Amelogenin

Gender identification (sex-typing) in commonly performed in conjunction with STR typing kit using PCR products generated from the amelogenin gene that occurs on both the X- and Y-chromosome. A commonly used PCR primer set first published by Sullivan et al. (1993) BioTechniques 15:637-641 targets a 6 bp deletion that occurs on the X-chromosome, which enables amplicons generated from the X- and Y-chromosome to be distinguished from one another when electrophoretic separation is performed to separate STR alleles. Most commercial STR kits utilize the Sullivan et al. (1993) primers or minor modifications. Since females are X,X, only a single peak is observed when testing female DNA whereas males, which possess both X and Y chromosomes, exhibit two peaks with a standard amelogenin test.

The ratio of amelogenin X and Y PCR products can be helpful in deciphering mixtures involving male and female DNA such as sexual assault evidence. However, as noted below, there have been reports in the literature for anomalous amelogenin results due to primer binding site mutations or deletions of sections of the Y-chromosome.

Alignment of Amelogenin Reference Sequences from the X and Y Chromosomes

Reports of Anomalous Amelogenin Alleles:

• Males possessing only a single X amelogenin amplicon (Y null) - a male DNA sample will falsely look like a female DNA sample:

  • Santos et al. (1998) reported a rare deletion of the amelogenin gene on the Y-chromosome

  • Y-STR typing can be performed to verify that other portions of the Y-chromosome are present

• Males possessing only a single Y amelogenin amplicon (X null):

  • Shewale et al. (2000) observed loss of the X chromosome amplicon in three out of almost 7,000 males examined

  • while this phenomenon should not result in a gender misclassification (as the Y null situation might), its occurrence can impact the expected X and Y amplicon ratios in a mixture (see NIST MIX05 interlab study, case #3)

Helpful References Regarding Amelogenin:

Akane, A. (1998) Sex determination by PCR analysis of the X-Y amelogenenin gene. Methods Mol.Biol. 98: 245-249.

Brinkmann, B. (2002) Is the amelogenin sex test valid? Int.J.Legal Med. 116(2): 63.

Budowle, B., Koons, B. W., and Errera, J. D. (1996) Multiplex amplification and typing procedure for the loci D1S80 and amelogenin. J.Forensic Sci. 41(4): 660-663.

Buel, E., Wang, G., and Schwartz, M. (1995) PCR amplification of animal DNA with human X-Y amelogenin primers used in gender determination. J.Forensic Sci. 40(4): 641-644.

Cadenas, A. M., Regueiro, M., Gayden, T., Singh, N., Zhivotovsky, L. A., Underhill, P. A., and Herrera, R. J. (2007) Male amelogenin dropouts: phylogenetic context, origins and implications. Forensic Sci.Int. 166(2-3): 155-163.

Cerri, N., Ricci, U., Sani, I., Verzeletti, A., and De Ferrari, F. (2003) Mixed stains from sexual assault cases: autosomal or y-chromosome short tandem repeats? Croat.Med J. 44(3): 289-292.

Chang, Y. M., Perumal, R., Keat, P. Y., Yong, R. Y., Kuehn, D. L., and Burgoyne, L. (2007) A distinct Y-STR haplotype for Amelogenin negative males characterized by a large Y(p)11.2 (DYS458-MSY1-AMEL-Y) deletion. Forensic Sci.Int. 166(2-3): 115-120.

Chang, Y. M., Both, K., and Burgoyne, L. A. (2002) Genetic variation at three DYS-STR loci and amelogenin between the Australian Caucasians and three Asian ethnic groups. J.Forensic Sci. 47(4): 918-921.

Chang, Y. M., Burgoyne, L. A., and Both, K. (2003) Higher failures of amelogenin sex test in an Indian population group. J Forensic Sci. 48(6): 1309-1313.

Cirigliano, V., Sherlock, J., Conway, G., Quilter, C., Rodeck, C., and Adinolfi, M. (1999) Rapid detection of chromosomes X and Y aneuploidies by quantitative fluorescent PCR. Prenat.Diagn. 19(12): 1099-1103.

Clayton, T. M., Whitaker, J. P., and Maguire, C. N. (1995) Identification of bodies from the scene of a mass disaster using DNA amplification of short tandem repeat (STR) loci. Forensic Sci.Int. 76: 7-15.

Clisson, I., Crubezy, E., Midant-Reynes, B., and Ludes, B. (2000) Megaplex analysis of brain remains from the necropolis of Adaima (Egypt, 3700-2800 BC). Progress in Forensic Genetics 8 (1999). 8: 555-557.

Collins, P. J., Hennessy, L. K., Leibelt, C. S., Roby, R. K., Reeder, D. J., and Foxall, P. A. (2004) Developmental validation of a single-tube amplification of the 13 CODIS STR loci, D2S1338, D19S433, and amelogenin: the AmpFlSTR Identifiler PCR Amplification Kit. J Forensic Sci. 49(6): 1265-1277.

Cotton, E. A., Allsop, R. F., Guest, J. L., Frazier, R. R., Koumi, P., Callow, I. P., Seager1 A, and Sparkes, R. L. (2000) Validation of the AMPFlSTR((R)) SGM plus system for use in forensic casework. Forensic Sci.Int. 112(2-3): 151-161.

Eng, B., Ainsworth, P., and Waye, J. S. (1994) Anomalous migration of PCR products using nondenaturing polyacrylamide gel electrophoresis: the amelogenin sex-typing system. J.Forensic Sci. 39(6): 1356-1359.

Evett, I. W., Gill, P. D., and Lambert, J. A. (1998) Taking account of peak areas when interpreting mixed DNA profiles. J.Forensic Sci. 43(1): 62-69.

Gill, P., Ivanov, P. L., Kimpton, C. P., Piercy, R., Benson, N., Tully, G., Evett, I., Hagelberg, E., and Sullivan, K. M. (1994) Identification of the remains of the Romanov family by DNA analysis. Nature Genetics. 6: 130-135.

Haas-Rochholz, H. and Weiler, G. (1997) Additional primer sets for an amelogenin gene PCR-based DNA-sex test. Int.J.Legal Med. 110(6): 312-315.

Hayashi, K., Hanaoka, Y., Matsumura, S., Takagi, T., Kajiwara, M., Tamaki, N., Minaguchi, K., and Sato, Y. (9-11-2000) An autopsy case of Klinefelter's syndrome suspected and its DNA analysis. Forensic Sci.Int. 113(1-3): 119-125.

Jobling, M. A., Lo, I. C., Turner, D. J., Bowden, G. R., Lee, A. C., Xue, Y., Carvalho-Silva, D., Hurles, M. E., Adams, S. M., Chang, Y. M., Kraaijenbrink, T., Henke, J., Guanti, G., McKeown, B., van Oorschot, R. A., Mitchell, R. J., de Knijff, P., Tyler-Smith, C., and Parkin, E. J. (2-1-2007) Structural variation on the short arm of the human Y chromosome: recurrent multigene deletions encompassing Amelogenin Y. Hum Mol.Genet. 16(3): 307-316.

Kimpton, C. P., Oldroyd, N. J., Watson, S. K., Frazier, R. R. E., Johnson, P. E., Millican, E. S., Urquhart, A., Sparkes, B. L., and Gill, P. (1996) Validation of highly discriminating multiplex short tandem repeat amplification systems for individual identification. Electrophoresis. 17: 1283-1293.

Komuro, T., Nakamura, M., Tsutsumi, H., and Mukoyama, R. (1998) Gender determination from dental pulp by using capillary gel electrophoresis of amelogenin locus. J.Forensic Odontostomatol. 16(2): 23-26.

Krenke, B. E., Tereba, A., Anderson, S. J., Buel, E., Culhane, S., Finis, C. J., Tomsey, C. S., Zachetti, J. M., Masibay, A., Rabbach, D. R., Amiott, E. A., and Sprecher, C. J. (2002) Validation of a 16-locus fluorescent multiplex system. J.Forensic Sci. 47(4): 773-785.

LaFountain, M. J., Schwartz, M., Cormier, J., and Buel, E. (1998) Validation of capillary electrophoresis for analysis of the X-Y homologous amelogenin gene. J.Forensic Sci. 43(6): 1188-1194.

Mannucci, A., Sullivan, K. M., Ivanov, P. L., and Gill, P. (1994) Forensic application of a rapid and quantitative DNA sex test by amplification of the X-Y homologous gene amelogenin. Int.J.Leg.Med. 106: 190-193.

Martinez-Gonzalez, L. J., Lorente, J. A., Martinez-Espin, E., Carlos, Alvarez J., Lorente, M., Villanueva, E., and Budowle, B. (2007) Intentional mixed buccal cell reference sample in a paternity case. J Forensic Sci. 52(2): 397-399.

McKeown, B., Stickley, J., and Riordan, A. (2000) Gender assignment by PCR of the SRY gene: an improvement on amelogenin. Progress in Forensic Genetics 8 (1999). 8: 433-435.

Michael, A. and Brauner, P. (2004) Erroneous gender identification by the amelogenin sex test. J Forensic Sci. 49(2): 258-259.

Mitchell, R. J., Kreskas, M., Baxter, E., Buffalino, L., and van Oorschot, R. A. (2006) An investigation of sequence deletions of amelogenin (AMELY), a Y-chromosome locus commonly used for gender determination. Ann.Hum Biol. 33(2): 227-240.

Mukherjee, K. K. and Biswas, R. (2005) Short tandem repeat (STRs) and sex specific Amelogenin analysis of blood samples from neurosurgical female transfused patients. J Clin.Forensic Med. 12(1): 10-13.

Pouchkarev, V. P., Shved, E. F., and Novikov, P. I. (1998) Sex determination of forensic samples by polymerase chain reaction of the amelogenin gene and analysis by capillary electrophoresis with polymer matrix. Electrophoresis. 19(1): 76-79.

Roffey, P. E., Eckhoff, C. I., and Kuhl, J. L. (2000) A rare mutation in the amelogenin gene and its potential investigative ramifications. J.Forensic Sci. 45(5): 1016-1019.

Santos, F. R., Pandya, A., and Tyler-Smith, C. (1998) Reliability of DNA-based sex tests. Nature Genetics. 18(2): 103-103.

Shadrach, B., Commane, M., Hren, C., and Warshawsky, I. (2004) A rare mutation in the primer binding region of the amelogenin gene can interfere with gender identification. J Mol.Diagn. 6(4): 401-405.

Shewale, J. G., Richey, S. L., and Sinha, S. K. (2000) Anomalous amplification of the amelogenin locus typed by AmpFlSTR Profiler Plus amplification kit. Forensic Science Communications. 2(4); http://www.fbi.gov/hq/lab/fsc/backissu/oct2000/shewale.htm

Sparkes, R., Kimpton, C. P., Watson, S., Oldroyd, N. J., Clayton, T. M., Barnett, L., Arnold, J., Thompson, C., Hale, R., Chapman, J., Urquhart, A., and Gill, P. (1996) The validation of a 7-locus multiplex STR test for use in forensic casework: (I) Mixtures, ageing, degradation and species studies. Int.J.Legal Med. 109: 186-194.

Steinlechner, M., Berger, B., Niederstatter, H., and Parson, W. (2002) Rare failures in the amelogenin sex test. Int.J.Legal Med. 116(2): 117-120.

Sullivan, K. M., Mannucci, A., Kimpton, C. P., and Gill, P. (1993) A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. BioTechniques. 15(4): 637-641.

Thangaraj, K., Reddy, A. G., and Singh, L. (2002) Is the amelogenin gene reliable for gender identification in forensic casework and prenatal diagnosis? Int.J.Legal Med. 116(2): 121-123.

Vauhkonen, H., Hedman, M., Vauhkonen, M., Sipponen, P., and Sajantila, A. (2004) Typing of XY (male) genotype from malignant neoplastic tissue by the amelogenin-based sex test. J Forensic Sci. 49(2): 222-226.

Zehner, R. and Bohrer, U. (1998) DYS19 and amelogenin in artificial blood stains with defined amounts of male and female cells. Int.J.Legal Med. 111(6): 340-342.

RETURN TO HOMEPAGE

Last Updated: 03/10/2008