Abstract
Sex identification of ancient individuals is important to understand aspects of the culture, demographic structure, religious practices, disease association, and the history of the ancient civilizations. Sex identification is performed using anthropometric measurements and molecular genetics techniques, including quantification of the X and Y chromosomes. These approaches are not always reliable in subadult, or fragmented, incomplete skeletons or when the DNA is highly degraded. Most of the methods include the identification of the male and female sexes, but the absence of a specific marker for the males does not mean that the sample obtained was from a female. This study aims (1) to identify new male-specific regions that allow male identification; (2) to contrast the effectiveness of these markers against AMELX/AMELY and anthropometric measurement procedures; and (3) to test the efficacy of these markers in archaeological samples. For the first two aims, we used known sex samples, and for the third aim, we used samples from different archaeological sites. A novel molecular technique to identify male-specific regions by amplification of TTTY7, TSPY3, TTTY2, and TTTY22 genes of the human Y chromosome was developed. The results showed amplification of the specific DNA regions of Y chromosome in male individuals, with no amplification being observed in any of the female samples, confirming their specificity for male individuals. This approach complements the current procedures, such as the AMELX/AMELY test and anthropometric principle.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
All data generated or analysed during this study are included in this published article.
Abbreviations
- Chr-X:
-
X chromosome
- Chr-Y:
-
Y chromosome
References
Adler CJ, Haak W, Donlon D, Cooper A (2011) Survival and recovery of DNA from ancient teeth and bones. J Archaeol Sci 38(5):956–964. https://doi.org/10.1016/j.jas.2010.11.010
Álvarez-Sandoval BA, Manzanilla LR, Montiel R (2014) Sex determination in highly fragmented human DNA by high-resolution melting (HRM) analysis. PLoS One 9:e104629. https://doi.org/10.1371/journal.pone.0104629
Bashamboo A, Giran HM, Azfer MA et al (2003) Genomics of the human Y chromosome and molecular diagnosis. Proc Indian Natl Sci Acad B Biol Sci, B69:525–538
Bass WM (1987). Human osteology: a laboratory and field manual. Missouri Archaeological Society
Blau S, Ubelaker DH (2009) Handbook of forensic anthropology and archaeology. Left Coast Press
Briggs AW, Stenzel U, Johnson PLF, Green RE, Kelso J, Prufer K, Meyer M, Krause J, Ronan MT, Lachmann M, Paabo S (2007) Patterns of damage in genomic DNA sequences from a Neandertal. Proc Natl Acad Sci 104:14616–14621. https://doi.org/10.1073/pnas.0704665104
Butler EK, Li R (2014) Genetic markers for sex identification in forensic DNA analysis. J Forensic Investig 2(3)
Campos PF, Craig OE, Turner-Walker G, Peacock E, Willerslev E, Gilbert MTP (2012) DNA in ancient bone—where is it located and how should we extract it? Ann Anat 194(1):7–16. https://doi.org/10.1016/j.aanat.2011.07.003
Candelas-González N, Rascón Pérez J, Chamero B et al (2017) Geometric morphometrics reveals restrictions on the shape of the female os coxae. J Anat 230:66–74. https://doi.org/10.1111/joa.12528
Dabney J, Meyer M, Pääbo S (2013) Ancient DNA Damage. Cold Spring Harb Perspect Biol 5. https://doi.org/10.1101/cshperspect.a012567
Dutta P, Bhosale S, Singh R et al (2017) Amelogenin gene—the pioneer in gender determination from forensic dental samples. J Clin Diagn Res 11(2):ZC56–ZC59. https://doi.org/10.7860/JCDR/2017/22183.9407
Esteve-Codina A, Niederstätter H, Parson W (2008) “GenderPlex” a PCR multiplex for reliable gender determination of degraded human DNA samples and complex gender constellations. Int J Legal Med 123:459–464. https://doi.org/10.1007/s00414-008-0301-z
Fazekas IG, Kósa F (1978) Forensic fetal osteology. Akadémiai Kiadó, Budapest
García-Campos C, Martinón-Torres M, Martín-Francés L, Martínez de Pinillos M, Modesto-Mata M, Perea-Pérez B, Zanolli C, Labajo González E, Sánchez Sánchez JA, Ruiz Mediavilla E, Tuniz C, Bermúdez de Castro JM (2018a) Contribution of dental tissues to sex determination in modern human populations. Am J Phys Anthropol 166:459–472. https://doi.org/10.1002/ajpa.23447
García-Campos C, Martinón-Torres M, de Pinillos MM et al (2018b) Modern humans sex estimation through dental tissue patterns of maxillary canines. Am J Phys Anthropol 167:914–923. https://doi.org/10.1002/ajpa.2371
Gaudio D, Fernandes DM, Schmidt R, Cheronet O, Mazzarelli D, Mattia M, O’Keeffe T, Feeney RNM, Cattaneo C, Pinhasi R (2019) Genome-wide DNA from degraded petrous bones and the assessment of sex and probable geographic origins of forensic cases. Sci Rep 9:8226. https://doi.org/10.1038/s41598-019-44638-w
GeneCards-Human Genes (n.d.) | Gene Database | URL https://www.genecards.org/
Gibbon V, Paximadis M, Strkalj G et al (2009) Novel methods of molecular sex identification from skeletal tissue using the amelogenin gene. Forensic Sci Int Genet 3(2):74–79. https://doi.org/10.1016/j.fsigen.2008.10.007
Graw M, Wahl J, Ahlbrecht M (2005) Course of the meatus acusticus internus as criterion for sex differentiation. Forensic Sci Int 147:113–117. https://doi.org/10.1016/j.forsciint.2004.08.006
Green RE, Krause J, Briggs AW, Maricic T, Stenzel U, Kircher M, Patterson N, Li H, Zhai W, Fritz MHY, Hansen NF, Durand EY, Malaspinas AS, Jensen JD, Marques-Bonet T, Alkan C, Prufer K, Meyer M, Burbano HA, Good JM, Schultz R, Aximu-Petri A, Butthof A, Hober B, Hoffner B, Siegemund M, Weihmann A, Nusbaum C, Lander ES, Russ C, Novod N, Affourtit J, Egholm M, Verna C, Rudan P, Brajkovic D, Kucan Z, Gusic I, Doronichev VB, Golovanova LV, Lalueza-Fox C, de la Rasilla M, Fortea J, Rosas A, Schmitz RW, Johnson PLF, Eichler EE, Falush D, Birney E, Mullikin JC, Slatkin M, Nielsen R, Kelso J, Lachmann M, Reich D, Paabo S (2010) A draft sequence of the Neandertal genome. Science 328(5979):710–722. https://doi.org/10.1126/science.1188021
Halder A, Kumar P, Jain M, Iyer VK (2017) Copy number variations in testicular maturation arrest. Andrology 5(3):460–472. https://doi.org/10.1111/andr.12330
Harris SM, Case DT (2012) Sexual dimorphism in the tarsal bones: implications for sex determination. J Forensic Sci 57:295–305. https://doi.org/10.1111/j.1556-4029.2011.02004.x
Hora M, Sládek V (2018) Population specificity of sex estimation from vertebrae. Forensic Sci Int 291:279.e1–279.e12. https://doi.org/10.1016/j.forsciint.2018.08.015
Integrative Genomics Viewer (2018) | Nature Biotechnology | URL https://www.nature.com/articles/nbt.1754
Kaestle FA, Horsburgh KA (2002) Ancient DNA in anthropology: methods, applications, and ethics. Am J Phys Anthropol Suppl 35:92–130. https://doi.org/10.1002/ajpa.10179
Kashyap V, Sahoo S, Sitalaximi T, Trivedi R (2006) Deletions in the Y-derived amelogenin gene fragment in the Indian population. BMC Med Genet 7:37. https://doi.org/10.1186/1471-2350-7-37
Kim K-Y, Kwon Y, Bazarragchaa M, Park AJ, Bang H, Lee WB, Lee J, Lee KH, Kim BJ, Kim K (2013) A real-time PCR-based amelogenin Y allele dropout assessment model in gender typing of degraded DNA samples. Int J Legal Med 127:55–61. https://doi.org/10.1007/s00414-011-0663-5
Krishan K, Chatterjee PM, Kanchan T et al (2016) A review of sex estimation techniques during examination of skeletal remains in forensic anthropology casework. Forensic Sci Int 261:165.e1–165.e8. https://doi.org/10.1016/j.forsciint.2016.02.007
Lin Z, Kondo T, Minamino T, Ohtsuji M, Nishigami J, Takayasu T, Sun R, Ohshima T (1995) Sex determination by polymerase chain reaction on mummies discovered at Taklamakan desert in 1912. Forensic Sci Int 75:197–205. https://doi.org/10.1016/0379-0738(95)01789-5
Lindahl T (1993) Instability and decay of the primary structure of DNA. Nature 362:709–715. https://doi.org/10.1038/362709a0
Llamas B, Valverde G, Fehren-Schmitz L, Weyrich LS, Cooper A, Haak W (2017) From the field to the laboratory: controlling DNA contamination in human ancient DNA research in the high-throughput sequencing era. STAR 3:1–14. https://doi.org/10.1080/20548923.2016.1258824
Luna LH, Aranda CM, Santos AL (2017) New method for sex prediction using the human non-adult auricular surface of the ilium in the collection of identified skeletons of the University of Coimbra. Int J Osteoarchaeol 27:898–911. https://doi.org/10.1002/oa.2604
Moore M (2013) Sex estimation and assessment. Research methods in human skeletal biology, In, pp 91–116
Ovchinnikov IV, Ovtchinnikova OI, Druzina EB et al (1998) Molecular genetic sex determination of Medieval human remains from North Russia: comparison with archaeological and anthropological criteria. Anthropol Anz 56(1):7–15
Quincey D, Carle G, Alunni V, Quatrehomme G (2013) Difficulties of sex determination from forensic bone degraded DNA: a comparison of three methods. Sci Justice J Forensic Sci Soc 53:253–260. https://doi.org/10.1016/j.scijus.2013.04.003
Quintana-Murci L, Fellous M (2001). The human Y chromosome: the biological role of a “functional wasteland.” J Biomed Biotechnol 1:18–24. https://doi.org/10.1155/S1110724301000080
Rohland N, Hofreiter M (2007) Ancient DNA extraction from bones and teeth. Nat Protoc 2:1756–1762. https://doi.org/10.1038/nprot.2007.247
Schutkowski H (1987) Sex determination of fetal and neonate skeletons by means of discriminant analysis. Int J Anthropol 2:347–352. https://doi.org/10.1007/BF02443994
Skoglund P, Malmström H, Raghavan M et al (2012) Origins and genetic legacy of Neolithic farmers and hunter-gatherers in Europe. Science (New York, N.Y.) 336(6080):466–469. https://doi.org/10.1126/science.1216304
Skoglund P, Storå J, Götherström A, et al. Accurate sex identification of ancient human remains using DNA shotgun sequencing, vol. 40; 2013.
Stelzer G, Rosen N, Plaschkes I, Zimmerman S, Twik M, Fishilevich S, Stein TI, Nudel R, Lieder I, Mazor Y, Kaplan S, Dahary D, Warshawsky D, Guan-Golan Y, Kohn A, Rappaport N, Safran M, Lancet D (2016) The GeneCards suite: from gene data mining to disease genome sequence analyses. Curr Protoc Bioinformatics 54:1.30.1–1.30.33. https://doi.org/10.1002/cpbi.5
Stevens AJ, Taylor MG, Pearce FG, Kennedy MA (2017) Allelic dropout during polymerase chain reaction due to G-quadruplex structures and DNA methylation is widespread at imprinted human loci. G3 (Bethesda) 7:1019–1025. https://doi.org/10.1534/g3.116.03868
Stone AC, Milner GR, Pääbo S, Stoneking M (1996) Sex determination of ancient human skeletons using DNA. Am J Phys Anthropol 99:231–238. https://doi.org/10.1002/(SICI)1096-8644(199602)99:2<231::AID-AJPA1>3.0.CO;2-1
Takayama T, Takada N, Suzuki R, Nagaoka S, Watanabe Y, Kumagai R, Aoki Y, Butler JM (2009) Determination of deleted regions from Yp11.2 of an amelogenin negative male. Legal Med 11:S578–S580. https://doi.org/10.1016/j.legalmed.2009.01.049
Thorvaldsdóttir H, Robinson JT, Mesirov JP (2013) Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform 14:178–192. https://doi.org/10.1093/bib/bbs017
Tschentscher F, Frey UH, Bajanowski T (2008) Amelogenin sex determination by pyrosequencing of short PCR products. Int J Legal Med 122:333–335. https://doi.org/10.1007/s00414-008-0228-4
Ubelaker DH (2014) Osteology reference collections. In: Smith C (ed) Encyclopedia of global archaeology. Springer New York, New York, pp 5632–5641
Ubelaker DH, DeGaglia CM (2017) Population variation in skeletal sexual dimorphism. Forensic Sci Int 278:407.e1–407.e7. https://doi.org/10.1016/j.forsciint.2017.06.012
Veroni A, Nikitovic D, Schillaci MA (2010) Brief communication: sexual dimorphism of the juvenile basicranium. Am J Phys Anthropol 141:147–151. https://doi.org/10.1002/ajpa.21156
White TD, Folkens PA (2005) The human bone manual. Elsevier Academic Press, San Diego
Willerslev E, Cooper A (2005) Ancient DNA. Proc Biol Sci 272:3–16. https://doi.org/10.1098/rspb.2004.2813
Acknowledgements
We thank the Architect Engineer Verónica Alejandra Roman-Navarro for the advice provided to develop Figs. 1 and 3. In addition, we specially acknowledge the Puxcatan, Abrigo Rocoso Fidencio López, and Sima Cuesta Chica native communities (Tacotalpa, Tabasco) for their collaboration in this project. We sincerely thank reviewers for their careful reading of our manuscript and their many insightful comments and suggestions.
Code availability
Not applicable.
Funding
We thank Consejo Nacional de Ciencia y Tecnología (CONACYT) for the financial support of the scholarship awards to M.T.N.-R (Becas Nacionales 2014_Primer Periodo-380118 and Becas Mixtas_2015-Marzo 2016-290936) and E.D.-de-la-C. (CVU 485179, CB 2015/258103, and AYTE. 177559; and COMECYT 18BTD0020). We also thank the FIS IMSS for financial support (FIS/IMSS/PROT/PRIO/17/063, 2017-785-071) and the research scholarship of IMSS Foundation A. C. given to Dr. Normand García Hernández.
Author information
Authors and Affiliations
Contributions
MTN-R and M-d-LM conceived and designed the research; MTN_R performed the research; EA- and ET-E contributed with the data of the ancient bones; ED-de-la-Cruz and MAM-G performed amplification experiments; NG-H obtained the contemporary DNA and contributed with data analysis; MTN-R and M-d-LM wrote the paper; all authors read and approved the manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Ethics approval
The samples were registered to project number 2017-785-071, and the National Ethics Commission (IMSS) ethics committee approved the study.
Consent to participate
The participants provided their written consent to participate.
Consent for publication
The participants provided their written consent for publication.
Additional information
Responsible Editor: Irina Solovei
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
ESM 1
(XLSX 12 kb)
Rights and permissions
About this article
Cite this article
Navarro-Romero, M.T., Muñoz, M.d.L., Alcala-Castañeda, E. et al. A novel method of male sex identification of human ancient skeletal remains. Chromosome Res 28, 277–291 (2020). https://doi.org/10.1007/s10577-020-09634-1
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10577-020-09634-1