Tetsushi Kitayama

A D19S433 Primer Binding Site Mutation and the Frequency in Japanese of the Silent Allele It Causes

Abstract:  Short tandem repeat studies are powerful tools for parentage analysis and for identification of missing persons, victims of murder, and victims of mass fatalities when reference samples are unavailable. The primer in the Identifiler® kit failed to amplify an allele at the D19S433 locus, producing a silent (“null”) allele. The causal mutation is a base change (G>A) 32 nucleotides downstream from the 3′ end of the AAGG repeats. The silent alleles are problematical in parentage analysis because when transmitted, they can cause a parent–child inconsistency that is unrelated to Mendelian genetics. The inconsistency is sometimes termed an “apparent opposite homozygosity” and it produces false evidence of nonparentage. Alternative primers were designed to amplify the D19S433 locus alleles and they detect the silent allele. Frequencies of the (no longer) silent allele were determined to be 0.0114 in 176 people from Shizuoka (Honshu) and 0.0128 in 156 people from Okinawa.

Allele frequencies for 22 autosomal short tandem repeat loci obtained by PowerPlex Fusion in a sample of 1501 individuals from the Japanese population.

Allele frequencies for 22 autosomal short tandem repeat loci (D3S1358, D1S1656, D2S441, D10S1248, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433, FGA, and D22S1045) were obtained from 1501 unrelated individuals sampled from the Japanese population.

Evaluation of a new experimental kit for the extraction of DNA from bones and teeth using a non-powder method.

An experimental DNA extraction kit (new kit) was recently developed to extract DNA from degraded skeletal remains without the need for powdering the samples. We compared the utility of the new kit with the conventional phenol/chloroform method using real-time quantitative PCR and multiplex STR analysis. The new kit yielded large amounts of DNA from a compact bone fragment compared with the conventional phenol/chloroform method. We were able to extract sufficient DNA for STR analysis from 75% (3 of 4) and 60% (3 of 5) of the un-powdered tooth and bone samples, respectively, using the new kit. We were able to perform mini-STR analysis of the remaining samples using DNA extracted with the new kit. Furthermore, we successfully performed mitochondrial DNA sequencing of every sample. The new kit simplifies the DNA extraction procedure as it does not require powdering samples. Decreasing the number of procedural steps in DNA extraction will be beneficial in controlling DNA contamination in laboratories. Our results suggest that the new kit may be used for the simple, simultaneous extraction of DNA from multiple samples.

Estimation of the detection rate in STR analysis by determining the DNA degradation ratio using quantitative PCR.

Performing short tandem repeat (STR) analysis from degraded DNA is a challenge for forensic biologists. For assessing the quality and quantity of DNA, we developed quantitative PCR assays to determine the extent of DNA degradation. Quantitative PCR assays using primers that generate two sizes of amplicons from the same region of genomic DNA were used to determine the extent of DNA degradation. These quantitative PCR assays were used with artificially degraded DNA and degraded DNA extracted from aged bloodstains. Increased DNA degradation correlated with a decrease in the number of detectable loci in STR analysis. The extent of DNA degradation and the number of loci detected by STR analysis varied depending on the method of degradation. The extent of degradation of DNA extracted from aged bloodstains correlated well with that of DNA artificially degraded by DNase I in the presence of Mn(2+). Thus, determination of the extent of DNA degradation was helpful for estimating the number of detectable loci. Furthermore, this estimation method is expected to save time and labor, and is particularly suitable when only a limited amount of DNA can be extracted from casework samples.

Comparison of automated and manual purification of total RNA for mRNA-based identification of body fluids

Silica column-based RNA purification procedures have widespread use in mRNA profiling for body fluid identification in forensic samples. Also, automated RNA purification systems employing magnetic bead technology have recently become available. In this preliminary study, to ascertain which RNA purification technology is more suitable for the identification of body fluids by real-time reverse transcription polymerase chain reaction (RT-PCR), comparative analyses of the yield and quality of total RNA were performed between automated purification using an EZ1 Advanced Instrument and manual purification using an RNeasy Mini Kit. The yield and size distribution of total RNA were compared by gene expression analysis of two different sized fragments of the β-actin gene. In addition, the relative amounts of several target genes were compared between the purification methods, and the integrity of total RNA was determined by chip-based electrophoresis. The results of this study suggest that RNeasy can purify higher-quality RNA as compared with automated purification using EZ1. The sensitivity of the RT-PCR analysis, however, was higher in the EZ1-purified samples, likely due to the relative efficiency of EZ1 in extracting short-length RNA from degraded samples. We also show that the quantification of relative levels of body fluid-specific genes could be influenced by the purification procedure. Our results indicate that although use of high-quality RNA is generally required for reproducible results in gene expression analysis, the forensic relevance of short RNA fragments in highly degraded samples cannot be ruled out. Furthermore, our results suggest that automated purification procedures as well as silica column-based manual purification procedures can be used for mRNA-based body fluid identification in forensic samples.

Allele frequencies for 21 autosomal short tandem repeat loci obtained using GlobalFiler in a sample of 1501 individuals from the Japanese population.

Allele frequencies for 21 autosomal short tandem repeat loci (D3S1358, vWA, D16S539, CSF1PO, TPOX, D8S1179, D21S11, D18S51, D2S441, D19S433, TH01, FGA, D22S1045, D5S818, D13S317, D7S820, SE33, D10S1248, D1S1656, D12S391, and D2S1338) were obtained using the GlobalFiler kit from 1501 unrelated individuals sampled from the Japanese population.

A Comparison of DNA Extraction Using AutoMate Express™ and EZ1 Advanced XL from Liquid Blood, Bloodstains, and Semen Stains

In this study, DNA was extracted using an AutoMate Express™ and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real‐time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpFℓSTR® Identifiler kit. The yields of DNA obtained by the AutoMate Express™ were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler™ lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler™ lysis buffer is the main contributor to the higher DNA yields of the AutoMate Express™ for fresh bloodstains.

Mitochondrial DNA population data of HV1 and HV2 sequences from Japanese individuals

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were determined for 1204 unrelated Japanese individuals. A total of 741 different mtDNA haplotypes were found, 157 of which were seen in multiple individuals. Twenty-seven of these individuals showed point heteroplasmy. The most frequent haplotype (16223T-16362C-73G-263G-315.1C) was found in 31 individuals and the second most frequent haplotypes (16129A-16223T- 16362C-73G-152C-263G-309.1C-315.1C) was found in 24 individuals. The haplotypes diversity and random match probability were calculated to be 0.9969 and 0.0040, respectively.

Next-generation sequencing analysis of off-ladder alleles due to migration shift caused by sequence variation at D12S391 locus

In short tandem repeat (STR) analysis, length polymorphisms are detected by capillary electrophoresis (CE). At most STR loci, mobility shift due to sequence variation in the repeat region was thought not to affect the typing results. In our recent population studies of 1501 Japanese individuals, off-ladder calls were observed at the D12S391 locus using PowerPlex Fusion in nine samples for allele 22, one sample for allele 25, and one sample for allele 26. However, these samples were typed as ordinary alleles within the bins using GlobalFiler. In this study, next-generation sequencing analysis using MiSeq was performed for the D12S391 locus from the 11 off-ladder samples and 33 other samples, as well as the allelic ladders of PowerPlex Fusion and GlobalFiler. All off-ladder allele 22 in the nine samples had [AGAT]11[AGAC]11 as a repeat structure, while the corresponding allele was [AGAT]15[AGAC]6[AGAT] for the PowerPlex Fusion ladder, and [AGAT]13[AGAC]9 for the GlobalFiler ladder. Overall, as the number of [AGAT] in the repeat structure decreased at the D12S391 locus, the peak migrated more slowly using PowerPlex Fusion, the reverse strand of which was labeled, and it migrated more rapidly using GlobalFiler, the forward strand of which was labeled. The allelic ladders of both STR kits were reamplified with our small amplicon D12S391 primers and their mobility was also examined. In conclusion, off-ladder observations of allele 22 at the D12S391 locus using PowerPlex Fusion were mainly attributed to a relatively large difference of the repeat structure between its allelic ladder and off-ladder allele 22.

D5S818 Typing Discrepancy Between PowerPlex Fusion and Other STR Kits Including GlobalFiler Caused by a One-base Deletion in 31 Nucleotides Upstream of the Repeat Region

Short tandem repeat (STR) typing is widely used in forensic investigation. When the same DNA sample is analyzed with different STR typing kits, a typing discrepancy is occasionally observed. In this study, we examined the cause of a typing discrepancy in a sample at D5S818 locus. This sample was designated as 10, 12 using Identifiler®, Identifiler® Plus, GlobalFiler®, PowerPlex® 16HS, and PowerPlex® 18D, but as 9.3, 12 using PowerPlex® Fusion. Sequencing results indicated that the shorter allele in the sample had a deletion (U31Tdel) at 31 nucleotides upstream of the repeat region (AGAT)10. This deletion was located in the binding site of the published D5S818 forward primer in PowerPlex® 16 and was only 9 and 11 nucleotides downstream of our estimated 5′ end position of D5S818 forward primer in GlobalFiler® and PowerPlex® 18D, respectively. We also examined the effect of primer length on the heterozygous peak balance in this sample.