X-chromosomal short tandem repeats (X-STRs) are short, highly polymorphic genetic markers characterised by a unique inheritance pattern: males are hemizygous, inheriting a single X chromosome from their mother, while females inherit one from each parent. This makes X-STRs particularly advantageous for complex kinship cases, such as deficiency paternity testing involving female offspring. Despite their utility, there is currently a paucity of population-specific X-STR data for the Maltese population. This pilot study aimed to characterize X-STR variation within the Maltese archipelago to address this gap and support the development of a representative national X-STR genetic database. Buccal swabs were collected from 150 unrelated Maltese males and pseudo-anonymized. The MagMAX™ DNA Multi-Sample Ultra 2.0 kit was used for DNA extraction, and the NanoDrop™ 2000 for quantification. Multiplex amplification of the 12 X-STR markers in the Investigator® Argus X-12 QS kit (QIAGEN) and the 15 X-STR markers in the GT XDetector® kit (GENETEK BIOPHARMA) was performed using a Biometra® TProfessional thermal cycler. The GT XDetector® kit was subsequently validated against the Argus X-12 kit to ensure concordance and sensitivity. The Spectrum® Compact CE system was used for fragment separation and sizing, and GeneMarker® HID for haplotyping. Statistical parameters, including haplotype frequencies and population genetic metrics, were calculated using Excel, and SPSS. Preliminary results across the four X-chromosomal linkage groups (LG1–LG4) demonstrate a high degree of genetic diversity. The majority of observed alleles and haplotypes were unique to individual samples, resulting in high haplotype diversity (HD). While most haplotypes were sporadic, specific shared lineages were identified in each linkage group, most notably 11-24-26.1 (LG1), 13-16-17 (LG2), 12-30.2-19 (LG3), and 14-36-44.2 (LG4). High-frequency alleles were documented at key loci, including allele 26.1 (DXS10148), 14 (DXS7132), 19 (DXS10103), and 15 (DXS7423). These distribution patterns align with established X-STR population databases, confirming the forensic utility of this marker set for kinship testing and human identification. This study generated the first X-STR haplotype dataset for the Maltese population, contributing to global databases and improving the statistical reliability of forensic and genetic analyses involving individuals of Maltese origin.

This research was supported by the FUSION: R&I Research Excellence Programme CAREOMICS (REP-2024-067, Xjenza Malta), the Research Networking Scheme (RNS-2026-036I, Xjenza Malta), COST Action CA22106 MDVI, QIAGEN GmbH, and GENETEK BIOPHARMA GmbH.

Authors

• Dr Joanna Vella (Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Malta)
• Sarah Theuma (Department of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, Malta)