Allele Frequency Database for 21 Short Tandem Repeats in the Ugandan Population

Keywords

Allele frequency • Autosomal short tandem repeats • Ugandan population • Forensic statistics parameters • Globalfiler

Introduction

Short Tandem Repeats (STRs) are the most common genetic markers, well widespread in the human genome and have a broad range of applications in DNA profiling [1-3]. The relatively short length of STRs is critical because it makes them the most amenable to multiplex amplification by Polymerase Chain Reaction (PCR) and therefore ideal forensic markers [4]. Short tandem repeat loci’s importance as the most informative genetic markers providing high statistical capability of discrimination and individualization is widely acknowledged, documented and the data generated is currently being used in various forensic and judicial settings [5-7]. Genetic studies on Ugandan population to date are very limited and yet knowledge of any such structure is important in the interpretation of the significance of DNA-based analysis for human identification in parentage testing as well as forensic investigations [8].

Prior to the application of any DNA based identification method, it is essential to estimate the allele frequencies and forensic statistical parameters of targeted STR loci in each population in order to provide a more precise reference database for forensic investigation. Following the expansion of the Combined DNA Index System (CODIS) core loci number from 13 to 20 [9], no population studies have been conducted on the overall Ugandan population. The paucity of data on autosomal STR markers of Forensic and Paternity interest in the Ugandan population prompts this study to be carried out following the recommendations of the DNA commission of ISFG (International Society for Forensic Genetics; 2020) [10].

Genetic studies done show allele frequencies differ worldwide, some alleles are more frequent while others are absent in certain populations. For this reason, the aim of the present study is to determine allele frequencies for 21 short tandem repeat loci in the Ugandan population. The results indicate that all loci are informative, highly polymorphic and discriminative. Combined, the 21 STRs are resourceful in paternity and forensic investigations.

Materials and Methods

Sample collection and DNA extraction

Ethical approval to conduct this research was granted by the Makerere University School of Biomedical Sciences Research and Ethics committee, (Research file reference: SBS-2023-478) and Uganda National Council of Science and Technology (Research file reference: NS716ES). Blood samples were collected from 1301 unrelated consenting individuals in the four major regions of the country, namely; Central, Eastern, Western and Northern region. For each sample, the identity of the participants was dissociated from the sample collected to respect confidentiality.

DNA extraction and quantitation

Genomic DNA was extracted following standard operating procedure of extracting DNA using Prepfiler Express on Automate Express DNA extraction system [11]. Following the manufacturer’s guide [12], the quantities of extracted DNA samples were determined using Quantifiler™ Trio DNA Quantification Kit on the 7500 Real Time PCR machine.

Multiplex PCR amplification

Multiplex amplification of genomic DNA after normalisation was performed as per GlobalFiler™ and GlobalFiler™ IQC PCR Amplification Kits protocol [12] on a Proflex PCR machine.

Genotyping

The amplicons were separated by capillary electrophoresis on the ABI 3500xL genetic Analyser (Thermal Fisher Scientific) using POP-4™ polymer and LIZ-600 as internal size standard (Life Technologies) and raw data was captured with 3500 Series Data Collection software 2 (Thermal Fisher Scientific) [13,14]. GeneMapper ID-X version 1.5 software (Thermal Fisher Scientific) was used to genotype the raw data and the corresponding allelic ladder employed as per the manufacturer’s guide [15].

Quality control

The negative control and the target were checked for peaks in order to discount contamination. The sizes of the observed peaks (above 150 RFU), quality, presence or absence of interferences and possible null alleles were assessed. After successfully passing the quality control tests, these profiles were used for statistical analysis. Quality control guidelines outlined by Schneider PM [16] were followed while performing the laboratory work. Our laboratory participates in the quality control proficiency tests annually organised by Forensic Assurance Proficiency Testing Provider (www.forensicassurance.com).

Statistical analysis

Genetic diversity, forensic and population statistical parameters including allele frequency, Observed Heterozygosity (Ho), expected Heterozygosity (He), Observed Homozygosity (H obs), Power of Discrimination (PD), Polymophic Information Content (PIC), March Probability (MP), Power of Exclusion (PE), were estimated using STRAF 2.0 [17,18]. Minimum allele frequency (MAF) was computed according to the NRC recommendations as 5/2N [19]. Exact tests were carried out with the Arlequin 3.5 software [20] to check Hardy-Weinberg Expectations (HWE) by locus. Bonferroni correction Weir BS [21] was applied to the probability of HWE to determine significant deviations.

Results

Allele frequency, rare alleles, microvariant alleles and tri-allelic patterns

A total of 1301 unrelated adult individuals were successfully genotyped for the STR loci CSFIPO, D10S1248, D12S391, D13S317, D16S539, D18S51, D19S433, D1S1656, D21S11, D22S1045, D2S1338, D2S441, D3S1358, D5S818, D7S820, D8S1179, FGA, SE33, TH01, TPOX, vWA, DYS391 and the Amelogenin loci yielding 2602 gene copies. A total of 342 alleles were detected and these ranged from allele 4.2 at SE33 to allele 43.2 at FGA as showed in Table 1.

Table 1. Uganda allele frequencies database and forensic statistics parameters.

The allele frequencies obtained from 21 autosomal STR markers in the Ugandan population are indicated in Table 1 with the least allele frequency of 0.0004 for rare alleles observed at many loci except at D8S1179 and THO1 and the highest frequency of 0.3797 at allele 12 of D5S818.

The common microvariants observed were of two incomplete repeats on loci namely SE33, D19S433, FGA, D21S11, D18S51and D3S1358 though microvariants with three incomplete repeats were also observed at D1S1656, THO1, D2S441, D7S820 and FGA. Rare microvariants of one incomplete repeat were as well observed in the Ugandan population at D21S11, D12S391, D7S820 and D5S818. Rare microvariants and tri-allelic patterns were genotyped twice and the same allele calls were generated. The minimum allele frequency for the Ugandan population is 0.0019 to be used were rare alleles were observed that did not meet 5 occurances.

Tri-allelic patterns, a rare occurance, were observed at TPOX and D7S820 loci and the genotypes of the 10 individuals who displayed these patterns are shown in Table 2. Type 2 tri-allelic pattern were the only variants in this study.

Table 2. Tri-allelic patterns observed in the Ugandan population.

Population genetic indices of the Ugandan population parameters

As shown in Table 1, all loci were in Hardy-Weinberg equilibrium except at D3S1358 (p=0.0020). However, after applying the Bonferroni correction, the departures observed were not significant. The expected heterozygosity values were very close to the observed heterozygosity value with the least observed at D13S317 (Hexp.=0.7134, Hobs=0.7002) and the highest at SE33 locus (Hexp.=0.9269, Hobs=0.9131). Total heterozygosity ranged from 0.7134 at D13S317 to 0.9270 at SE33. Low values of inbreeding coefficient (Fis), a measure of genetic relatedness, in the Ugandan population ranged from -0.0229 at D10S1248 to 0.0197 at vWA were observed. Values of genetic differentiation (Fst) were low ranging from 0.00096 at D16S539 to 0.00456 at D2S441.

Genetic diversity and forensic efficiency parameters

The computed forensic metrics are shown in Table 1 with the most and least polymorphic loci being SE33 and D13S317 with PIC values of 0.9221 and 0.6657 respectively. Matching Probability (PM) values ranged from 0.010 (SE33) to 0.096 (TPOX) with the probability of obtaining a random match between individuals; Combined Match Probability (CMP) of 6.76703 × 10²⁷. All loci were highly discriminatory with high Power of Discrimination (PD) values ranging from 0.8725 at D13S317 to 0.9899 at SE33 locus.

The Ugandan population is a more diverse one based on the observed proportions of heterozygous and homozygous individuals (Power of Exclusion; PE). High PE values were associated with SE33 (PE=0.8223) and the lowest with D13S317 (PE=0.4286). Typical Paternity Index (TPI) values measuring how many times more likely that a possible father is the actual father than a randomly selected man in the population ranged from 1.6679 at D13S317 locus to 4.3953 at FGA locus.

All the 21 autosomal markers were highly informative and genetically diverse with the most informative locus in our data set being SE33 (Ho=0.9134, Hexp=0.9269, PIC=0.9221, PE=0.8223 and PD=0.9899) and the least informative locus was D13S317 (Ho=0.7002, Hexp=0.7134, PIC=0.6657, PE=0.4286 and PD=0.8725). When combined, the Combined Power of Exclusion (CPE), Combined Match Probability (CMP) and Combined Power Of Discrimination (CPD) and Combined Typical Paternity Index were 0.999980519, 1 in 6.76703 × 10²⁷, 1 and 695669937.8 respectively. The small CMP values and large CPE values support the use of these 21 autosomal markers in individual identification and discrimination within the Ugandan population.

Discussion and Data Analysis

This is the first study in the Ugandan population on the 21 STR markers with frequencies generated at all loci below 50% as shown by the predominant alleles at each locus tested. These low frequency values reflect the usefulness and validity of these loci in discriminating individuals [21].

The 21 STR markers in the Ugandan population are in Hardy-Weinberg equilibrium (p- values > 0.05) since no deviation was significant after applying Benferroni correction (0.05/21=0.0024) as is in previous studies from other populations such as Arabian populations from Morocco and Syria [22], African-American, US Caucasian and US Hispanic [23], NorthWest Spain (Gacilia) [24], Angola [25], Tigray population of Ethiopia [26] and Nigeria [27] which showed no deviations from Hardy-Weinberg equilibrium from all tested loci. Observed heterozygosity values were very close to the expected heterozygosity values at all loci indicating that the Ugandan population is in Hardy-Weinberg equilibrium and both heterozygosities were above 70% at all loci as in other published data [28-30]. This suggested that all these populations are not small, not closed and that there is minimal inbreeding if any [31].

TPOX loci had the highest number of tri-allelic patterns observed in the Ugandan population and these were of Type 2. Tri-allelic patterns are extensively studied [32-34] and may be as a result of duplication located on the same chromosome, translocation or chromosome aneuploidy (trisomy). Since TPOX occurs near the end of chromosome 2, it is more likely to duplicate for telomeres maintenance in order to keep intact the chromosome end [35].

The most informative and discriminatory locus in our data set was SE33 (PD =0.9900, PIC=0.9221, Ho =0.9131, He=0.9266, 49 alleles) while the lowest D13S317 (PD =0.8723, PIC=0.6656, Ho =0.7000, He=0.7131, 9 alleles) similar to other studied populations [27,36-41]. This dataset indicates a high degree of gene diversity detected and that Uganda’s population maintains a high degree of genetic variation with a history of high miscegenation. Similar results were obtained for the Power of Exclusion (PE) and Typical Paternity Index (TPI) with the highest PE and TPI values being associated with SE33 (PE=0.8223, TPI=5.7566) proving to be a powerful marker for human identification and paternity testing within the Ugandan population.

According to Kimura M [42], alleles with a frequency of less than 0.01 in a population database are regarded as rare alleles. The Ugandan population database as well had rare alleles with the lowest frequency of 0.004 and these were mainly microvariants; alleles with incomplete repeats [31]. Locus that displayed the highest number of microvariants was SE33 and these had two incomplete repeats only while at TH01 a 9.3 microvariant was observed and this was found to have been caused by a missing adenine in the seventh repeat [43]. The same study points out that the cause of a .2 microvariant at D21S11 locus is a -TA- dinucleotide partial repeat before the last full TCTA repeat and a .2 microvariant at FGA locus is caused by a -TT- dinucleotide partial repeat after the fifth full repeat and before the variable CTTT repeat motif.

The large Combined Power of Discrimination (CDP), Combined Power of Exclusion (CPE) and Combined Paternity Index (CPI) values and low value of Combined Matching Probability (CMP) support the use of the 21 STR loci in individual identification and paternity testing within the Ugandan population. These values were remarkably higher than that of the 15 autosomal loci obtained for the studied populations [44,45].

Conclusion

This study confirms that the 21 Short Tandem Repeat loci is a suitable tool for human identification in forensic, parentage and kinship analysis in the Ugandan population due to their genetic variability and combined discriminatory power. Since these loci studied represent a combination of CODIS core and expanded European loci, this dataset and the allelic frequencies calculated from it can help international authorities with Ugandan victims or suspects.

Acknowledgement

We gratefully acknowledge the study participant who actively and willingly contributed to this research. We also would like to thank Mr. Kepher Kuchan Kateu the Chief Government Chemist and Mr. Tarsisius Byamugisha, the Commissioner at the Government Analytical Laboratory, Ministry of Internal Affairs for their continued technical guidance and professional suggestions all the way through the demanding moments of this research. We thank all Forensic Biology laboratory staff for their continued generosity in offering the information required as well as the enthusiasm they had for this research. Funding for this research was provided by the Government of Uganda.

Conflict of Interest

The authors declare no potential conflicts of interest.

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