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International Journal of Forensic Sciences Research Article 11 min read

X-STR Allele and Linkage Haplogroup Frequencies in the Lebanese Population and the Potential of X-STR Polymorphism in Forensic Casework

Obeid M#, El Andari A#, Outhman H and Mansour I*
* Corresponding author
ISSN: 2573-1734  10.23880/ijfsc-16000145  Received: October 24, 2018  Published: November 05, 2018
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Keywords
X-STR Allele Frequency Linkage Group Lebanese Population
Abstract

When autosomal STR profiling fails to supply satisfactory outcome in human identification and/or in forensic casework, scientists may use Y-STR and/or X-STR profiling. However, representative X-STR allele and haplogroup frequencies should be established in the studied population for proper use of X-STR profiles. In this study, 507 non-related males and females representative of the Lebanese population as to the religious and geographic distributions were tested to establish the X-STR allele frequencies. 500 non-related males were tested to establish the X-STR haplogroup frequencies. DNA was amplified using Investigator™ Argus X-12 multiplex PCR system (Qiagen). Results were analyzed using in-house software (FIMS), Arlequin v3.5 software, MEGASTAT software and an online software: http://www.chrx-str.org. System DXS10135 showed to be the most informative while system DXS8378 was the least informative. The combined discrimination power (CDP) in both males and females was equal to one. The combined mean exclusion chance (CMEC) was 0.999999548 in duo paternity cases and 0.999999998 in trio paternity cases. The haplotype assessment showed that all 500 tested haplotypes were unique. The haplogroups assessment using the Investigator™ Argus X-12 kit, which determines four haplogroups named haplogroup one, two, three and four, showed high number of haplogroups 329, 194, 185 and 257 respectively, which reflects a relatively high level of X-STR polymorphism in the Lebanese population compared to Y-STR. The most frequent linkage group showed a relatively low frequency of 0.048. However, when compared to the most frequent linkage group in the German population (0.037), it showed a higher frequency, Results indicate high potentials in the field of forensic science and mandate the use of X-STR analysis for complex cases based on their ability to complement autosomal and Y-STR analysis.

Introduction

Many polymorphic DNA loci have been utilized for PCR-based typing, including variable number of tandem repeat (VNTR) markers and short tandem repeat (STR) loci [1, 2]. The latter are tandem repeats of 2-6 bp which have become a wide-spread routine in human identification as well as forensic case work and kinship testing owing to their high individualization power and practicality [3, 4, 5].

However, sometimes autosomal STRs may show microsatellite mutations (gain or loss of repeats in the alleles) at one or more loci. Knowledge about mutation rates and the mutational process of STRs used in forensic analysis is crucial for the correct interpretation of resulting genetic profiles. Moreover, when there are one or two alleles mismatch, data from other DNA markers become essential to resolve the relatedness between the parent and offspring especially in paternity deficient cases. The most widely used markers in combination with the autosomal STR in such aberrant situations are mtDNA and Y-STR to resolve maternity and paternity disputes, respectively [6].

Although mitochondrial DNA is highly polymorphic, yet the mitochondria DNA-based assay is time consuming. That is why, Typing of X chromosomal STRs (X-STRs) could be highly useful for establishing paternity when the child is a female because fathers transmit their X-STRs to all their daughters.

X-chromosomal short tandem repeats (STRs) have a wide range of applications such as solving complex kinship cases and deficiency paternity cases especially when the disputed child is a female [7, 8, 9]. The utility of such loci comes from the fact that males are hemizygous for all X chromosomal markers reveals their haplotypes directly from STR typing. In other words, men transmit to his daughters one X-chromosome they received from their mothers. Therefore, in the absence of the father, paternal grandmother and uncles can be used to infer the father’s profile [10]. Moreover, in pedigrees with proved kinship relations ChrX haplotypes can also be determined for females in many instances. True daughters carry always the whole paternal X-chromosome (ChrX) and therefore both of the daughters ChrX haplotypes can also be completely recognized when a father and his daughter are submitted to a jointed typing [11, 12]. Other cases include when the alleged father is related to the true biological father, and the exclusion power of the genetic systems is greatly diminished, may be of special interest [13]. In population and forensic genetics, many studies have also focused on the analysis of X-chromosome short tandem repeats (X-STRs) based on the comparison with first- or second-degree relatives and on the collection of frequency and forensic efficiency information [14, 15, 16, 17, 18, 19].

So far, there are a reduced number of population genetic studies regarding X-STR markers. Lebanon for example, has just recently developed an autosomal and Y- STR allele frequency database [20, 21]. Lebanon is a small country of around 4.5 million. Moreover, practices such as endogamy and consanguinity have reached high rates of 36% and 88% respectively [22, 23]. Results of these databases have shown so far that there might be an effect of consanguinity and endogamy on these population and subpopulation genetics. Such data are not yet available regarding X-STR analysis for the Lebanese population, and it would be highly informative to assess these frequencies where consanguinity and endogamy are widely spread practices. Moreover, in order to increase the knowledge and applicability of X-STRs in the forensic genetics field and before a new locus can be introduced in the forensic current practices, further population genetic database for the relevant population must be established in order to evaluate its effectiveness.

Materials and Methods

Population

EDTA blood tubes or buccal swabs (from right cheek, left cheek and tongue) from 507 unrelated healthy volunteers were collected with informed consent for X- STR allele frequency assessment in the Lebanese population. In addition, 99 unrelated male individuals were sampled to reach 502 samples for the haplogroup studies. The donors were selected based on religious and geographic distribution as described by Ansar, et al. [20].

DNA Extraction and Quantitation

DNA was extracted from whole blood leukocytes using the salting out method and from buccal swabs using a modified phenol-chloroform-isoamyl alcohol method. DNA quantification was performed using Nanodrop 2000C spectrophotometer equipment (Thermo Fisher

Scientific Inc.) and diluted accordingly to approximately 0.5 - 1 ng/μl.

PCR Amplification

Investigator Argus X-12 PCR amplification kit (Qiagen) was used to amplify 12 X-Chromosomal STR systems (DXS7132, DXS7423, DXS8378, DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, HPRTB and Amelogenin) according to the manufacturer's recommendations [24, 25].

DNA Typing

The amplified PCR products were electrophoresed on an ABI Prism 3130 Genetic Analyzer with POP-7 polymer using BTO 500 as a size standard. The data was analyzed with GeneMapper _ ID Software v4.0 (Applied Biosystems, Foster City, CA, USA).

Data Analysis

All genotypes were transferred from the Genemapper to in-house software Forensic Information and Management System (FIMS) in Addition to Arlequin v3.5 to determine the allele frequencies. ANOVA test was performed using MEGASTAT software to determine if the difference in allele frequency between males and females is statistically significant.

In order to evaluate the usefulness and potential applications of these twelve X-chromosome markers in forensic practice we performed HWE using chi-square test in female samples, and calculated GD and LD values. For the evaluation of forensic efficiency, various statistical parameters (CMECD, CMECT, PDM, PDF and CPD) were calculated using Chromosome X website software (http://www.chrx-str.org).

Results and Discussion

A Lebanese population sample of genetically unrelated individuals was studied for twelve X chromosome markers DXS7132, DXS7423, DXS8378, DXS10074, DXS10079, DXS10101, DXS10103, DXS10134, DXS10135, DXS10146, DXS10148, HPRTB and Amelogenin. Tables 1a, 1b, 1c and 1d describe the allele frequencies for each X- STR marker.

AlleleDXS10074DXS10079DXS10101DXS10103DXS7132
70.03105
80.1879
90.01471
100.00163
110.001630.01307
120.001630.12582
130.006540.28595
140.014710.33824
150.07680.014710.00490.20425
160.179740.017970.055560.03105
16.20.01471
170.233660.07190.101310.00163
180.156860.174840.21732
190.065360.276140.48529
200.011440.25490.12418
210.001630.129080.01144
220.04739
230.01307
24.20.00327
26.20.01144
270.00327
27.20.04085
280.02451
28.20.12092
290.01634

Table 1: Allele frequency for DXS10074, DXS10079, DXS10101, DXS10103 and DXS7132 Systems.

29.20.15686
300.07516
30.20.17647
310.08333
31.20.10294
320.0866
32.20.04248
330.03431
33.20.01307
340.00817

Table 2: Allele frequency for DXS10074, DXS10079, DXS10101, DXS10103 and DXS7132 Systems.

AlleleDXS10134DXS10135DXS10146DXS10148
13.30.01471
160.00327
170.00980.00654
17.10.00327
17.30.00163
180.049020.11601
18.10.0098
190.063730.02778
19.10.02451
200.063730.00817
20.10.008170.00163
210.09150.0049
21.10.01471
220.073530.01307
22.10.00980.02451
230.055560.001630.01797
23.10.008170.06863
23.30.00163
240.08660.013070.01471
24.10.12418
250.075160.04575
25.10.1781
260.001640.08660.09804
26.10.14869
270.078430.09641
27.10.10131
280.003270.058820.1781
28.10.07843
290.006550.039220.137250.00163
29.10.03595
300.003270.035950.099670.00163
30.10.00654
310.004910.021240.03268
AlleleDXS10134DXS10135DXS10146DXS10148
31.10.00163
320.014730.013070.0098
32.10.00327
32.20.00164
330.081830.008170.00654
33.20.00164
340.144030.001630.00163
350.22095
35.20.00327
360.153850.00327
370.13748
37.30.00164
380.07365
38.20.003270.00163
38.30.03764
390.00982
39.20.001630.02288
39.30.03601
400.00164
40.20.01961
40.30.02619
41.20.02124
41.30.01964
42.20.04248
42.30.01146
43.20.06373
43.30.00327
44.20.0719
45.20.01961
46.20.00654
47.10.0049

Table 3: Allele frequency for DXS7423, DXS8378 and HPRTB Systems.

Table 2,3: Allele frequency for DXS100103, DXS10135, DXS10146 and DXS10148 Systems.

AlleleDXS7423DXS8378HPRTB
80.00163
90.0098
100.393790.01307
110.383990.12745
120.199350.37255
130.035950.00980.30065
140.406860.003270.13399
150.325160.04575
160.194440.0049
170.03758

Table 4: Allele frequency for DXS7423, DXS8378 and HPRTB Systems.

After determining the allele frequencies, the gene diversity values of the tested X-STR systems in Lebanese population were calculated (Table 3). The most polymorphic X-STR marker was DXS10135 while the least polymorphic marker was DXS8378.

In order to evaluate the usefulness and potential application of these twelve X-STR systems in a forensic practice in the Lebanese population, we performed HWE (Hardy-Weinberg Equilibrium) using Chi-square test on female samples, and no significant deviations (P < 0.05) were observed at the twelve studied loci except for the DXS10148 locus Table 5 [26, 27].

X-STR LocusHWE P value
DXS101030.81649
DXS83780.78279
DXS71320.23935
DXS101340.11926
DXS100740.12904
DXS101010.59922
DXS101350.45176
DXS74230.77072
DXS101460.15791
DXS100790.60388
HPRTB0.37241
DXS101480.0097

Table 5: HWE results 12 X-STR in the Lebanese females population.

Moreover, in paternity testing the usefulness of a genetic marker is measured through the probability of finding, in randomly chosen individuals, inconsistencies with parent to child Mendelian rules of transmission. This parameter is called power of exclusion (PE), paternal exclusion chance or probability, can be defined for duos (mother not typed) or trios (random false fathers are matched against mother/child pairs) and performed both for autosomal and X-chromosomal markers (restricted to paternity testing involving daughters). PE is an a priori statistic, in the sense of not depending on the individual’s genetic data of a case, being dependent however on the estimates of genetic markers allele (or haplotype) frequencies [28, 29, 30].

Mean exclusion chance in trios involving daughters (MECT) and in father/daughter duos (MECD) as well as power of discrimination in females (PDF) and in males (PDM) were calculated using formulae according to Desmarais, et al. [31].

The polymorphism information content was calculated according to the formula stated in Botstein, et al. [32]. Unbiased estimate of expected heterozygosity was computed as described by Edwards, et al. Table 6 summarizes the above mentioned parameters [33].

PForensicr
DXS8378DXS7423DXS7132DXS10148DXS10146DXS10135DXS10074DXS10079DXS10101DXS10103DXS10134HPRTB
aramete
MECDTrio0.649250.587630.701990.858680.822870.883640.933990.631030.898770.776050.691770.88288
MECDDuo0.504360.440680.562950.763910.713540.800140.879620.485630.823590.652440.552120.79911
PDF0.863780.812810.891880.970740.955910.979160.992550.845610.983960.933990.886860.97892
PDM0.688340.657540.745170.871410.841990.893030.937550.688220.905970.803420.734330.89238
PIC0.649250.587630.701990.858680.822870.883640.933990.361030.898770.776050.691770.88288
HET0.688340.657540.745170.871410.841990.893030.937550.688220.905970.803420.734330.89238
PE0.410450.365670.501530.737460.679140.781210.872570.410280.807640.605390.483340.77988

Table 6: Forensic parameters for 12 X-STRs assessed on the Lebanese population.

PIC, polymorphic information content; HET, observed heterozygozity; PE, power of exclusion; PD, power of discrimination in females and males; MEC, mean exclusion chance calculated according to Desmarais in trios and duos. Table 6: Forensic parameters for 12 X-STRs assessed on the Lebanese population.

LebanonCPD femalesCPD malesCombined MEC Desmerais trioCombined MEC Desmerais duo
Lebanese Population110.9999999980.999999548

Table 7: X-STR Kit Assessment.

A high power of discrimination in females and males as well as high CMEC in trios and duos was encountered which highlights the efficiency of all the 12 X-STR systems combined in our Lebanese population. Haplotypes assessment revealed 500 unique male haplotype using Argus X-12 kit.

Haplogroups assessment using the Investigator™ Argus X-12 kit showed high number of haplogroups 329,

194, 185 and 257 respectively. The most frequent haplogroup showed a relatively low frequency of 0.048.

Conclusion

High level of CPD (1) and CMEC (1) for Argus X-12 kit (Qiagen) in the lebanese population, shed the light on the importance it adds to forensic case work especially when an incomplete autosomal profile is recorded. Despite males being hemizygous, high levels of CPDmales and CMEC for duos and trios were observed which is directly related to the high level of polymorphism encountered due to the presence of new alleles and micro variants.

Haplotypes and haplogroups result come to back-up our previous results and stress on the power Argus X-12 adds to forensic cases.

These facts are of high significance in the field of forensic science and mandate the use of X-STR analysis for complex cases based on their ability to conclude where autosomal and Y-STR fail to prevail.

Acknowledgment

The authors wish to thank and acknowledge: all the Lebanese volunteers for participating in this study, and Qiagen for granting and supporting this project.

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@article{obeid2018,
  title   = {X-STR Allele and Linkage Haplogroup Frequencies in the Lebanese Population and the Potential of X-STR Polymorphism in Forensic Casework},
  author  = {Obeid M#, El Andari A#, Outhman H and Mansour I},
  journal = {International Journal of Forensic Sciences},
  year    = {2018},
  volume  = {3},
  number  = {3},
  doi     = {10.23880/ijfsc-16000145}
}
Obeid M#, El Andari A#, Outhman H and Mansour I (2018). X-STR Allele and Linkage Haplogroup Frequencies in the Lebanese Population and the Potential of X-STR Polymorphism in Forensic Casework. International Journal of Forensic Sciences, 3(3). https://doi.org/10.23880/ijfsc-16000145
TY  - JOUR
TI  - X-STR Allele and Linkage Haplogroup Frequencies in the Lebanese Population and the Potential of X-STR Polymorphism in Forensic Casework
AU  - Obeid M#, El Andari A#, Outhman H and Mansour I
JO  - International Journal of Forensic Sciences
PY  - 2018
VL  - 3
IS  - 3
DO  - 10.23880/ijfsc-16000145
ER  -