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Deletions in the Y-derived amelogenin gene fragment in the Indian population

BMC Medical Genetics20067:37

DOI: 10.1186/1471-2350-7-37

Received: 21 November 2005

Accepted: 10 April 2006

Published: 10 April 2006

Abstract

Background

Rare failures in amelogenin-based gender typing of individuals have been observed globally. In this study, we report the deletion of a large fragment of the amelogenin gene in 10 individuals out of 4,257 male samples analyzed from 104 different endogamous populations of India.

Methods

Samples were analyzed using commercial genetic profiling kits. Those that exhibited failures in amelogenin-based gender identification were further analyzed with published as well as newly designed primers to ascertain the nature and extent of mutation.

Results

The failure rate among Indian males was 0.23 %. Though the exact size and nature of the deletion (single point mutations at a number of positions or a single large deletion) could not be determined in the present study, it is inferred that the deletion spans a region downstream of the reverse primer-binding site of commercially available amelogenin primer sets. Deletions were conspicuously absent among the Mongoloid tribes of Northeast India, while both caste and tribal groups harbored these mutations, which was predominantly among the Y-chromosomes belonging to J2 lineage.

Conclusion

Our study indicates that the different amelogenin primer sets currently included in genetic profiling multiplex kits may result in erroneous interpretations due to mutations undetectable during routine testing. Further there are indications that these mutations could possibly be lineage-specific, inherited deletions.

Background

Genotyping the X-Y homologous amelogenin gene segment for gender identification is widely used for DNA profiling in DNA databasing, forensic casework, archeological specimens, preimplantation and prenatal diagnoses [14]. The amelogenin gene is a single copy gene, homologues of which are located on Xp22.1-Xp22.3 and Yp 11.2 [5]. Regions on this gene that are sufficiently conserved are amplified for simultaneous detection of the X and Y alleles in gender identification procedures. Primers bind to the first intron region of the amelogenin gene on the X and Y-chromosomes [6] and amplify regions that differ in base sequence, hence resulting in products that are easily distinguishable by differences in size and sequence. The most widely used primer set [6] delimits a 6 bp deletion on the X-chromosome and produces fragments of 106 bp and 112 bp for the X and Y chromosomes respectively. Presence of two amplified products indicates a male genotype, while a single amplicon implies female genotype. However, mutations in the Y-derived fragment of the gene may result in amplification failure of the Y-allele, causing misidentification of the biological sample as of a female. Similarly, mutations on the X homologue would also result in non-amplification of the X-derived fragment although the genotype would still be identified as male due to amplification of the Y-amelogenin allele.

Recently, a few studies have revealed misidentification of the male genotype while employing the amelogenin gender test [[79]]. Failures in accurate determination of gender have been reported to be particularly high among individuals of Indian origin. The frequency of failure was observed to be 8 % by Santos et al. [7], while Thangaraj et al. [9] reported 5 cases of amelogenin failure (1.85 %) among the 270 Indian males studied. The failure rate of the amelogenin sex test was particularly high (3.6 %) in an Indian population group from Malaysia [10]. However, a parallel study testing a larger number of individuals from the Austrian National DNA database reported a failure rate of 0.018 % [11]. The high frequency in incidence of failures in the Indian sub-continent prompted us to scrutinize the amelogenin typing results of 7,214 individuals (including 4,257 males) belonging to 104 different endogamous populations that were genotyped as part of our DNA databasing project. Individuals were sampled from diverse geographic regions across India such that all existing socio-ethnic groups and linguistic families were represented. In this paper, we report failures in genotyping of male individuals due to mutations originating in the Y-homologue of the amelogenin gene. We have further characterized the nature and extent of mutations and provide evidence for a plausible inherited mode of transmission of the mutation.

Methods

Population samples analyzed

DNA was isolated by standard organic extraction method [12] either from blood or buccal swabs of consenting 4,257 male and 2,957 female individuals belonging to 104 different endogamous groups. Individuals represented major caste and tribal groups of India, which were sampled from across 20 geographical regions of India (Table 1, Figure 1).
https://static-content.springer.com/image/art%3A10.1186%2F1471-2350-7-37/MediaObjects/12881_2005_Article_145_Fig1_HTML.jpg
Figure 1

Map depicting the regions covered in the study and location of samples where amelogenin deletion were observed.

Table 1

Incidences in failures of gender testing among the 104 endogamous populations of India tested for amelogerin gene efficacy

S.No

State

Population

Social Group

Individuals Tested

Failure in Gender Analysis (No of samples)

1

Jammu & kashmir

Balti

Tribe

67

Nil

  

Drokpa

Tribe

38

Nil

  

Argon

Tribe

51

Nil

  

Buddhist

Religious Group

156

Nil

2

Himachal Pradesh

HPRajput

Caste

50

Nil

3

Uttaranchal

Tharu

Tribe

45

Nil

  

Jaunsari

Tribe

45

Nil

  

Bhoksha

Tribe

42

Nil

4

Uttar Pradesh

Kanyakubj Brahmin

Caste

98

Nil

  

UP Jat

Caste

48

Nil

  

UP Thakur

Caste

48

Nil

  

Khatri

Caste

47

1

  

UP Kurmi

Caste

45

Nil

5

Bihar

Bihar Brahmin

Caste

59

Nil

  

Bhumihar

Caste

65

Nil

  

Rajput

Caste

58

Nil

  

Kayasth

Caste

53

Nil

  

Yadav

Caste

44

Nil

  

Kurmi

Caste

50

Nil

  

Baniya

Caste

45

Nil

6

Gujarat

Gujarat Patel

Caste

45

Nil

7

Maharastra

Desasth Brahmin

Caste

70

1

  

Chitpavan Brahmin

Caste

78

Nil

  

Maratha

Caste

65

1

  

Dhangar

Caste

150

Nil

  

Pawara

Tribe

82

Nil

  

Katkari

Tribe

95

Nil

  

Madia Gond

Tribe

45

Nil

  

Mahadeo Koli

Tribe

45

Nil

8

Chattisgarh

Brahmin

Caste

51

Nil

  

Satnami

Caste

50

Nil

  

Teli

Caste

50

Nil

  

Dheria Gond

Tribe

35

Nil

  

Agharia

Tribe

70

1

  

Oroan

Tribe

42

Nil

9

Jharkhand

Ho

Tribe

50

Nil

  

Bhumij

Tribe

56

Nil

  

Kharia

Tribe

83

Nil

  

Munda

Tribe

64

Nil

  

Birhor

Tribe

61

Nil

  

Santhal

Tribe

61

Nil

  

Oroan

Tribe

60

Nil

10

Westbengal

Brahmin

Caste

110

Nil

  

Kayasth

Caste

103

Nil

  

Mahishya

Caste

60

Nil

  

Namasudra

Caste

55

Nil

  

Bauri

Caste

54

Nil

  

Maheli

Tribe

49

Nil

  

Karmali

Tribe

51

Nil

  

Kora

Tribe

59

Nil

  

Lodha

Tribe

99

Nil

11

Orrisa

Oriya Brahmin

Caste

57

Nil

  

Karan

Caste

62

Nil

  

Khandayat

Caste

62

1

  

Gope

Caste

60

Nil

  

Paroja

Tribe

78

1

  

Juang

Tribe

50

Nil

  

Saora

Tribe

35

Nil

12

Andhra Pradesh

Andhra Brahmin

Caste

106

Nil

  

Raju

Caste

66

Nil

  

Kappu Naidu

Caste

104

Nil

  

Kamma Chaudhary

Caste

106

Nil

  

Reddy

Caste

107

Nil

  

Komati

Caste

104

Nil

  

Yerukula

Tribe

101

Nil

  

Chenchu

Tribe

100

Nil

  

Naikpod Gond

Tribe

104

Nil

  

Lambadi

Tribe

107

Nil

  

Golla

Caste

65

Nil

  

Sakunupakshollu

Caste

30

Nil

13

Tamil Nadu

Chakkiliar

Caste

49

Nil

  

Tanjore Kallar

Caste

101

1

  

Vanniyar

Caste

87

1

  

Pallar

Caste

33

Nil

  

Gounder

Caste

56

Nil

  

Irular

Tribe

54

Nil

  

Paraiyar

Caste

21

Nil

14

Kerala

Nair

Caste

87

Nil

15

Karnataka

Iyenger Brahmin

Caste

65

1

  

Lingayat

Caste

98

Nil

  

Gowda

Caste

56

Nil

  

Bhovi

Caste

52

Nil

  

Christian

Religious Group

55

Nil

  

Muslim

Religious Group

65

Nil

  

Kuruva

Tribe

60

1

16

Sikkim

Bhutia

Tribe

75

Nil

  

Nepali

Caste

110

Nil

  

Lepcha

Tribe

48

Nil

17

Mizoram

Mara

Tribe

90

Nil

  

Hmar

Tribe

80

Nil

  

Lai

Tribe

92

Nil

  

Lusei

Tribe

92

Nil

  

Kuki

Tribe

105

Nil

18

Arunachal Pradesh

Adi Pasi

Tribe

203

Nil

19

Manipur

Garo

Tribe

110

Nil

  

Meitei

Tribe

105

Nil

  

Naga

Tribe

106

Nil

  

Hmar

Tribe

101

Nil

  

Manipuri Muslim

Religious Group

101

Nil

20

Andaman & Nicobar Islands

Great Andamanese

Tribe

24

Nil

  

Jarawa

Tribe

50

Nil

  

Onge

Tribe

16

Nil

  

Nicobarese

Tribe

28

Nil

  

Shompen

Tribe

33

Nil

* NUMBER OF POPULATIONS ANALYZED: 104; 4257 MALES OUT OF 7214 INDIVIDUALS ANALYZED IN GENETIC PROFILING

Amelogenin typing using commercial genotyping kits

The DNA samples were amplified using commercial multiplex short tandem repeat (STR) kits; PowerPlex® 16 system (Promega Corporation, Madison, USA) and Identifiler™ (Applied Biosystems, Foster City, CA), which include the amelogenin marker for gender determination. Genotyping of the amplified products was performed on an ABI Prism™ 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). The amelogenin profile was determined from the electropherograms by comparing the presence or absence of 106 and 112 bp peaks with known male and female controls. Females exhibit a single peak of 106 bp while males exhibit two peaks of 106 and 112 bps.

Amelogenin typing using newly designed primers and other published primers

Samples that showed abnormal amelogenin peak profiles with the commercial kits were reamplified with primers described by Steinlechner et al. [11] followed by genotyping as described above. Male samples exhibit two peaks of 219 and 225 bp, while female samples exhibit a single peak at 219 bp.

Additional primers were designed for amplifying the region identified by Roffey et al. [8] to decipher the origin of mutations. The sequences of new primers designed to facilitate detection of mutation are as follows:

1. #P1: 5'- TTACGGCCATATTTAGGA-3' (for amplification of X and Y homologues)

2. #P2: 5'- GAAAGAGTCAATCCGAATGGT-3' (for amplification of Y homologue)

Analysis of SRY, Y-STRs and Y-SNPs

To confirm the gender of the studied samples, a sex-determining locus (SRY) [13] specific to males, was amplified. Occurrence of a single 93 bp amplicon would distinguish an authentic male DNA sample from a female DNA sample. Further, four Y- short tandem repeats (Y-STRs) [14, 15] (DYS19, DYS389I, DYS389II and DYS390) were amplified to determine the extent of deletion of the Y-chromosome and to determine if Y-STR haplotype profiles were shared between individuals. Y-STR amplification was carried out in a single tube multiplex reaction [14] and genotyped on an ABI Prism™ 377 DNA Sequencer (PE Applied Biosystems, Foster City, CA). Y-single nucleotide polymorphisms (Y-SNPs) (M89, M9, M172, 92R7, M45, M20, M70, M214, M69, M124, M173, M17) [16] were profiled hierarchically to identify the lineage of the test samples.

Results and Discussion

Out of the 4,257 males analyzed with either PowerPlex® 16 or Identifiler™ multiplex system, 10 confirmed male samples exhibited a dropout of the 112 bp amelogenin Y-allele (Table 1). To verify the cause of observed abnormalities, we tested such samples with alternate primer pairs that encompassed the region amplified with primers reported by Sullivan et al. [6] and, are also typically used in the commercial kits. Amplification of the test samples with the primer set described by Steinlechner et al. [11] resulted in complete absence of the 225 bp Y-specific product.

All 10 ambiguous samples were confirmed to be from male individuals on testing with male-specific SRY locus, which yielded the characteristic 93 bp amplicon reconfirming the gender of these subjects as males.

Additional analysis with four Y-chromosomal STR markers, DYS19, DYS389I, DYS389II and DYS390, yielded complete and different Y-STR haplotype profiles. Amplification of Y-STR indicates that these samples had failed the amelogenin typing either due to mutation in the primer-binding region [17] or due to deletions in the amelogenin region (11.2p) on the Y-chromosome [11]. Among the 10 samples, eight distinct Y-STR haplotype profiles were observed; one was shared between Khatri and Kuruva and, another one by Agharia and Kallar (Table 2). Further amplification and sequencing was carried out using the forward primer of Steinlechner et al. [11] and a set of newly designed reverse primers spanning the hypothetical region of mutation – #P1, which is 62 bp downstream, and #P2, a Y-specific primer that is 43 bp downstream to the Steinlechner's reverse primer binding region, in order to determine the nature and extent of mutation. The newly designed primers depicted in Fig.2, result in 287 bp and 268 bp amplicon for #P1 and #P2 respectively, for the Y-chromosome, when used along with the forward primer of Steinlechner et al., #P1 results in a 281 bp product for the X-chromosome. However, the newly designed and validated primer sets also failed to amplify the Y-homologue in test samples suggesting deletion of a significant portion of the amelogenin region in male samples. The deletion in amelogenin gene has recently been mapped to span around 2.5 Mb [18].
Table 2

Y-chromosome profiles of the amelogenin-deletion individuals*

S. No

Sample

Y-SNP

DYS 19

DYS 389I

DYS 389II

DYS 390

1

Kallar

NA

15

12

29

25

2

Vanniyar

J2

15

13

29

24

3

Agharia

NA

15

12

29

25

4

Khatri

NA

15

13

30

25

5

Iyenger Brahmin

J2

14

13

29

24

6

Kuruva

NA

15

13

30

25

7

Khandayat

J2

15

11

30

23

8

Paroja

J2

15

13

26

25

9

Desasth Brahmin

J2

14

13

30

23

10

Maratha

J2

15

11

28

25

NA: Samples not be examined for Y-SNPs

* Integers in bold represent shared haplotype between Kallar and Agahria; while those in bold italics represent haploype sharing between Khatri and Kuruva

https://static-content.springer.com/image/art%3A10.1186%2F1471-2350-7-37/MediaObjects/12881_2005_Article_145_Fig2_HTML.jpg
Figure 2

Y-chromosome nucleotide sequence of the human amelogenin gene (GenBank Accession Number M55419) showing the location of annealing regions of the primer sets used in this study.

The overall rate of failure among the Indian population was found to be 0.23%. Table 1 shows the frequency of failure among different endogamous groups. Failures were exhibited by both caste groups (Khatri, Desasth Brahmin, Maratha, Khandayat, Tanjore Kallar, Vanniyar and Iyenger Brahmin) and tribal populations (Agharia, Paroja, and Kuruva) while Mongoloid and Negrito populations were not found to harbor the deletions (Fig. 1). Interestingly, we observed that these deletions were present predominantly in individuals belonging to the J2 Y-chromosomal lineage. J2 is found in approximately 5.1% of the Indian population, while majority of the Indian males harbor H (25%), R1a1 (19%) and R2 (16%) haplogroups in their Y-chromosomes [19]. Probably originating in the Middle East [20], the J2 lineage has been found distributed across southeastern Europe and Asia with frequencies of 6.5% in Central Asians [16], 23.8% in Sephardic Jews, 20% in Lebanese, 17.8% in Konyan Turks, 16.3% among Italians of Apulia, 13.6% in French Basque [20], 10.2% in Moroccan Arabs [21]. The above observations and positive amplification of the SRY gene and the appearance of discrete Y-STR haplotypes, suggests that the mutations probably arose independently on a J2 Y chromosome lineage background.

Of the endogamous populations screened in this study, ~10 % exhibited failures in the amelogenin gender test. Since the extent of deletion is large to avoid amplification dropout of the Y-homologue with currently available commercial primer sets, we suggest it would be prudent to include an additional gender test such as SRY and/or Y-STR testing for accurate gender identification of biological specimens.

Conclusion

Earlier studies have reported high failure rates in amelogenin-based gender testing of individuals from the Indian sub-continent. In this study, we have analyzed 4,257 male samples and report a failure rate of 0.23%. Due to ease of typing, this test has gained wide acceptance and has been integrated into routine automated genetic profiling procedures. However, the fallibility of the amelogenin test raises concern over its continued use especially in medical and forensic sciences.

Although our study indicates that individuals belonging to the J2 lineage are more prone to deletion in Y-derived amelogenin gene, further corroborating studies are desired. The amelogenin-based gender test thus needs to be applied with caution, with supplementation with other Y-chromosome specific analyses for reliable gender identification.

Declarations

Acknowledgements

This study was supported by a research grant under the X Five Year Plan to CFSL, Kolkata. The authors are thankful to Rajkumar R, Gaikwad S, Sarkar N, Tandon M, Guha S, Maity B, Ashma R, Banerjee J, Singh A and G. Hima Bindu, research scholars of the National DNA Analysis Centre, Kolkata and S. Krittika, JRF (Indian Statistical Institute, Kolkata) for providing their data for use in this study. We also thank the two reviewers for their valuable suggestions. SS and ST were assisted with MHA and CSIR fellowships, respectively.

Authors’ Affiliations

(1)
National DNA Analysis Centre.Central Forensic Science Laboratory
(2)
National Institute of Biologicals

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  22. Pre-publication history

    1. The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/7/37/prepub

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© Kashyap et al; licensee BioMed Central Ltd. 2006

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