In 1990, a 4-year-old girl was murdered and abandoned at the bank of a river (known as the Ashikaga case in Japan). The short-sleeved shirt of the victim found at the river side was an important piece of evidence. It was confirmed by microscopic examination that a lot of sperm stuck to the shirt.
Two similar cases occurred in a residential area in 1979 and 1984. This suggested that the Ashikaga case was suspected as a set of multiple murders of young girls. Numerous homicide detectives were involved in the search of the criminal, however; no suspect was identified for a period of more than six months.
Eventually, “Mr. S”, the bus driver for kindergarten students, was identified as a suspect. One and a half years later, the National Research Institute of Police Science (NRIPS) stated that the D1S80 [
- Budowle B.
- Chakraborty R.
- Giusti A.M.
- Eisenberg A.J.
- Allen R.C.
Analysis of the VNTR locus DIS80 by the PCR followed by high-resolution PAGE.
Am. J. Hum. Genet. 1991; 48: 137-144
2] type and ABO blood type of the sperm on the shirt were identical to those of Mr. S. As a result, he was arrested and was forced to confess to the crime. According to the NRIPS original report, both the sperm on the shirt and Mr. S had 16-26 (D1S80) and B (ABO blood typing). This was the first case that DNA testing was used for a criminal arrest in Japan.
- Kasai K.
- Nakamura Y.
- White R.
Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science.
J. Forensic Sci. 1990; 35: 1196-1200
However, a serious problem concerning the D1S80 typing method was reported just after an appraisal of the original DNA test was submitted. It had been observed in other studies that DNA molecular size standard (such as a 123 bp ladder marker) did not simply migrate according to its molecular size in native polyacrylamide gel, so that the amplicon size cannot be determined precisely only by comparing it to a standard marker [
- Sugiyama E.
- Honda K.
- Katsuyama Y.
- Uchiyama S.
- Tsuchikane A.
- Ota M.
- Fukushima H.
Allele frequency distribution of the D1S80 (pMCT118) locus polymorphism in the Japanese population by the polymerase chain reaction.
Int. J. Legal Med. 1993; 106: 111-114
4]. After presentation of this problem in the court room, the NRIPS changed the original typing and revised the results from 16-26 to 18-30 during trial. Nevertheless NRIPS still insisted that the DNA type of the sperm is the same as that of Mr. S also for the revised DNA type (18-30).
- Marini J.C.
- Levene S.D.
- Crothers D.M.
- Englund P.M.
Bent helical structure in kinetoplast DNA.
Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 7664-7668
However, a very terrible thing occurred subsequently. Five years after Mr. S was arrested, a similar murder case of a 4-year-old girl occurred in the neighbouring city of Ashikaga, and her body has never been discovered. Obviously, Mr. S was not involved in this case because he had already been taken into custody.
Nevertheless, the Supreme Court accepted the NRIPS's DNA test at last and rejected the defense's appeal for retrial. In the end, the original life imprisonment sentence was confirmed on July 17, 2000.
Mr. S. has repeatedly demanded DNA re-testing, but his request was denied by the court of law. In December 2008, the first DNA re-test was granted by the Tokyo High Court. The Tokyo High Court nominated two forensic scientists for the re-test: Dr. S (prosecution recommendation) and Dr. H (defense recommendation). Nineteen years had passed since the crime had occurred, the new DNA analysis was considered difficult and challenging. The shirt was divided between the two experts. Dr. H selected the piece of shirt with greenish discoloration parts indicating the presence of semen in which moss and mould could easily grow.
As reference samples, blood and oral swab were taken from Mr. S who was incarcerated at the Chiba prison. Unfortunately, the differential DNA extraction method [
] was not applicable because the crime scene sample was very old. Instead, total DNA extraction from each sample was performed using conventional methods of organic extraction. This technique is suitable for old and contaminated samples [
- Gill P.
- Jeffreys A.J.
- Werrett D.J.
Forensic application of DNA ‘fingerprints’.
Nature. 1985; 318: 577-579
]. D1S80 amplification was performed as previously described [
- Butler J.M.
Fundamentals of Forensic DNA Typing.
Academic Press, Elsevier, 2009
- Honda K.
- Nakatome M.
- Islam M.N.
- Bai H.
- Ogura Y.
- Kuroki H.
- Terada M.
- Yamazaki M.
- Misawa S.
- Wakasugi C.
Detection of D1S80 (pMCT118) locus polymorphism using semi-nested polymerase chain reaction in skeletal remains.
J. Forensic Sci. 1995; 40: 637-640
In addition, Y-chromosomal STR testing was performed instead of autosomal STR to exclude potential cross-contamination by the DNA of the victim and to improve the efficiency of the amplification [
]. The DNA extracts were typed using Y-STR markers with the addition of VCl4 in the reaction buffer for PCR enhancer. The sizing of PCR products was performed by capillary electrophoresis with an ABI PRISM 3130xl Genetic Analyser. The typing results using PowerPlex™ Y System (Promega) are shown in Table 1. The Y-STR results with seven exclusion constellations between the evidence and Mr. S are fully conclusive and exclude the suspect as the donor of the stain.
- Honda K.
- Roewer L.
- de Knijff P.
Male DNA typing from 25-year-old vaginal swabs using Y chromosomal STR polymorphisms in a retrial request case.
J. Forensic Sci. 1999; 44: 868-872
Table 1Results of Y-STR typing.
|Match or no match||Match||Match||No match||No match||No match||Match|
|Match or no match||Match||No match||No match||No match||No match|
The frequency of the perpetrator's haplotype found in the sperm trace was calculated as follows: no observations in 1503 Japanese reference haplotypes from 21 populations (YHRD release 39 from 17.02.12; www.yhrd.org [
]). Using the counting method (1/database size + 1) the frequency is 6.6 × 10−4. In the full East Asian metapopulation including the Japanese (n = 14,012, YHRD release 39) the haplotype is observed 2 times resulting in a frequency of 3.8 × 10−5 [confidence interval limits (CI): 4.6 × 10−6 to 1.4 × 10−4].
- Willuweit S.
- Roewer L.
Y chromosome haplotype reference database (YHRD): update.
Forensic Sci. Int. Genet. 2007; 1: 83-87
On the other hand, the DYS393 allele 18 from Mr. S was extremely rare; it has only been observed two times in YHRD release 39 (n = 101,055 haplotypes; [f = 1.979 × 10−5 (95% CI: 2.397 × 10−6 to 7.149 × 10−5)]) in 2 of 766 populations, once in Mongolia, and once in South Korea. Even the difference only for this locus (DYS393) represents strong evidence of exclusion.
The Y-STR haplotype of the sperm stain clearly indicates a single male (or male lineage) origin that did not match with Mr. S's haplotype. Similarly, the other expert (Dr. S) also confirmed that the DNA type of the evidence was different from that of Mr. S. It was therefore concluded that Mr. S was not the true perpetrator. On June 4, 2009, the public prosecutor accepted the results of the new DNA test done by Dr. S, so that Mr. S was released from the prison.
In addition, D1S80 re-typing by Dr. H showed that the type of Mr. S. was 18-29. So, the D1S80 type defined by NRIPS of the sperm (18-30) did not match with Mr. S's (18-29). Therefore, the DNA typing results using D1S80 could have been inconclusive in this case since the beginning.
Furthermore, the victim's umbilical cord and an oral swab from her mother were obtained by Dr. H as reference samples. The data indicated that the D1S80 types of victim and mother were the 18-31 and 30-31, respectively, and it was suspected that the 18 and 30 alleles detected in the original D1S80 type done by NRIPS could have been the result of a cross contamination from the victim's and her mother's DNA.
The public prosecutor did not easily accept the results of the new DNA test including D1S80 typing done by Dr. H. At the Utsunomiya District Court (re-trial court), the prosecutor argued that the new DNA test by Dr. H had detected contaminated DNA that was not from the true criminal. For the objection, Dr. H stated that the major DNA types attached to the sample were highly conserved, and that this indicated that the DNA types came from the true criminal.
The Utsunomiya District Court accepted DNA re-test as a crucial evidence, which revoked the results of the old DNA analysis (D1S80 typing) that was performed by the NRIPS. The retrial request was admitted and Mr. S was finally declared innocent on March 26, 2010. Similar exoneration cases have been published recently by the “Innocence Project” [
- Hampikian G.
- West E.
- Akselrod O.
The genetics of innocence: analysis of 194 U.S. DNA exonerations.
Annu. Rev. Genomics Hum. Genet. 2011; 12: 97-120
These results also show that Y-STR typing proved to be a very powerful tool for exonerations. The results of this study show that Y-STR typing is possible, even from a 19-year-old sperm stain. At the same time, it revealed that the previous D1S80 typing by the NRIPS yielded false typing results, and that an innocent man was imprisoned for 17.5 years. The results of DNA re-testing in the Ashikaga case indicate that multiplex STR typing may be a useful tool in the review of other retrial cases.
10 July 2012
Appendix A. Supplementary data
The following are supplementary data to this article:
- Analysis of the VNTR locus DIS80 by the PCR followed by high-resolution PAGE.Am. J. Hum. Genet. 1991; 48: 137-144
- Amplification of a variable number of tandem repeats (VNTR) locus (pMCT118) by the polymerase chain reaction (PCR) and its application to forensic science.J. Forensic Sci. 1990; 35: 1196-1200
- Allele frequency distribution of the D1S80 (pMCT118) locus polymorphism in the Japanese population by the polymerase chain reaction.Int. J. Legal Med. 1993; 106: 111-114
- Bent helical structure in kinetoplast DNA.Proc. Natl. Acad. Sci. U.S.A. 1982; 79: 7664-7668
- Forensic application of DNA ‘fingerprints’.Nature. 1985; 318: 577-579
- Fundamentals of Forensic DNA Typing.Academic Press, Elsevier, 2009
- Detection of D1S80 (pMCT118) locus polymorphism using semi-nested polymerase chain reaction in skeletal remains.J. Forensic Sci. 1995; 40: 637-640
- Male DNA typing from 25-year-old vaginal swabs using Y chromosomal STR polymorphisms in a retrial request case.J. Forensic Sci. 1999; 44: 868-872
- Y chromosome haplotype reference database (YHRD): update.Forensic Sci. Int. Genet. 2007; 1: 83-87
- The genetics of innocence: analysis of 194 U.S. DNA exonerations.Annu. Rev. Genomics Hum. Genet. 2011; 12: 97-120
Published online: August 13, 2012
Received: August 29, 2011
© 2012 Elsevier Ireland Ltd. Published by Elsevier Inc. All rights reserved.