INTRODUCTION

In forensics, sex determination is critical and inescapable step in DNA profiling of cases such as mass disaster victim identification, investigation of missing persons, solving murder mysteries and sexual assault cases [1]. Previously, sex determination of skeletal remains was done on the basis of its morphological examination. Nevertheless, this technique of gender determination fails while examining fragmentary remains especially in case of mass disaster victim identification. However, with the evolution of molecular biology, a gender can be determined from the traces of skeleton with DNA profiling [2]. In gender identification through DNA profiling, Amelogenin is most common sex determining marker. Amelogenin is encoded by two single copy genes i.e. AMELX (Xp22.1–Xp22.3) and AMELY (Yp11.2) [3]. For amelogenin typing, the universal set of primers is available which depicts six base pair deletion on AMELX [4–6]. In 1998, first time sex determination based on the amelogenin marker was questioned [7]. Afterword various studies have shown AMELY deletion in many of the population studies around the globe [8, 9]. AMELY is now been well characterized and its deletion mapping data is available [10, 11]. In present study, we also experienced AMEL Y deletion in the Brahmin population of Rajasthan and Haryana state, India. Further, we also compare our findings with the other available population’s data.

MATERIALS AND METHODS

Blood samples of 102 and 100 unrelated Brahmin males from Haryana and Rajasthan states were collected on FTA Cards with informed consent after ethical approval. DNA was extracted from these samples using a novel DNA purification buffer given by Sahajpal et al. [12]. Autosomal STR and Amelogenin were typed using Powerplex 21 Autosomal kit (Promega, Madison, WI, USA) according to the corresponding manufacturer’s recommendations. Further Global filer PCR amplification was performed on these samples as it contains the 3 markers of Y chromosomes. In addition to this, further amplification was carried out with Y Chromosome specific Kits like Y Filer, Powerplex Y23 (Promega, Madison, WI, USA), and Y Filer plus PCR amplification kit (Applied Biosystem) to know the number of markers deleted in the null Y allele males. PCR was done according to the recommended protocol using GeneAmp® PCR System 9700 (Applied Biosystems). The amplified products of Powerplex 21 Autosomal STRs, Y filer and Powerplex Y23 were detected through capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems) while Global Filer and Y filer Plus amplified products were separated on a 3500xl Genetic Analyzer (Applied Biosystems).

Statistical Analysis

The analysis was performed using GeneMapper ID v3.2 and GeneMapper ID-X (Applied Biosystems) to generate DNA profiles. Alleles were assigned according to the International Society of Forensic Genetics (ISFG) guidelines for STR analysis.

RESULTS

In amplification with Powerplex 21, three samples out of 102 of Haryana Brahmin and one out of 100 of Rajasthan Brahmin showed Y deletion at amelogenin and exhibited a female genotype as shown in Fig. 1. All these samples were further typed with Global Filer autosomal STRs kit, which contains three respective gender-determining markers like amelogenin, Y Indel, and DYS390. Except for amelogenin Y, the other two markers showed their presence as shown in Fig. 2.

Fig. 1.
figure 1

Male Y missing profile (AMELY) with Powerplex 21.

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Fig. 2.
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Male Y missing profile (AMELY) with Global Filer.

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DISCUSSION

This study analyzed the Brahmin population of Rajasthan (100) and Haryana (104) state. The observed rate of amelogenin Y failure was high in Haryana Brahmin population compared to various other published studies. In earlier Indian population studies, the samples represented the state’s whole population, but our study is specially focused on the state’s Brahmin population. In addition to this, these samples were typed with a different increased number of Y chromosome-specific marker kits to check the number of loci missing in a Y amelogenin deletion male. In genotyping with Y filer marker DYS458 was missing in all three samples and similar results were found in the Japanese population [3]. With Powerplex Y 23, kit markers DYS458, DYS481, DYS570, and DYS576 were missing while Yfiler plus showed two more dropout markers DYS449 and DYS627. Dropout of markers with Powerplex Y23 was concordant with the global study conducted on 19 300 samples by Purps et al [13]. In this study, only nine samples showed deletion at Yp11.2 AMELY with the same four markers dropout and all the samples were of Asian ancestry, namely Tamils from Southern India, Indians from Singapore, and British Asians with India or Pakistan origin. A similar various study conducted around the world and India is given in Tables 1 and 2. In comparison with 104 endogamous populations tested [4] and the Delhi population [14]. Brahmins of the Haryana population showed the highest amelogenin Y deletion frequency. In comparison with other Brahmin population, only Desasth Brahmin from Maharashtra and Iyenger Brahmin from Karnataka showed Y deletion.

Table 1. Frequency of amelogenin Y deletion in different Indian populations
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Table 2. Frequency of globally observed Amelogenin Y deletion populations
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CONCLUSIONS

DNA is the workhorse for sex identification for many years, but sex determination based on amelogenin marker has some discrepancies. In present study, confirmed known males were mistyped as female samples because of Y amelogenin deletion. Modern multiplex PCR amplification kits with single sex-determining marker even fail to identify the gender of a person if the identity of the sample is unknown. Failure of sex determination based on Amelogenin is the most common in forensic science laboratories throughout the world. Therefore, whenever any case shows deletion or negative result with AMELY typing can never be conclusive to confirm sample of female origin. It is therefore, Y-STR typing becomes mandatory in such cases. The deletion of DYS458 locus in Y‑STR profile also serve as indication for AMELY deletion. In the various studied Indian populations, Brahmin of Haryana showed the maximum AMELY deletion. Although the frequency of AMELY deletion is less in other, worldwide population groups in comparison to Indian population. That is why gender testing in human identification needs high reliability and other methods like sex-determining region Y (SRY), Y-encoded testis-specific protein (TSPY), locus DXYS156, and steroid sulfatase (STS). All these markers have properties that could be used for developing more rigorous methods of testing forensic DNA samples for a Y chromosome. In this connection, advanced multiplex STRs kits like Global Filer, PowerPlex Fusion 6C and Investigator 24Plex QS should be used for cases like unidentified dead bodies or mass disaster victim identification. In Indian population, where AMEL Y deletion is present, use of these advanced multiplex PCR amplification kit is must.