Xiu-Lin Huang

ALLELE DISTRIBUTION AT NINE STR LOCI-D3S1358, VWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 AND D7S820-IN THE JAPANESE POPULATION BY MULTIPLEX PCR AND CAPILLARY ELECTROPHORESIS

Nine tetranucleotide short tandem repeat (STR) loci, D3S1358, vWA, FGA TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820, were analyzed in the Japanese population with a newly released kit for personal identification using multiplex PCR with fluorescent-labeled primers following capillary electrophoresis. The observed heterozygosities were 0.67, 0.77, 0.82, 0.61, 0.62, 0.73, 0.78, 0.81 and 0.74, respectively, and the combined discrimination power of the nineplex was 0.9999999991. None of the nine loci deviated from Hardy-Weinberg equilibrium expectations using the chi-square test, homozygosity test, likelihood ratio test and exact test after the grouping of the alleles. The nine STR loci allele frequencies were significantly different from those of other ethnic populations.

Maternal Identification from Skeletal Remains of an Infant Kept by the Alleged Mother for 16 Years with DNA Typing

This is a case study concerning maternal identification by DNA typing at various loci. An infant skeleton was found in the alleged mothers apartment after it was kept for 16 years. We obtained the skeletal remains as well as saliva stains from the alleged mother. DNA typing was conducted for three loci in the HLA class II region (HLA-DQA1, -DPB1, and DRB1), five loci with the AmpliType PM kit (LDLR, GYPA, HBGG, D7S8, and GC), five STR loci (LPL, vWA, F13B, TH01, and TPOX) and D-loop region in mtDNA for maternal identification. Sex determination was accomplished using fluorescent DNA capillary electrophoresis typing. Approximately 5 ng of human DNA was recovered from 1 g of femur bone retrieved from the infant skeletal remains. The probability of two unrelated Japanese sharing the same genotypes was estimated as 7.2 x 10(-11). The combined probability of exclusion that an individual is not the mother was also calculated at 0.998. We therefore conclude that the skeleton is from a female infant, and that there is no inconsistency in the claim that the infant was a daughter of the alleged mother.

A case of fatal methemoglobinemia of unknown origin but presumably due to ingestion of nitrate.

Abstract A case of fatal methemoglobinemia (MetHb-emia) which was presumably due to ingestion of nitrate is presented. An unidentified man was taken to a local emergency hospital suffering from deep cyanosis and 7 h later he was found dead in the hospital bed. The post-mortem examination of the blood revealed a methemoglobin (MetHb) concentration of 78% and the concentrations of nitrate and nitrite were 1.50 and 0.76 μg/mL, respectively. Capillary gas chromatography coupled to mass spectrometry (GC-MS) and capillary gas chromatography with a nitrogen-phosphorus detector (NPD) were used to detect nitrates and nitrites in the blood.

Determination of chronic methamphetamine abuse by hair analysis

A 30-year-old male truck driver, known to be asthmatic, was found dead at the roadside lying near his car. A nebulizer bottle of Berotec (fenoterol hydrobromide) was found near his hand. The anatomic cause of death was suspected to be asthma. Toxicological screening of urine using Triage demonstrated the presence of methamphetamine. The blood concentration of methamphetamine was 0.4 microg/ml, and fenoterol was not detected. Hair analysis clearly indicated chronic methamphetamine abuse and medium dependency during the 2 months before death. We conclude that death might have been induced by the interaction of fenoterol and methamphetamine.

Applications of Minisatellite Variant Repeat (MVR) Mapping for Maternal Identification from Remains of an Infant and Placenta

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) at D1S8 (MS32) was applied to a practical forensic case of an infant and placenta found in an incinerator. They were thought to be left for a few days postmortem, and the infant was severely burnt when found. DNA was extracted from the infantile muscle and maternal placental hematoma. MVR-PCR analysis as well as other common DNA typing (D1S80, HLA-DQA1) were performed on both DNA samples. Both MVR diploid codes were matched although some extra faint bands in the ladder were observed from the maternal placental sample, which probably indicated superimposing of an allele derived only from the mother, and not the infant. In order to detect the original maternal alleles, three flanking polymorphic sites were typed and allele-specific MVR-PCR was performed. Finally, one maternal allele not inherited by the infant and two alleles from the infant were typed. Two alleles suggested the infant and/or mother was Japanese. The two diploid codes including one possibly from the mother were deduced and compared with other codes in the databases for evaluating the discriminating power.

The application of minisatellite variant repeat mapping by PCR (MVR-PCR) in a paternity case showing false exclusion due to STR mutation.

A boy and a girl with their mother brought a paternity suit against an alleged but deceased father. We tested six conventional genetic markers, the AmpliType PM+ DQA1 and twelve STR loci the children and mother together with the alleged paternal grandparents. We also DNA typed the bloodstain found later in the alleged fathers medical record. Only the result at D3S1358 in a nineplex STR system excluded the alleged father from parentage of the boy, whereas all markers were inclusive for the girl. Accordingly, we performed sequence analysis at D3S1358 to confirm the presence of a paternal exclusion or mutation. The sequence analysis indicated that the boys allele 17 could have originated from either of the alleged fathers allele 16 or 18 by a single-step mutation associated with slippage mutation in STR loci. We carried out minisatellite variant repeat mapping by PCR (MVR-PCR) at loci D1S8 (MS32) and D7S21 (MS31A) and mapped allele haplotypes of all individuals except the deceased alleged father. The MVR-PCR analysis showed that the boy has no inconsistency with the relationship between the alleged grandparents, and was very effective at increasing the paternity index (PI) value. We conclude that there is biological relationship between not only the girl but also the boy and the alleged father.

Analysis of allelic structures at the D7S21 (MS31A) locus in the Japanese, using minisatellite variant repeat mapping by PCR (MVR-PCR).

To sample the diversity of allelic structures at the D7S21 (MS31A) locus in the Japanese, allele‐specific minisatellite variant repeat mapping using polymerase chain reaction (MVR‐PCR) was performed on genomic DNA from a number of Japanese individuals. Three polymorphic positions in the MS31A 5′ flanking DNA were typed from 214 un‐related Japanese, and the distribution of haplotypes was analysed. Allele‐specific MVR‐PCR, using primers that discriminate between different alleles at these polymorphic positions in heterozygous individuals, allows single alleles to be mapped from genomic DNA in approximately 80% of Japanese. 149 Japanese alleles have been mapped to date and all of them, except for two pairs of indistinguishable alleles, have different internal structures. More than half of the mapped alleles showed similar regions of internal structure to other alleles and were classified into groups on this basis.

Sequence analysis of alleles at a microsatellite locus D14S299 (wg1c5) and population genetic comparisons.

Abstract In order to increase the discriminating power of DNA analysis in personal identification, we evaluated the forensic utility of the microsatellite locus D14S299 (wg1c5) in the Japanese population and also in the Chinese and Caucasian populations. Twelve different alleles were identified in length by gel electrophoresis with silver staining. The major alleles in Japanese were sequenced and designated as the numbers of the variable repeats (GGAT or GGAA). There were five variable regions and extensive homoplasy was found. However, the allele fragment lengths were in 4 bp increments and no “interalleles” were found. The estimated heterozygosity and the polymorphism information content (PIC) were 0.726 and 0.689, respectively in Japanese. Those in Chinese (0.743 and 0.704) were similar to those in Japanese, while those in Caucasians (0.812 and 0.781) were much higher. After adjacent alleles were combined to yield at least five entries, statistical analysis was performed. The power of discrimination (PD) was 0.887 in Japanese, 0.895 in Chinese and 0.935 in Caucasians and no significant deviations from the Hardy-Weinberg equilibrium were found in the three populations. We retyped all apparently homozygous samples using an alternative pair of flanking primers and found them to be true homozygotes. D14S299 appears to be a useful STR locus for forensic practice.

Large-scale preparation of high-molecular weight DNA from buccal mucosa

Four non-invasive methods of sampling DNA from buccal mucosa, simple rinses, scrubbing with cotton balls, scrubbing with toothbrushes and rinses after scrubbing with toothbrushes, were investigated. Scrubbing with toothbrushes yielded 5.79 +/- 5.56 microg of DNA rich in high-molecules, while less than one eighth the amount was recovered by scrubbing with cotton balls. Rinses after scrubbing with toothbrushes gave 50.0 +/- 46.0 microg of DNA and simple rinses 34.4 +/- 35.7 microg, although the DNA was considerably degraded. DNA specimens obtained from buccal cells were shown to be more or less in the process of degradation including apoptosis. For minisatellite analysis, only DNA prepared by scrubbing with toothbrushes could be used, while all specimens could be applied to PCR analyses. Since scrubbing with toothbrushes is painless and harmless, we recommend this method. Subsequent rinsing will yield a large amount of DNA suitable for many PCR analyses.

The potential contribution of MVR-PCR to paternity probabilities in a case lacking a mother.

Minisatellite variant repeat (MVR) mapping using the polymerase chain reaction (PCR) was applied to a paternity case lacking a mother to evaluate the paternity probability. After three flanking polymorphic sites at each of MS31A and MS32 loci were investigated from the child and alleged father, allele-specific MVR-PCR was performed using genomic DNA. It was confirmed that one allele in the child was identical to that in the alleged father at both loci. Mapped allele codes were compared with allele structures established from population surveys. No perfect matches were found although some motifs were shared with other Japanese alleles. The paternity index and probability of paternity exclusion at these two MVR loci were then estimated, establishing the power of MVR-PCR even in paternity cases lacking a mother.