Jianhui Xie

Mutation analysis of 19 autosomal short tandem repeats in Chinese Han population from Shanghai.

The mutation of short tandem repeat (STR) loci is affected by several factors, such as sex, age, and DNA architectures. Previous studies have shown a different profile of mutation rates at autosomal STR loci among populations. It is important to provide population data and reveal underlying factors influencing the evaluation of STR mutation rates. In this study, we performed a comprehensive analysis on the mutation of 19 autosomal STR loci through 124,773 parent-child allelic transfers from 5846 paternity testing cases. A total of 197 mutations were observed including 187 single-step mutations. The observed mutation rates ranged from 0.15 × 10−3 (TH01) to 4.57 × 10−3 (FGA), and the average mutation rate across all the 19 loci was 1.58 × 10−3. Furthermore, the average mutation rate of STR loci increases with the paternal conception ages and remains relatively stable in different maternal age groups, which suggest the profile of paternal conception ages as a potential factor influencing the evaluation of STR mutation rates and the ratio of paternal versus maternal mutation rate in populations. Multidimensional scaling analysis (MDS) shows a difference in the profile of mutation rates at 13 CODIS STR loci among ethnical groups. Based on our data, our results support that short alleles are biased towards expansion mutation and longer alleles favor contraction mutation. In conclusion, our results provide useful information for further investigation on STR mutation in forensic genetics and population genetics.

Null alleles and sequence variations at primer binding sites of STR loci within multiplex typing systems

Rare variants are widely observed in human genome and sequence variations at primer binding sites might impair the process of PCR amplification resulting in dropouts of alleles, named as null alleles. In this study, 5 cases from routine paternity testing using PowerPlex®21 System for STR genotyping were considered to harbor null alleles at TH01, FGA, D5S818, D8S1179, and D16S539, respectively. The dropout of alleles was confirmed by using alternative commercial kits AGCU Expressmarker 22 PCR amplification kit and AmpFℓSTR®. Identifiler® Plus Kit, and sequencing results revealed a single base variation at the primer binding site of each STR locus. Results from the collection of previous reports show that null alleles at D5S818 were frequently observed in population detected by two PowerPlex® typing systems and null alleles at D19S433 were mostly observed in Japanese population detected by two AmpFℓSTR™ typing systems. Furthermore, the most popular mutation type appeared the transition from C to T with G to A, which might have a potential relationship with DNA methylation. Altogether, these results can provide helpful information in forensic practice to the elimination of genotyping discrepancy and the development of primer sets.

Genetic analysis of 29 Y-STR loci in the Chinese Han population from Shanghai

The analysis of Y chromosomal short tandem repeats (Y-STRs) provides important information that can be used to forensic investigation and population studies. In this study, typing of 29 Y-STRs included in the PowerPlex® Y23 system (PPY23) and Yfiler™ Plus system (Yfiler plus) was performed on 843 unrelated male samples from Han population in Shanghai. Besides null, duplicate, and intermediate alleles reported in previous studies, an allele of 10 at DYS643 with a 2-base deletion in the flanking region was initially observed. The gene diversity (GD) values of the 29 Y-STRs range from 0.4186 at DYS438 to 0.9653 at DYS385a/b. The haplotype diversity of two commonly used haplotype sets, PPY23 set and Yfiler plus set is 0.999980 and 0.999997, respectively. Pairwise genetic distances between Han population in Shanghai and other Han populations estimated using Yfiler plus set are slightly larger than that between other Han populations. Visualization of pairwise genetic distances between 17 worldwide populations using multidimensional scaling (MDS) demonstrates the distribution of populations according to their ethno-geographic patterns. Compared with Yfiler set, Yfiler plus set appears to have a relatively high population discrimination capacity for these tested populations. Altogether, these results can provide useful information of Y-STRs for forensic investigation and population studies.

Applying massively parallel sequencing to paternity testing on the Ion Torrent Personal Genome Machine

Massively parallel sequencing (MPS) is a promising supplementary method for forensic genetics and has gradually been applied to forensic casework. In this study, we applied MPS to forensic casework on an Ion Torrent Personal Genome Machine to evaluate its performance in paternity testing with mismatched STR loci. A total of 15 samples from seven cases containing one mismatched locus by capillary electrophoresis typing were analyzed. Combined paternity index (CPI) and relative chance of paternity were calculated according to the International Society for Forensic Genetics guidelines and the Chinese national standards recommended for paternity testing. With simultaneous analysis of enough STR loci, the results support the certainty of paternity, and the mismatched alleles were considered to be mutations (CPI>10,000). With the detection of allele sequence structures, the origins of the mutations were inferred in some cases. Meanwhile, nine STRs (CSF1PO, D1S1656, D2S441, D2S1338, D3S1358, D8S1179, D12S391, D21S11 and D4S2408) were found in an increased number of unique alleles and three new alleles in three STRs (D2S441, D21S11, and FGA) that have not been reported before were detected. Therefore, MPS can provide valuable information for forensic genetics research and play a promising role in paternity testing.

Evaluation of the inclusion of circular RNAs in mRNA profiling in forensic body fluid identification

The use of messenger RNA (mRNA) profiling is considered a promising method in the identification of forensically relevant body fluids which can provide crucial information for reconstructing a potential crime. However, casework samples are usually of limited quantity or have been subjected to degradation, which requires improvement of body fluid identification. Circular RNAs (circRNAs), a class of products from the backsplicing of pre-mRNAs, are shown to have high abundance, remarkable stability, and cell type-specific expression in human cells. In this study, we investigated whether the inclusion of circRNAs in mRNA profiling improve the detection of biomarkers including δ-aminolevulinate synthase 2 (ALAS2) and matrix metallopeptidase 7 (MMP7) in body fluid identification. The major circRNAs of ALAS2 and MMP7 were first identified and primer sets for the simultaneous detection of linear and circular transcripts were developed. The inclusion of circRNAs in mRNA profiling showed improved detection sensitivity and stability of biomarkers revealed by using serial dilutions, mixed samples, and menstrual bloodstains as well as degraded and aged samples. Therefore, the inclusion of circRNAs in mRNA profiling should facilitate the detection of mRNA markers in forensic body fluid identification.

Identification of mammalian species using the short and highly variable regions of mitochondrial DNA

Abstract The mitochondrial DNA (mtDNA) typing is useful for the species determination of degraded samples and the nucleotide diversity of target fragments across species is crucial for the discrimination. In this study, the short and highly polymorphic regions flanked by two conserved termini were sought by the sequence alignment of mtDNA across species and two target regions located at 12S rRNA gene were characterized. Two universal primer sets were developed that appear to be effective for a wide variety of mammalian species, even for domestic birds. The two target regions could be efficiently amplified using their universal primer sets on degraded samples and provide sufficient information for species determination. Therefore, the two short and highly variable target regions might provide a high discriminative capacity and should be suitable for the species determination of degraded samples.

Identification and characterization of the highly polymorphic locus D14S739 in the Han Chinese population

Aim To systemically select and evaluate short tandem repeats (STRs) on the chromosome 14 and obtain new STR loci as expanded genotyping markers for forensic application. Methods STRs on the chromosome 14 were filtered from Tandem Repeats Database and further selected based on their positions on the chromosome, repeat patterns of the core sequences, sequence homology of the flanking regions, and suitability of flanking regions in primer design. The STR locus with the highest heterozygosity and polymorphism information content (PIC) was selected for further analysis of genetic polymorphism, forensic parameters, and the core sequence. Results Among 26 STR loci selected as candidates, D14S739 had the highest heterozygosity (0.8691) and PIC (0.8432), and showed no deviation from the Hardy-Weinberg equilibrium. 14 alleles were observed, ranging in size from 21 to 34 tetranucleotide units in the core region of (GATA)9-18 (GACA)7-12 GACG (GACA)2 GATA. Paternity testing showed no mutations. Conclusion D14S739 is a highly informative STR locus and could be a suitable genetic marker for forensic applications in the Han Chinese population.

Deletion mapping of the regions with AMELY from two Chinese males

The amelogenin (AMEL) is widely used in many multiplex PCR kits for gender determination. However, the null of amelogenin Y (AMELY) can result in the incorrect genotyping of male samples as females. In this study, we report the deletion of AMELY in two cases with a deletion frequency of 0.019% (2/10526) in our laboratory. The deletion region with AMELY was mapped by using other twelve loci, which shows the class I deletion pattern. Further, the Y chromosome short tandem repeat (Y-STR) typing shows that these two cases share the same haplotype with other two cases from previous reports. The haplogroup of the two cases was predicted as O3 haplogroup with a 100% probability. Altogether, this study will provide evidence to further demonstrate the deletion of AMELY in Chinese population.