Keywords

Variant Alleles; CODIS STR; Forensics; TPOX; TH01; vWA.

Introduction

Polymorphic Short Tandem Repeat (STR) loci have become useful tool for DNA analysis and typing for human identification and paternity testing for forensic purposes in most of the forensic laboratories in the world [1, 2]. STR alleles of a questioned individual are designated by comparing with alleles of allelic ladder, provided by manufacturer of commercial kit [3]. Allelic ladder is the combination of alleles based on population genetic variations observed in developmental process. STR alleles of most of the DNA profiles represented with in allelic ladder are found in a small number of individuals.

For proper casework interpretations, the forensic scientists should be aware of the existence of rare alleles so that the variants can be recognised. Correctly designated alleles can sometimes greatly increased power of discrimination of STR analysis. It is, therefore, important that forensic scientists should share information on the occurrence of these variants in different populations.

India is culturally and geographically a highly diverse country. Human Diversity in India is defined by 4693 different documented population groups that include 2205 major communities, 589 segments and 1900 territorial units spread across the country [4]. This diversity is due to different cultural heritage (linguistic, ethnic etc.), history of different waves of migration and impact of deep seated caste systems (or practice of strict endogamy/marriage rule). In this study, 3 variant alleles were found at 3 GenePrint STR loci, viz. TPOX, TH01 and vWA (Promega Corporation, Madison, US) during Indian population studies.

Materials and Methods

Samples and DNA extraction

A total of 244 blood samples were collected from unrelated individuals of two populations namely Rajputs and Ramgaria-Sikhs from Delhi, India. Genomic DNA from each sample was extracted by Phenol: Chloroform extraction method [5]. Whole blood sample was used as a DNA source. Extracted DNA samples were quantified by using NanoDrop spectrophotometer.

PCR Amplification and STR typing

About 2-5 ng of total DNA was used as template DNA in a total reaction volume of 25 μl for each amplification process. Initially, 244 samples were screened for 12 STR loci namely CSF1PO, D16S539, D7S820, D13S317, F13A01, F13B, FESFPS, HPRTB, LPL, TPOX, TH01 and vWA using GenePrint STR system (Promega Corporation, Madison, US) and polyacrylamide gel electrophoresis.

Thermal cycling parameters were setup according to the manufacturer’s protocol (Promega Corporation, Madison, US). Amplification products (2.5 μl per sample mixed with an equal volume of STR 2X loading solution) were electrophoresed at 50 Watt for 2-4 h on a 40cmX20cm, 0.35mm thick 6% denaturing polyacrylamide gel by manual GenoSequencer (Atto Corporation, Japan) with 0.5X TBE as gel running buffer. The bands were visualized by staining with silver stain [6]. Direct comparison between the allelic ladders and amplified samples of the same locus allowed for determination of alleles. Variant alleles were designated according to PCR product size, calculated relative to a molecular marker (pBR322 DNA/MspI digest) with Image master 1D Elite v3.01 (Amersham Biosciences, USA).

The genotype data was compiled for twelve STR markers in Excel sheets to facilitate for statistical treatment for generation of allele frequencies of the 12 STR loci. The Allele frequencies and statistical population parameters were calculated using “PowerStats” Microsoft Excel workbook template provided by Promega Corporation ( www.promega.com/geneticidtools/).

Results

This study screened a total of 244 individuals of two different Indian populations namely Rajputs and Ramgaria-Sikhs from Delhi, for variant alleles not included in the allelic ladder provided by the manufacturer. DNA samples were initially screened for 12 STR loci by silver stain detection method. A total of 3 variant alleles were recognized at 3 STR loci viz. TPOX, TH01 and vWA and determined by a visual comparison of sample band position relative to those of known to allelic ladder. Each variant allele was confirmed by re-extraction and amplification of each locus. Allelic ladders from kit contain alleles 5-11 at TH01, 6-13 at TPOX, whereas at vWA alleles ranging from 13 to 20 (Table 1). All these variants were not included in the allelic ladder provided by Gene- Print STR system amplification kit. All three variants were heterozygotes, paired with well characterized sister allele represented in allelic ladder in each case. The TH01 variant TH018.3 (allele 8.3) was found with TH019 allele (Figure 1) as heterozygous condition in Ramgaria-Sikh population. While variants TPOX14 (allele 14 at TPOX) (Figure 2) and vWA21 (allele 21 at vWA) (Figure 3) were paired with TPOX9 and vWA18 alleles, respectively in Rajput population. Variant of TPOX and vWA loci contains an allele that was larger than 4 repeats which was largest in their respective ladder (Table 2).

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Figure 1:

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Figure 1. Variant TH08.3 paired with allele TH09 (sample 1) at TH01 locus. 1-3 are the samples amplified and A is the Allelic ladder of respective locus.

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Figure 2:

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Figure 2: Variant TPOX14 paired with alleleTPOX9 (sample 4 and 5) at TPOX locus. 1-8 are the samples amplified and A is the Allelic ladder of respective locus.

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Figure 3:

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Figure 3: Variant vWA21 paired with vWA18 allele (sample 5) at vWA locus. 1-8 are the samples amplified and A is the Allelic ladder of respective locus.

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Table 1:

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Table 1. Characteristics of STR loci that showed variant alleles.

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Table 2:

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Table 2. Comparison of Allele frequencies of observed variant alleles in different populations.

Discussion

The STRBase website (http://www.cstl.nist.gov/biotech/strbase) lists only two variants TH018.3 and TPOX14. Alleles TH018.3 and TPOX14 have been separately reported 26 and 7 times respectively in STRBase. Allele vWA21 was not reported previously in STRBase. Variant vWA21 has previously been reported in Turks and Chinese population with a frequency of 0.004 and 0.006, respectively [7]. While Lins et al. [8] reported this variant in Africans- Americans with a frequency of 0.002. This variant also been reported in Germens, Moroccans, Japanese and Papuans with a frequency of 0.001, 0.004, 0.004 and 0.009, respectively [9]. Variant vWA21 also been reported in Caucasian, African-Americans as well as Hispanics [10] with a frequency of 0.002, 0.004 and 0.004, respectively. African-Jordanians population has a frequency 0.005 of vWA21 variant allele [11]. Variant allele TH018.3 has been reported with frequency of 0.001 in German population [7]. This variant also been reported in European-Americans and Bosnia population with a frequency of 0.003 and 0.050, respectively [12,13]. TPOX14 has previously been reported in Chinese population with a frequency of 0.004 [14]. Berardi et al. [15] reported this variant allele in Argentina population with a frequency of 0.0071 (Table 2).

A literature search shows that variant alleles vWA21 and TPOX14 have been reported in different Indian populations also (Table 2). Variant allele TPOX14 has been reported in a Khandayat (Orissa), Marathas and Brahmins (Maharashtra) populations with a frequency of 0.008. 0.11. 0.008 respectively [16, 17]. Variant allele vWA has been reported in Rajputs (U.P.), Reddy and Brahmins (A.P.) populations of with a frequency of 0.010, 0.016 and 0.019, respectively [18-20].

Conclusion

This study identified 3 unusual variant alleles at 3 loci that are not represented in commercially available allelic ladder in a database of nuclear STR profiles from India. Variant alleles TH018.3 and TPOX14 have been separately reported in STRBase. While allele vWA21 was not reported previously in STRBase. The variant allele TH018.3 was not previously reported in any population of India. Dissemination of the information will assist Forensic DNA analysis and other human identification laboratories in an Awareness of anomalies due to variants off-ladder alleles encountered during analysis.

Acknowledgements

This study was supported by funds obtained from the Indian Ministry of Homes. Author thanks Dr. K. P. S. Kushwaha for their valuable guidance.

References

1. Chakraborty R, Stivers DN, Su B, Zhong Y, Budowle B (1999) The utility of short tandem repeat loci beyond human identification: Implication for development of new DNA typing system. Electrophoresis 20(8): 1682-1696.
2. Butler JM (2001) Forensic DNA typing: Biology and Technology behind STR markers. (2nd edtn), London Academic Press, UK.
3. Griffith RA, Barber MD, Johnson PE, Gillbard SM, Haywood MD, et al. (1998) New reference allelic ladder to improve allelic designation in a multiplex STR system. Int J Legal Med 111(5): 267-272.
4. Singh KS (1998) India’s communities-People of India. National Series volume IV India, Oxford University Press, USA.
5. Shambrook J, Accallum P, Russell D (2001) Molecular cloning: A Laboratory manual. (3rd edtn), Cold Spring Harbour Laboratory Press, New York.
6. Bassam BJ, Caetaro-Anolles G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196(1): 80-83.
7. Brinkmann B, Sajantila A, Goedde HW, Matsumoto H, Kand N, et al. (1996) Population Genetic comparisons among eight population using allele frequencies and sequence data from three microsatellite loci. Eur J Hum Genet 4(3): 175-182.
8. Lins AM, Micka KA, Sprecher CJ, Taylor JA, Bacher JW, et al. (1998) Development and population study of an eight-locus Short Tandem Repeat (STR) multiplex system. J Forensic Sci 43(6): 1168-1180.
9. Brinkmann B, Junge A, Meyer E, Weigand P (1998) Population Genetic Diversity in relation to microsatellite heterogeneity. Hum Mutat 11(2): 135-144.
10. Butler JM, Schoske R, Vallone PM, Redman JW, Kline MC (2003) Allele Frequencies of 15 autosomal STR loci on U. S. Caucasian, African and Hispanic populations. J Forensic Sci 48(4): 908-911.
11. Yasin SR, Hamad MM, Elkarmi AZ, Jaran AS (2005) African Jordanian population genetic database on fifteen Short Tandem Repeat Genetic loci. Croat Med J 46(4): 587-592.
12. Budowle B, Moretti TR, Baumstark AL, Defenbaugh DA, Keys KM (1999) Population data on thirteen CODIS core STR loci in African Americans, US Caucasians Hispanics, Bahamians, Jamaicans and Trinidadians. J Forensic Sci 44(6): 1277-1286.
13. Huel RML, Basic L, Todorovie K, Smajlovic L, Eminovic I, et al. (2007) Variant alleles, Tri-allelic patterns and Point mutations observed in nuclear Short Tandem Repeat typing of populations in Bosnia and Serbia. Croat Med J 48(4): 494-502.
14. Huang NE, Schumm JW, Budowle B (1995) Chinese population data on 3 tetrameric STR loci (HumTH01, TPOX, CSF1PO) derived using multiplex PCR and manual typing. Forensic Sci Int 71: 131-136.
15. Berardi G, Toscanini U, Raimondi E (2003) STR data for Powerplex 16 system from Buenos Aires population, Argentina. Forensic Sci Int 134(2-3):222-224.
16. Sahoo S, Kashyap VK (2002) Allele frequency data for powerplex 16 loci in four major populations of Orissa, India. J Forensic Sci 47(4): 912-915.
17. Gaikwad S, Kashyap VK (2002) Polymorphism at fifteen hypervariable microsatellite loci in four populations of Maharashtra, India. Forensic Sci Int 126(3): 267-271.
18. Tandon M, Trivedi R, Kashyap VK (2002) Genomic diversity at 15 fluorescent labeled short tandem repeat loci in few important populations of State of Uttar Pradesh, India. Forensic Sci Int 128(3): 190-195.
19. Kashyap VK, Ashma R, Gaikwad S, Sarkar BN, Trivedi R (2004) Deciphering diversity in populations of various linguistic and ethnic affiliations of different geographical regions of India: analysis based on 15 microsatellite markers. J Genet 83(1): 49-63.
20. Reddy BM, Naidu VM, Madhavi VK, Thangaraj K, Langstieh BT, et al. (2005) STR data for the Amp FISTR Profiler Plus loci among 27 populations of different social hierarchy from southern part of Andhra Pradesh, India. Forensic Sci Int 149(1): 81-97.