implied that single locus frequencies rather than haplotype frequencies should be applied for forensic practice in the Chinese population.     

Resolving the recombination pattern of 38 X-STRs from Chinese Han three-generation pedigrees

X-chromosomal markers have been proved as a useful tool for solving complex kinship cases due to its sex-linked inheriting feature. Among these markers, tightly linked X-STR clusters forming haplotypes are highly informative. The analysis of the haplotypes requires determination of linkage disequilibrium. In this study, genetic linkage, recombination fractions and mutation rates of 38 X-STR loci in 177 three-generation pedigrees were investigated. Genetic linkage analysis and calculation of recombination fractions were performed within each pair of markers and clusters. Then mutation rates were calculated. The results showed that, a) 22 recombination events happened within the tightly linked X-STR clusters, which span<1.0 Mb; b) significantly linked marker pairs were observed with the LOD (logarithm of the odds) scores > 2.0 (2.0104 to 54.8316); c) the average mutation rate of the 38 X-STR loci was 1.32 × 10⁻³ per meiosis in the Chinese Han population, with DXS10135 and DXS8377 presenting notably high mutation rate (6.5 × 10⁻³). Our results confirmed that meiotic recombination was not a simple function of physical distance, so that whether recombination occurred at the closely clustered X-STRs or not should be assumed cautiously considering the stability of haplotypes in inheritance process for kinship analysis. This study supplemented the existing database and laid an experimental foundation for the future study on genetic characteristics, recombination, and mutation of the X-STRs.     

Forensic parameters of 19 X-STR polymorphisms in two Chinese populations

Application of X-STRs as complements of autosomal STR application in the forensic genetics has become a tendency for kinship testing, especially in deficiency paternity cases. Recently, a novel kit of 19 X-STR loci was developed, which permitted the analysis of 19 STR in the same PCR reaction, and these markers can be clustered into seven groups for the physical linkage. The objective of this study was to evaluate the allele and haplotype diversity of 19 X-STR loci in the Uygur (n = 220) and Tibetan nationality (n = 270) and to estimate the usefulness for complex kinship analysis. In the Tibetan and Uygur populations, a total of alleles of all loci were 188 and 212, with the allele frequencies ranged from 0.0037 to 0.5593 and from 0.0045 to 0.5409, respectively. Compared with previous studies, DXS10135 was the most polymorphic locus in the two population groups, whereas the least variant locus was DXS10164 in the Uygur population and DXS7423 in the Tibetan nationality. Haplotype diversity obtained in this investigation was greater than 0.9 across all LGs. This study indicated the new kit could be used as a supplementary tool in kinship testing in China. In addition, the data sets can be used as supplementary national X-STR references to enlarge the database.

     

A reference database of forensic autosomal and gonosomal STR markers in the Tigray population of Ethiopia

Allele frequencies of 21 autosomal STR markers (AmpF/STR GlobalFiler) and haplotype frequencies of 27 Y- and 12 X-STR markers (AmpF/STR YFiler Plus and Investigator Argus X-12, respectively) were investigated in the Tigray population of Ethiopia, representing the main population group in the Tigray regional state of Ethiopia and neighboring Eritrea. For autosomal STR allele frequencies, the average random match probability in the Tigray sample was 2.1 × 10^-27. The average locus by locus F_ST distance calculated comparing autosomal STR allele frequencies from Tigray and from a broad regional reference dataset currently available for the Horn of Africa was 0.003. The Tigray male sample displayed high Y-STR diversity, with complete individualization of haplotypes using the AmpF/STR YFiler Plus panel. Analysis of molecular variance did not detect significant heterogeneity between Y-STR haplotypes observed in the present study and those previously reported in the literature for other Tigray population samples from Ethiopia and Eritrea. Study of the X-STR landscape in Tigray evidenced several distinctive features including: the molecular characterization of a novel null allele at locus DXS10146 with frequency > 1%; allele dependency between loci within linkage groups I and III; significant differences in haplotype distribution compared to other Horn of Africa populations, that should be taken into account in kinship analysis. The collected data can be used as a reference STR database by local forensic genetics services and in genetic identification procedures of victims of human trafficking in the Mediterranean Sea, which frequently involve individuals originating from the Horn of Africa.     

Genetic polymorphisms of twelve X-STRs of the investigator Argus X-12 kit and additional six X-STR centromere region loci in an Egyptian population sample

Recently, many researchers have focused on analysis of different X-chromosomal STRs as they bear the potential to efficiently complement the analysis of autosomal and Y-chromosomal STRs in solving special complex kinship deficiency cases. In the current study we examined a sample of 250 unrelated Egyptian males with the Investigator Argus X-12 kit (Qiagen GmbH, Hilden, Germany) which detects 12 X-STR markers distributed over the entire X-chromosome as four closely linked clusters. Microvariant off ladder alleles as well as null alleles have been detected in some loci. Furthermore, discordant results were observed between the Investigator Argus X-12 and the Mentype^® Argus X-8 kits (Biotype AG, Dresden, Germany). New primers were designed for loci DXS10101, DXS10146 and DXS10148 to correct the allele drop outs observed in these loci with the Investigator Argus X-12 kit. Additionally, DNA sequence analysis revealed the polymorphisms responsible for the allele drop outs. Furthermore, six additional X-STRs (DXS10161, DXS10159, DXS10162, DXS10163, DXS10164 and DXS10165) located in the centromere region at Xp11.21–Xq11.1 were examined in a single multiplex reaction. Allele and haplotype frequencies as well as different forensic statistical parameters of the 18 X-STR loci tested indicated that they are highly informative in different forensic applications in the Egyptian population. However, some modifications still need to be performed on the Investigator Argus X-12 kit before its use in forensic casework is validated.     

Allele frequency distribution of 13 X-chromosomal STR loci in Pakistani population

Short tandem repeat (STR) markers are extensively being used for human identification as well as paternity and forensic case work. X-chromosome STR (X-STR) markers are a powerful complementary system especially in deficiency paternity testing. Many X-linked microsatellites have been evaluated but further studies are required to determine population specific statistics. Here, we report allele frequencies of 13 X-linked microsatellites (DXS8378, DXS9902, DXS6810, DXS7132, DXS981, DXS6793, DXS6801, DXS6789, GATA172D05, HPRTB, GATA31E08, DXS8377, and DXS7423) in the Pakistani population. Blood samples were collected from individuals representing all major ethnic groups of the Pakistan population. A total of 5–18 alleles were observed for each locus and altogether 109 alleles for all 13 X-STR loci. Heterozygosity in females ranged from 0.524 to 0.884. No significant deviation was observed from Hardy–Weinberg equilibrium for all 13 microsatellites. In addition, there was no evidence of linkage disequilibrium in any pairs of these markers. These results strongly suggest that the X-linked microsatellites described here can potentially serve as an extension to autosomal systems currently used in parentage analysis and forensic case work. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A multiplex PCR for 4 X chromosome STR markers and population data from Beijing Han ethnic group

STR multiplex is a practical and simple method to obtain large amounts of important information in forensic and population genetic studies. The present work describes a new multiplex system that allows the simultaneous analysis of 4 X-STR markers, namely DXS9902, DXS6800, DXS6799 and DXS7132, as the tool of approach for X-STR studies. In addition, this work presents the genotyping results obtained for a sample 400 individuals (200 males and 200 females) from Beijing Han ethnic group in China.     

Population genetic evaluation of eight X-chromosomal short tandem repeat loci using Mentype Argus X-8 PCR amplification kit

The evaluation of four pairs of X-chromosomal short tandem repeats (STRs), i.e. DXS10135–DXS8378, DXS7132–DXS10074, HPRTB-DXS10101 and DXS7423–DXS10134 was carried out using the Argus X-8 Multiplex amplification kit. These eight STRs are distributed as four closely linked pairs over the entire X-chromosome (ChrX), and for practical reasons they are assigned to four linkage groups 1–4. The genetic distance within the STR pairs is assumed to be <1 cM, whereas the pair to pair space is about 50 cM or more. Here, we present single STR allele frequencies, haplotype frequencies of the respective STR pairs and further population genetic parameters of forensic interest. Most data refer to a German population, however small samples from Ghana and Japan were also investigated. Furthermore, sequencing of all STR loci displayed the presence of microvariant alleles and variations in the repeat flanking region. A total of 350 meioses investigated here revealed only one sperm DXS7132 mutation. For analysis of linkages within the STR pairs a study involving 104 female meiosis with respect to recombination events was performed. The STR panel presented here provides a powerful tool for solving complex kinship in the case that X-chromosomal lineages can be taken under investigation.     

Application of a novel multiplex polymerase chain reaction system for 12 X-chromosomal short tandem repeats to a Japanese population study

The analysis of X-chromosomal short tandem repeat (X-STR) polymorphisms has been the focus of attention in several researches, mainly due to its applicability in the investigation of complex kinship cases. A new 12 X-STR multiplex system (GATA172D05, DXS7423, DSX6809, DXS10134, DXS7132, DXS9902, DXS6789, DXS10074, DXS8378, DXS9898, DXS10147, and GATA31E08) was developed and applied to a Japanese population study. DNA samples from 290 males and 160 females were successfully analyzed using the 12 X-STR multiplex system. No mutation was detected in the kinship cases involving 34 family trios. The combined powers of discrimination of the 12 X-STR loci in males and females were 0.999997 and 0.9999999996, respectively. We conclude that the combined analysis of 12 X-STR loci using this single multiplex polymerase chain reaction system is a powerful tool in forensic DNA testing.     

Development of 11 X-STR loci typing system and genetic analysis in Tibetan and Northern Han populations from China

X-chromosomal short tandem repeats (X-STRs) loci are used for forensic practice in recent years which play increasingly important roles in some complex kinship cases. In this paper, a new multiplex polymerase chain reaction (PCR) system which can simultaneously analyze 11 X-STR markers (DXS8378, DXS6795, DXS7132, DXS6803, DXS9898, DXS6801, DXS7133, GATA165B12, HPRTB, DXS8377 and DXS7423) was developed. The samples of 1,605 (742 males and 863 females) unrelated individuals from Tibetan and Northern Han population were successfully analyzed using this multiplex system. A total of 103 alleles for all the loci were observed. Hardy–Weinberg equilibrium tests demonstrated no significant deviation from expected values (P > 0.05) for all of the 11 X-STR loci in the two studied populations. Polymorphism information contents of the loci were 0.3864–0.9013, and powers of discrimination in females of the loci were 0.6317–0.9845. There were no statistically significant differences between Tibetan and Northern Han populations in allele distribution of the 11 X-STR loci, in line with analysis of molecular variance (AMOVA) results. Our work indicates that this multiplex system is useful for forensic analysis for the two populations in China.     

Thirteen X-chromosomal short tandem repeat loci multiplex data from Taiwanese

Study results of variations in the X chromosome are useful tools in researching the genetic diversity of human populations and individual identification. We developed a 13 X chromosomal short tandem repeat (STR) multiplex system (DXS6807, DXS8378, DSX9902, DXS7132, DXS9898, DXS6809, DXS6789, DXS7424, DXS101, GATA172D05, HPRTB, DXS8377, DXS7423) amplified in one single polymerase chain reaction. DNA samples of 113 male and 108 female Taiwanese Han subjects were successfully analyzed using this 13 X-STR multiplex system. The distributions of allele frequencies were examined for independence. DXS8377, DXS101, DXS6789, and DXS6809 were found to be the most polymorphic markers in this study. High values of discrimination power and mean exclusion chance without significant evidence of association between these markers were obtained. In conclusion, this 13 X chromosomal STR multiplex system offers considerable forensic efficiency and may be useful in forensic identification casework.     

Development and characterization of a new 12-plex ChrX miniSTR system

Short tandem repeat (STR) markers are extensively used for human identification as well as paternity and forensic casework. X-chromosome STR (X-STR) markers are a powerful complementary system especially in deficiency paternity testing. This study presents the development and characterization of a new X-chromosomal short tandem repeat (STR) multiplex using short amplicon (<200 bp). A total of 366 samples from Punjabi population and 346 samples from Sindhi population were typed for 11 X-chromosomal STR markers: DXS101, DXS6789, DXS6793, DXS7132, DXS7423, DXS7424, DXS8378, DXS9902, GATA31E08, GATA172D05, and HPRTB along with sex-typing locus, amelogenin. Each marker showed a high degree of polymorphism, and the multiplex was sensitive down to 250 pg of human DNA. A total of 78 alleles were found with 5–11 alleles for each marker. The population data can be used as reference database for Sindhi and Punjabi populations.