Home About Us Laboratory Services Forensic Science Communications Back Issues April 2005 research Research and Technology - Forensic Science...

Research and Technology - Forensic Science Communications - April 2005

Research and Technology - Forensic Science Communications - April 2005

fsc_logo_top.jpg
fsc_logo_left.jpg

April 2005 - Volume 7 - Number 2

Research and Technology

Population Genetics of 17 Microsatellite Loci in Three Major Groups of Andhra Pradesh, India

G. Hima Bindu
Doctoral Student

Rajni Trivedi
Deputy Director

V. K. Kashyap
Director
Central Forensic Science Laboratory
Directorate of Forensic Sciences
Ministry of Home Affairs
Government of India
West Bengal, India

Abstract

Polymorphisms at 15 tetrameric short tandem repeat loci and two pentameric short tandem repeat genetic loci, Penta D and Penta E, were determined in 276 people belonging to three predominant caste populations of Andhra Pradesh, India. All the loci were found to be highly polymorphic in all studied populations. No significant deviations from the Hardy-Weinberg expectations were observed with exceptions at three loci. Although individual power of exclusion estimates varied at the studied loci, the combined exclusion value was greater than 0.9999 in all the populations. Combined power of discrimination was observed to be greater than 0.9757, whereas polymorphism information content was high in the selected microsatellites. Loci having alleles with higher repeat units exhibited high typical paternity index estimates. The discriminatory potential of the 17 short tandem repeat loci revealed Penta E and FGA to be the most informative loci in studied populations.

Introduction

Autosomal microsatellites due to their hypervariability have emerged as a potent tool for elucidating human identification. They involve a base motif of one to six base pairs, are highly polymorphic, and uniformly distributed throughout the human genome with high discriminatory power (Chakraborty and Jin 1993; Jefferys and Pena 1993; National Research Council 1996; Pena and Chakraborty 1994).

India is the second most populous country in the world and has populations with distinct ethnicity, culture, and religions. Databases for the diverse populations of this region where these genetic systems can be harnessed for match probability in human identification, population genetics, evolutionary, and disease management are essential.

The aim of the present study was to establish a database of upper caste groups of Andhra Pradesh—Brahmins, Komati, and Raju. These groups are globally dispersed and distinct from similar caste groups in other parts of India (Singh 1998) for forensic purposes, including parentage analysis. Therefore, two pentameric markers, Penta D and Penta E, and the recently introduced AmpFlSTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, California), which amplifies the 13 core CODIS short tandem repeat loci, seven European Network of Forensic Science Institute's short tandem repeat loci, and two newly introduced tetranucleotide loci, D2S1338 and D19S433, were evaluated.

This report describes the allele frequencies and the measure of forensic parameters for the 17 short tandem repeat loci in a sample of 276 unrelated people.

Materials and Methods

Samples and DNA Extraction

Peripheral blood samples were obtained by venipuncture from informed, consenting, healthy unrelated people of the three major populations of Andhra Pradesh, India. People belonging to the priestly group (Andhra Brahmins, 106), warrior community (Raju, 66), and traders (Komati, 104) participated in the study. Genomic DNA was extracted from blood using the standard phenol-chloroform extraction method (Sambrook et al. 1989), followed by ethanol precipitation. Extracted DNA was quantitated using Quantiblot Human DNA Quantitation Kit according to the manufacturer's instructions (Perkin Elmer, New York).

PCR Amplification

The AmpFlSTR Identifiler PCR Amplification Kit was used to coamplify 15 tetranucleotide repeat loci in one nanogram of individual genomic DNA samples.

The thermal cycling parameters were as follows: hold at 95°C for 11 minutes, followed by 28 cycles of 94°C for 1 minute, 59°C for 1 minute, 72°C for 1 minute, then hold at 60°C for 60 minutes, followed by soaking at 4°C.

The duplex PCR consisting of Penta E and Penta D was amplified using published primer sequences (Krenke et al. 2002) and the following cycling conditions: 28 cycles of 94°C for 1 minute, 57°C for 1 minute, 72°C for 1 minute, then hold at 72°C for 3 minutes, followed by soaking at 4°C.

All amplifications were carried out in a GeneAmp PCR 9700 thermal cycler (Applied Biosystems, Foster City, California).

Short Tandem Repeat Typing

DNA typing was performed using a five percent denaturing polyacrylamide gel electrophoresis on an ABI Prism 377 DNA Sequencer (Applied Biosystems, Foster City, California) with laser induced fluorescent detection. The allele classification was made in comparison with allelic ladders provided with AmpFlSTR Identifiler PCR Amplification Kit and PowerPlex 16 Kit (Promega, Madison, Wisconsin). The data analysis and allele designations were carried out using the GeneScan Analysis Software (Version 3.7; Applied Biosystems, Foster City, California) and GenoTyper Software (Version 3.7; Applied Biosystems, Foster City, California).

Statistical Analysis

Allele frequencies were computed using the gene counting method. Conformation to Hardy-Weinberg Equilibrium was tested by the exact test (Guo and Thompson 1992) and logarithmic likelihood ratio G test (Weir 1992) using the DNATYPE program (Chakraborty et al. 1998). The observed heterozygosity, probability of homozygosity (Nei 1978), and the number of alleles exhibited were also estimated. The determination of the power of discrimination and match probability (Jones 1972), polymorphism information content (Botstein et al. 1980), power of exclusion, and typical paternity index (Garber and Morris 1983) was performed using the software package PowerStats (Promega Corporation, Madison, Wisconsin).

Results and Discussion

The allele frequency distributions of 17 short tandem repeat loci and forensically important statistical parameters in the three caste populations of Andhra Pradesh, India, are shown in Tables 1, 2, and 3. Accordance with Hardy-Weinberg Equilibrium expectations was exhibited in all the 17 short tandem repeat loci (based on exact test and G test) with exceptions at Penta E for Andhra Brahmins and Komati, FGA for Raju, and D7S820 for Komati. The observed heterozygosities ranged from 0.943 (Penta E) for Andhra Brahmins, ≥ 0.892 (FGA) for Raju and Komati, to 0.625 (CSF1PO) for Komati, and ≥ 0.642 (TPOX) for Andhra Brahmins and Raju. A maximum number of alleles was detected at Penta E, 16 to 19, respectively, for Raju and Komati and at FGA, 16 alleles were exhibited for Andhra Brahmins, whereas a minimum of five alleles was observed at loci THO1 (Komati) and TPOX.

Significantly, combined probability of match for Andhra Brahmins is 1 in 1.752 x 1020, 1 in 1.886 x 1019 for Raju, and 1 in 1.601 x 1019 for Komati. Individual power of exclusion values ranged from 0.344 (TPOX) to 0.885 (Penta E) for Andhra Brahmins. Maximum power of exclusion value at 0.784 (FGA) and 0.780 (FGA), and minimum at 0.322 (CSF1PO) and 0.379 (TPOX) was observed for Komati and Raju, respectively. Combined power of exclusion was estimated to be greater than 0.9999 in all the studied populations. Among the 17 short tandem repeat loci evaluated, the highest power of discrimination was observed with Penta E in all three population groups (≥ 0.897) and lowest with D5S818 in Komati (0.644), TPOX in Raju (0.654), and TPOX in Andhra Brahmins (0.678). All the selected loci possessed a combined power of discrimination of 0.9848 for Komati, 0.9757 for Andhra Brahmins, and 0.9809 for Raju.

Relatively high polymorphism information content was exhibited among all populations at Penta E, whereas low values were observed at TPOX and D5S818. Loci having alleles with higher repeat units exhibited high typical paternity index values at Penta E (8.833) for Andhra Brahmins and at FGA (>4.643) for Komati and Raju. Selected short tandem repeat loci that exhibit Penta E and FGA are the most informative markers in the studied populations. This is the first study reporting polymorphisms at the two newly introduced tertrameric nucleotides, D2S1338 and D19S433, 13 core CODIS loci, and two pentameric loci in the studied populations. Evaluation of the forensic applicability of the 17 short tandem repeat loci, the genotype distributions and allele frequencies, and various statistical parameters would contribute to the database of global populations and increase the international short tandem repeat database of global populations.

Acknowledgments

The authors are thankful to the participants for volunteering their blood samples and various nongovernmental organizations for facilitating in this objective. The work was carried out with an X Plan Project Grant from the Central Forensic Science Laboratory, Kolkata, West Bengal, India. G. Hima Bindu was supported by a national fellowship from the Council of Scientific and Industrial Research, New Delhi, India.

References

Botstein, D., White, R. L., Skolnick, M., and Davis, R. W. Construction of a genetic linkage map in man using restriction fragment length polymorphisms, American Journal of Human Genetics (1980) 32:314-331.

Chakraborty, R. and Jin, L. Determination of relatedness between individuals by DNA fingerprinting, Human Biology (1993) 65:875-895.

Chakraborty, R., Strivers, D., and Zhong, Y. DNATYPE: Computer program. Version Windows 95/NT. University of Texas, Houston, Texas. 1998.

Garber, R. A. and Morris, J. W. General equations for the average power of exclusion for genetic systems of n co-dominant alleles in one-parent cases of disputed parentage. In: Inclusion Probabilities in Parentage Testing. R. H. Walker, ed. American Association of Blood Banks, Arlington, Virginia, 1983, pp. 277-280.

Guo, S. W. and Thompson, E. A. Performing the exact test of Hardy-Weinberg proportion for multiple alleles, Biometrics (1992) 48:361-372.

Jefferys, A. J. and Pena, S. D. J. Brief introduction to human DNA fingerprinting. In: DNA Fingerprinting: State of the Science. S. D. J. Pena, R. Chakraborty, J. T. Epplen, and A. J. Jefferys, eds. Birkhauser Verlag, Basel, Switzerland, 1993, pp. 1-20.

Jones, D. A. Blood samples: Probability of discrimination, Journal of the Forensic Science Society (1972) 12:355-359.

Krenke, B. E., Treba, A., Anderson, S. J., Buel, E., Culhane, S., Finnis, C. J., Tomsey, C. S., Zacheti, J. M., and Sprecher, C. J. Validation of a 16-locus fluorescent multiplex system, Journal of Forensic Sciences (2002) 47:773-785.

National Research Council. Evaluation of Forensic DNA Evidence, National Academy Press, Washington, DC, 1996.

Nei, M. Estimate of average heterozygosity and genetic distance from a small number of individuals, Genetics (1978) 89:583-590.

Pena, S. D. J. and Chakraborty, R. Paternity testing in the DNA era, Trends in Genetics (1994) 10:204-209.

Sambrook, J., Fritsch, E. F., and Maniatis, T. Molecular Cloning. A Laboratory Manual. 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989.

Singh, K. S. People of India. National Series Vol. IV, V, VI, India's Communities (A-Z), Anthropological Survey of India. Oxford University Press, New Delhi, India, 1998.

Weir, B. S. Independence of VNTR alleles defined as fixed bins, Genetics (1992) 130:873-887.