Personal identification from skeletal remain by D1S80, HLA DQA1, TH01 and polymarker analysis Shin-ichi Kubo1, Yoshihiko Fujita1, 2, Yasushi Yoshida2, Katsuya Kangawa2, Itsuo Tokunaga1, and Takako Gotohda1 1Department of Legal Medicine, The University of Tokushima School of Medicine, Tokushima, Japan;and 2Scientific Investigation Research Laboratory, Tokushima Prefectural Police Headquarters, Tokushima, Japan Abstract: The completely reduced skeleton was found in a mountain stream. We presumed that the skeleton was a woman from a morphology feature of the skull and the pelvis bone. The level of the suture of the skull indicated that the age was the first half of the 70-years old from the 60-years old. As a result of the police investigation, the possibility of 66 years old woman who was missing for about six months was suspected. To inquire into her identification, a skull and left thighbone were cut off, and blood was collected from the suspect's daughters. We examined blood (ABO) and DNA types (D1S80, HLA DQA1 TH01 and polymarker system) for the skeleton and the suspect's families. Blood and DNA types analysis of two daughters revealed that their patients have 19 alleles in 9 blood and DNA types. Twelve alleles were admitted in the skeletal remain among presumed 19 alleles. There was no blood and DNA type to deny the mother and daughter relation all of nine types. The skeletal remain was not contradicted from the above-mentioned result though thought daughters' mother. J. Med. Invest. 49:83-86, 2002 Keywords:forensic casework, personal identification, skeletal, DNA analysis INTRODUCTION DNA analysis demonstrates large powers in forensic caseworks, especially the personal identification (1-3). The completely reduced skeleton was found. From the morphological examination, the possibility of a missing woman of 66 years old had been suspected. In this paper, we report a personal identification by DNA analysis from this skeletal remains. CASE REPORT The completely reduced skeleton was found in a mountain stream. The bones were a skull, a pelvis bone, thighbones (right and left), and a finger bone. These bones were morphologically thought to be the one of the same individual. We presumed that the skeleton was a woman from a morphology feature of the skull and the pelvis bone. The level of the suture of the skull indicated that the age was the first half of the 70-years old from the 60-years old. It was impossible to presume her height, because the thighbone had been damaged. The postmortem duration was suspected to be about one year from half a year. As a result of the police investigation, the possibility of 66 years old woman who was missing for about six months was suspected. MATERIAL AND METHODS To inquire into her identification, a skull and left thighbone were cut off, and blood was collected from the suspect's daughters (relative no. 1 and 2). The suspect's pedigree is shown in Fig. 1. We examined blood and DNA types for the skeleton and the suspect's families. Blood type examination Absorption-elution method (4) and enzyme-linked immunosorbent assay (ELISA) method (5) were carried out for ABO blood typing from the skeletal samples. ABO blood type was detected from each blood by slide method (6). DNA type examination DNA was extracted from each material by phenol-chloroform method. DNA typing of D1S80 (MCT118), HLA DQA1, TH01 and polymarker (PM) system were carried out according to the manual of the National Research Institute of Police Science, Japan. 1) D1S80 type D1S80 typing was carried out according to the previous report (7, 8) using D1S80 Primer Mix (LifeCodes, USA) and 2 ng of extracted DNA as template for amplification. 2) HLA DQA1 type and PM system HLA DQA1 type and PM system were carried out according to the manufacture's recommendation using AmpliType PM+DQA1 PCR Amplification and Typing kit (Perkin-Elmer, USA) (9) and 4 ng of extracted DNA as template for amplification. 3) TH01 type TH01 typing was carried out according to the manufacture's instruction using GenePrint STR System-TH01 (Promega, USA) and 2 ng of extracted DNA as template for amplification. RESULTS In blood groups ABO blood typing carried out. DNA types of D1S80 (Fig. 2), HLA DQA1 (Fig. 3), TH01 (Fig. 4) and PM system (Fig. 5) were analyzed. Table 1 shows the results of blood and DNA typing. DISCUSSION DNA analysis is the useful technique in forensic caseworks, especially the personal identification (1-3) and various body fluids (10-12). D1S80 type is mini-satellite polymorphism, variable number of tandem repeat (VNTR), and its length is 16 base pair repeat. Since D1S80 population data are well characterized, D1S80 locus is used widely for forensic analysis (7, 8, 13). TH01 type is micro-satellite polymorphism, short tandem repeat (STR), and its length is 4 base pair repeat. TH01 locus is becoming more widely used for genetic characterization of forensic biological evidence (14, 15). From the reasons above, routinely, we carried out DNA analysis with combination of D1S80, TH01, HLA DQA1 and PM system. In this case, a skull and left thighbone were cut off for DNA sampling. DNA extracted from thighbone-marrow was highly fragmented because of putrefaction (16). So, we used for analysis DNA from a skull. The suspect's husband had already died. Materials from her brothers (sisters) were not obtained. Then, the individual was identified from the blood only of two daughters in the human race genetics. As a result, the personal identification was possible. Daughters' ABO type were A and O type, and their mother's ABO alelle was suspected to be A and/or O. Skeletal remain was corresponding by A (AA or AO) type. Daughters' D1S80 types were 24-25 and 24-30 type, respectively. Skeletal remain were 24-24 type, and 24 alleles were corresponding. When making it from daughters' HLA DQA1 types to mother, it was considered to have three alleles, such as 1.1, 1.2 and 3. Skeletal remain had two (1.1, 3) of three. It was suspected to have three alleles (8, 9, 10-1) when making it from daughters' TH01 types to mother. Skeletal remain had two (8 and 9) of three. The types of skeletal remain and daughters' types were completely corresponding to the LDLR, the GYPA, and the HBGG type among PM system. 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DNA Polymorphism 3:280-4, 1995 (in Japanese) Abbreviations:EGFP, enhanced green fluorescence protein;BEAS/WT, wild type BEAS-2B cells;BEAS/EGFP, BEAS-2B cells transduced with EGFP gene;BEAS/IL12, wild type BEAS-2B cells transduced with IL-12 gene;IFN-γ interferon-γ ICAM-1, intracellular adhesion molecule-1 ; VCAM-1, vascular cellular adhesion molecule-1 Received for publication December 3, 2001 ; accepted January 4, 2002. Address correspondence and reprint requests to Shin-ichi Kubo, Department of Legal Medicine, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima 770-8503, Japan and Fax:+81-88-633-7084.