1. PRESENCE OF CONSTITUTIONAL (GERMINAL) Y-CHROMOSOME SEQUENCES
IN DNA OF A YOUNG FEMALE VICTIM OF SEXUAL ASSAULT
Agnieszka Maciejewska1, Beata S. Lipska-Ziętkiewicz2, Magda Koczkowska2, Joanna Chamier-Ciemińska1, Janusz Limon2, Ryszard Pawłowski1
1Department of Forensic Medicine, Medical University of Gdańsk, Poland
2Department of Biology and Genetics, Medical University of Gdańsk, Poland
A thirteen-year-old girl (SW) was raped and forced to oral sex by a man she knew (GL). SW testified that the assailant used a condom but ejaculated to her mouth. Genital tract and mouth swabs were collected 18 hr after the rape. No spermatozoa were observed in any of SW swabs tested in a hospital laboratory. Then a private laboratory A (Lab A) performed SAP (seminal acid phosphatase) tests and DNA typing. According to the Lab A report, all swabs collected from SW were SAP negative (microscope searching for spermatozoa and PSA testing were not performed), the SGMPlus® profiling revealed the victim’s profile only, while Yfiler® typing gave negative results for all samples except for an oral swab, which revealed a haplotype of an unknown man. Inconsistency between DNA profiling results and the victim’s testimony resulted in the decision of the court to retest evidence samples.
INTRODUCTION
MATERIALS AND METHODS
DNA samples: DNA extracts isolated from SW genital tract swabs by Lab A, SW and GL buccal swabs (reference material).
Hematoxylin-Eosin stained microscopic slides with smears from oral and genital tract of SW obtained during gynecological examination.
DNA concentration was determined using Real Time PCR method and Alu Yb8 repetitive sequence specific for human DNA [1].
DNA profiling: PowerPlex ESX17 and PowerPlex Y23 (Promega), Investigator Argus X-12 (Qiagen).
Array-CGH was performed using CytoSure ISCA UPD 4x180k (Oxford Gene Technology Inc.). A pool of female DNA (Promega) was used as a reference. The array was scanned at 2 µm using MS200 Microarray Scanner (Roche). Feature Extraction (Agilent Technologies Inc.) and Nexus Copy Number 7.5 (BioDiscovery Inc.) were applied for data analysis.
Figure 1. Representative example of the PowerPlex® Y23 DNA profile obtained from SW’s genital tract swab. Nine of 23 amplified loci were present.
Figure 2. Relative positions of 23 Y-STR loci (Human Genome version 19) available in the PowerPlex® Y23 System. Loci shown in blue where detected in SW’s DNA.
RESULTS AND DISCUSSION
All DNA extracts obtained from Lab A were retested in our laboratory and gave identical amplification signals for 9 of 23 Y-STR loci tested (Fig. 1). Identified haplotype did not match either the suspect GL or an unknown man identified at Lab A. DNA testing of the victim reference sample with PowerPlex Y23 kit revealed the same Y-STR profile as obtained for all victim’s swabs and lack of Y allele when amelogenin locus was analyzed. From PowerPlex Y23 typing results it can be estimated that at least 7Mb of Y chromosome is present in victims DNA (Fig. 2). Analysis of X-chromosome specific loci identified 8 heterozygotes indicating the presence of two X chromosomes. No AMELY allele or DNA mixture was detected with PowerPlex ESX17 (not shown) and Argus X-12 typings (Fig. 3). Finally victim’s DNA was subjected to the array-CGH testing, which revealed the presence of Yq to Yq (12,500,000-28,784,530 bp); besides weaker signals at Yp11.31 (2,650,559-2,914,431 bp) and Yp11.2 (6,108,898-6,445,542 bp) were also noted. The analysis of Y-STR loci located within the Yq confirmed the array-CGH result. However, no Y-STR loci were amplified in the chromosome regions Yp11.31 and Yp11.2. The later will be worked out by analyzing aCGH profle of the victim’s probes against reference male DNA (analysis pending).
The result of this study indicates that high-resolution array-CGH technique may serve as extension of STR analysis (Fig.4).Because of the lack of good quality DNA and impossibility to obtain samples for karyotyping and FISH (blood leukocytes, fibroblasts etc.), we can only speculate whether the observed aberration results from a constitutional (germinal) translocation of Y chromosome fragment to other chromosome or from the presence of a structurally abnormal Y chromosome. Lack of amplification of DYS loci, in all the victim’s samples analyzed at the Lab A, remains unknown.
Figure 3. Argus X-12 DNA profile obtained from SW’s buccal swab sample. Heterozygotes in eight loci were observed, indicating the presence of two X chromosomes.
Figure 4. Array-CGH profiles obtained using genomic DNA isolated from SW’s buccal swab sample.
The log2 ratio of victim’s probes against female reference DNA is plotted as a function of chromosomal position according to NCBI37/hg19 assembly. Also, the mapping of selected Y-STR loci is shown.
Note: Even though the par value of resolution of aCGH experiments was 85 kbp, only gross aberrations could positively be detected because of fair average quality of DNA extracted (mad1dr 0.39) resulting in considerable signal scattering.
CONCLUSION
This case and previous observations of mutations at amelogenin gene [2-4] strongly indicates that Y-STR typing of female reference samples should be always performed routinely.
BIBLIOGRAPHY
[1] Mirowska A., Pawłowski R.: Determination of nuclear DNA concentration using Real-Time PCR technique. Problems of Forensic Sciences 68 (2006) 351–361.
[2] Maciejewska A. , Pawłowski R.: A rare mutation in the primer binding region of the Amelogenin X homologue gene. Forensic Science International: Genetics 3 (2009) 265–267.
[3] Shadrach B et al.: A rare mutation in the primer binding region of the amelogenin gene can interfere with gender identification. The Journal of Molecular Diagnostics 6 (2004) 401–405.
[4] Xueling Ou et al.: Null alleles of the X and Y chromosomal amelogenin gene in a Chinese population. International Journal of Legal Medicine 126 (2012) 513–518.