Population Genetic Analysis of 12 X-STRs in a Bahraini Population Sample

Introduction

The Investigator Argus X-12 QS Kit (Qiagen, Germany) is a multiplex solution that amplifies 12 segments of DNA located in the chromosomal X short tandem repeats (X-STRs). This solution is used for human identification, paternity testing, forensic cases and population genetic studies [1]. X-STR markers are very informative for some cases where autosomal STRs fail to provide crucial answers such as in kinship testing involving female offspring sharing the same father [2]. In this kit, the 12 X-STR markers are clustered into four distinct linkage groups (LG), each cluster includes three markers: LG1 (DX8378-DXS10135- DXS10148), LG2 (DXS7132-DXS100740-DXS10079), LG3 (DXS10101-DXS10103-HPRTB), LG4 (DXS7423-DXS10134- DXS10146) [3]. The dependency between markers is known as linkage and is the physical proximity of two loci on the same chromosome [2].

To date, there are few population genetic studies conducted on the Kingdom of Bahrain as well as in the Arabian Peninsula region. Knowledge of any such structure is crucial in the interpretation of DNA-based forensic evidence and in the construction of applicable databases. Recent studies were done using autosomal STR and YSTR kits [4, 5].

Kingdom of Bahrain is a small archipelago located in the Arabian Gulf with a total landmass of 760 square kilometers [6] consisting of 33 islands, only the five largest are inhabited. These islands are consisting of Bahrain, Muharraq, Umm and Nasan and Sitra. To the southeast of Bahrain is the State of Qatar, and to its west stands the Kingdom of Saudi Arabia, with which it is connected by a 25-kilometer causeway. To the north and east of Bahrain is the Islamic Republic of Iran [7].

Because of the geographical location of Bahrain, the diversity of the population had been affected which is generally divided into four main ethnic groups: Arabs, Baharna and Persians (Huwala and Ajam) [8, 9, 10]. This geographical and social association might be anticipated to have an effect on the patterns of the genetic variations [11]. Currently, the geographical distribution of Bahrain is included into 4 governorates: Northern, Muharraq, Capital and Southern Governorates.

This present study is to genetically characterize the Bahraini population, using Investigator Argus X-12 QS Kit (Qiagen, Germany). We present a database of 12 X-STRs allele and haplotype frequencies for Bahraini population sample including the high combined forensic statistical parameters of these loci, and collectively as haplotypes, illustrate that they are informative and discriminatory. The comparison with worldwide populations demonstrate that they generally reflect biogeography and historical relationships.

Twelve X-STRs were studied to characterize different genetic population and forensic parameters in 156 Bahraini males. These includes the following primers to co-amplify the Amelogenin (AM) used for gender determination, DXS7132, DXS7423, DXS8378, DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148 and HPRTB which have been proven to offer consistent DNA typing results and boost the power of discrimination (PD). Furthermore, the autosomal STR marker D21S11 is included as a reliable marker for common database kits and prevents sample mixup and was excluded from the genetic analysis for this study. In addition, the Kit Primer comprises of an internal PCR control (Quality Sensor QS1) to provide support about the efficacy of the PCR. The Quality Sensor is amplified along with the polymorphic X-STRs’ markers and was excluded from all the genetic analysis for this study.

Results

Allele Frequencies, Forensic Parameters and Efficiency: As for the allele frequency score, some alleles show very high frequencies in the Bahrain population such as allele 14 in DXS7423 with highest frequency of 0.462, followed by allele 19 in DXS10103 with frequency of 0.447 (Tables 1-4). The least frequent was 0.0064 for 12 different alleles. The probability that two randomly chosen person have the same unspecified genotype at a locus is the sum squares of the frequencies of all genotypes at that locus. The full set of Bahrain data is available in Table S1. No shared profiles were observed.

In this studied population, number of alleles per locus (Nall) was ranged from 5 for marker DXS8378 to 27 for DXS10135 with an average Nall per locus with 13.66, and a total number of alleles observed was 164. The most polymorphic locus was DXS10135 (Tables 1-4).

The highest gene diversity (GD) was observed for locus DXS10135 with 0.9467 while the smallest observed was for locus DXS7423 with 0.6693 (Tables 1-4).

Based on allele frequencies, we further determined the statistical parameters of forensic interest. Generally, the polymorphism degree of a specific locus can be measured by the Polymorphism Information Content (PIC) which indicates the degree of genetic polymorphism. We have found out that PIC values for all STR loci were highly informative (PIC≥0.6) with an average of 78.9% (Tables 1-4). PM was ranged from 0.0594 for DXS10135 to 0.3403 for DXS7423.

*N: Number of samples; PIC: Polymorphism information content; h: Homozygotie; HET: Heterozygotie; PE: Power of Exclusion; PD: Power of discrimination; cMEC: Combined Mean paternity exclusion change The PM was ranged from 0.0594 for DXS10135 to 0.3403 for DXS7423. The power of discrimination (PD) explains level of discriminating between members. As higher the discriminating power of a locus, the more efficient it can be used to discriminate between members. DXS10135 showed the greatest (PD) in Bahraini population with value of 0.9406, whereas DXS7423 gave the lowest value of (PD) with only 0.6597. The average (PD) of the tested loci was 0.815. The combined PD (CPD) and combined MP (CMP) for all the 12 X-STR loci were 99.99999997% and 3.02583E- 10 respectively. The combined values of each forensic parameter, cPDM, cMEC Krüger, cMEC Kishida, and cMEC Desmarais as well as cMEC Desmarais Duo, were 0.9999983, 0.9999979, 0.9999939, 0.9999996 and 0.9999514

respectively (Tables 1-4). The high values of the above- mentioned parameters indicated the usefulness of using the 12-XTRs markers included in the Investigator Argus X-12 QS Kit as a discrimination tool in complement with autosomal STRs [4] and can be utilized for genetic characterization of the Bahraini population to differentiate between individuals for forensic and kinship purposes.

Linkage Disequilibrium Analysis: As shown in Table 5, the study showed no significant deviation from linkage disequilibrium (LD) between pairwise STR loci after Bonferroni’s correction in Bahraini population except when plotting the following loci; DXS10074 - DXS10135, DXS10148 - DXS10148, DXS10134 - DXS10148, DXS7132 - DXS10146, DXS7132 - DXS7132 and DXS10103 – HPRTB. The highest pairwise LD was 0.999 when plotting DXS10101 with DXS10148 and also when plotting DXS10103 with DXS10146 and the lowest pairwise LD was 3.62E-07 when plotting DXS10148 with DXS10148.

The significance in LD was obtained between different loci in different LGs and within same LGs.

Haplotype Allele Frequencies: Haplotype allele frequencies were determined using the four LG clusters as shown in Tables 6-9. The numbers of observed haplotypes in each of the 4 linkage groups-LG1, LG2, LG3, LG4- were 148, 156, 149 and 153, respectively, while the Haplotype Diversity (HD) values were equal to 1.0000. The three most common haplotype for LG1 was 11-21-25.1 displaying 1.99% of haplotype frequency, in LG2 three sets of haplotypes 14-17-19, 14-7-20 and 15-8-19 were observed each with a frequency of 2.56 % LG3 presented haplotypes 29.2-19-12 4.46% and LG4 14-33- 29 and 15-37-27 with haplotype frequency 2.63%.

As for LG1, most of haplotype frequencies counted as 1 or 2 which gave very diverse combinations of haplotypes. Unique haplotypes, which were observed once, accounted for 55.9% of all LGs haplotype observed (339/606).

Rare Variants, Off-ladder and Null Alleles: Several cases showed off ladder (OL) in various loci; two allelic ladder variants were detected at the DXS10146; Sample#12 indicated OL in 223.05 bp and sample#34 showed OL in 243.87 bp. Four allelic ladder variants were detected at DXS10148; Sample#29 showed OL in 312.62 bp, sample#49 and sample#50 showed OL in 312.57, and sample#123 showed OL in 280.19 bp. One allelic ladder variants were detected with OL in DXS10134; Sample#72 showed OL with 325.61 bp. Null alleles were also observed in two loci; DXS10148 in sample#105 and in DXS10103 showed drop out allele in sample#135 and sample#119 (Figure 1).

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High frequent cases of non-standard ladders (OL) and null alleles were at DXS10148, which gave an indication that the Investigator Argus X-12 QS kit provided standard ladder which lacked significant coverage of bins at DXS10148 as it was confirmed in previous literature [17].

More than half of the samples (103/156) showed 0.1 variant in DXS10148 and 18 samples gave the same variant in DXS10135.

More than half of the samples (102/156) also showed 0.2 variant in DXS10101, 39 samples in DXS10146, one sample in DXS10134 and one in DXS10148. As for 0.3 variant; 9 samples were detected in DXS10134 and 3 samples in DXS10148.

Interpopulation Diversity: To measure the diversity between Bahraini population and other populations previously reported, we have constructed the phylogenetic tree from allelic frequencies data by using the neighbor- joining (NJ) method via MEGA X: Molecular Evolutionary Genetics Analysis (Figure 2).

We have used 9 populations including: Saudi [15], Filipino [16], Emiratis [17], Bengali [18], Egyptian [19], Turkish [20], Indian [21], Algerian [22] and Jewish [23]. Fst and p-values for allele frequency distribution between Bahraini population and the published groups are shown in (Table 10). It is shown that Bahraini and Emirati populations shared the most genetic relatedness than other populations, as the Saudi population considers being the most geographically close population, however gave more genetic relatedness with Egyptian population than with Bahraini population. The rest of populations stood distant of genetic association with the Bahraini population. We have also constructed the MDS plot using IBM SPSS Statistics v21.0 Software, and it gave correlating results with the phylogenetic tree. As Bahraini, Emirati and Algerian populations gave the same clusters and Saudi, Egyptian and Jewish in another cluster (Figure 3).

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Discussion

The observed deviation from LD (neglecting the Bonferroni’s correction) could be a result of the high diversity of the Bahraini population or caused by high polymorphism at the same loci investigated loci. This observation are likely to reflect the high level of inbreeding with consanguinity rates in Bahrain, with intra-familial unions accounting for 20–50% of all marriages compared to other Arab countries [27]. The PD in correlation with PM supports the high degree of polymorphism between Bahraini individuals.

As shown the significance of LD provided of loci in different LGs, suggesting of using individual allele frequency as well as haplotype allele frequencies for population database and likelihood studies.

We have compared Bahraini population data with other populations according to the available data using the accessible loci (Table S2).

It is shown that the Bahraini population shares similar results with the study conducted of Emirati populations using the X-STRs loci. Allele 14 in locus DXS7423 scored the highest frequency for Bahraini, Emirati, Egyptian and Indians populations [17, 19, 21] whereas allele 15 in same locus were the highest frequency for Filipino and Algerian populations [16, 22]. Turkish population gave the highest allele frequency in allele 13 (HPRTB) [20]. Jewish and the Saudi populations shared the highest frequency with allele 19 (DXS10103) [15, 23]. Bengali population showed the highest allele frequency in allele 11(DXS8378) [18].

Regarding the Interpopulation diversity, the phylogenetic tree was constructed based upon the data from the nine populations which were consistent with other population data from the region based upon the Fst values obtained. The obtained Fst value of Bahrain is <0.0000 which is less than the recommended value for casework statistics of Fst < 0.01 [28].

As shown, Bahraini and Emirati populations were more genetically related in terms of phylogenetic tree and MDS plot in contrary of Saudi population which was shown in previous papers published being more genetically related to Bahrain [4, 5]. This can be explained by studying the origins of the mothers of participants as it wildly affects the X-STRs results.

Once more studies of Arab populations in the region become accessible, it may be more probable to develop a greater understanding of the genetic associations between the different populations for the Arabian Peninsula. Further linkage studies must be conducted to determine if the loci are physically linked.

Conclusion

In conclusion, this is first study to report the allele frequencies and forensic statistical parameters of the X chromosomal STRs included in the Investigator Argus X-12 QS Kit in a sample population of Bahrain. X-STRs panels can be used for some cases for forensics investigations such as human identification and paternity testing.

It is shown that X-STRs included in the Investigator Argus X-12 kit can be utilized for forensic practice in Bahraini population. Our results demonstrate the importance of analyzing diverse populations using X-STRs markers.

Conflict of Interest

The authors declare that they have no conflict of interest

Acknowledgment

We would like to thank the authorities in General Directorate of Criminal Investigation and forensic Science in Bahrain, namely Mr. Abdulaziz Mayoof Alrumaihi, Mr. Raed Ali Almaeeli and Mr. Mohammed Abdulla Ghayyath for allowing us to utilize the Bahrain forensic Science Laboratory. Also, many thanks to Sabah Nazir and Meshael Ahmed Alqerainees for their technical support. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.