Validation of the Utility of the Genetically Shared Regions of Chromosomes (GD-ICS) Measuring Method in Identifying Complicated Genetic Relatedness

Background@#Relatives share more genomic regions than unrelated individuals, with closer relatives sharing more regions. This concept, paired with the increased availability of highthroughput single nucleotide polymorphism (SNP) genotyping technologies, has made it feasible to measure the shared chromosomal regions between individuals to assess their level of relation to each other. However, such techniques have remained in the conceptual rather than practical stages in terms of applying measures or indices. Recently, we developed an index called “genetic distance-based index of chromosomal sharing (GD-ICS)” utilizing large-scale SNP data from Korean family samples and demonstrated its potential for practical applications in kinship determination. In the current study, we present validation results from various real cases demonstrating the utility of this method in resolving complex familial relationships where information obtained from traditional short tandem repeats (STRs) or lineage markers is inconclusive. @*Methods@#We obtained large-scale SNP data through microarray analysis from Korean individuals involving 13 kinship cases and calculated GD-ICS values using the method described in our previous study. Based on the GD-ICS reference constructed for Korean families, each disputed kinship was evaluated and validated using a combination of traditional STRs and lineage markers. @*Results@#The cases comprised those A) that were found to be inconclusive using the traditional approach, B) for which it was difficult to apply traditional testing methods, and C) that were more conclusively resolved using the GD-ICS method. This method has overcome the limitations faced by traditional STRs in kinship testing, particularly in a paternity case with STR mutational events and in confirming distant kinship where the individual of interest is unavailable for testing. It has also been demonstrated to be effective in identifying various relationships without specific presumptions and in confirming a lack of genetic relatedness between individuals. @*Conclusion@#This method has been proven effective in identifying familial relationships across diverse complex and practical scenarios. It is not only useful when traditional testing methods fail to provide conclusive results, but it also enhances the resolution of challenging kinship cases, which suggests its applicability in various types of practical casework.

Investigation of Mosaicism Detected in STR Typing / 대한법의학회지

Short tandem repeats (STRs) are the most popular markers for human identification in forensics. These markers can be easily analyzed through a multiplex polymerase chain reaction and electrophoresis and provide high discrimination power. However, in STR analysis, several atypical phenomena can be observed such as allelic dropouts, drop-ins, or imbalance, which may be due to DNA polymerase slippage or DNA degradation effects. The observed atypical STR profiles can also provide information for mixed DNA samples or chromosomal abnormalities. In this study, we report a case of mosaicism detected in routine casework of paternity testing. Hair samples from a phenotypically normal male were tested, and the result presented a typical STR profile of a female for the amelogenin gene (XX). Through chromosome analysis using peripheral blood, it was found that 45,X/46,XY mosaicism resulted in the discrepancy between the genotype and the phenotype. In addition, the amount of Y chromosome detected was particularly low in hair compared to that in blood. This study shows that mosaicism can make interpretation difficult during STR analysis and suggests that sample types and repeated analysis should be considered even for routine STR testing.

Single Nucleotide Polymorphisms Associated with Alcohol-Induced Flushing Syndrome in Korean Population / 대한법의학회지

Alcohol-induced flushing syndrome is one of the alcohol hypersensitivity reactions commonly found among Asian population. This study was designed to find markers that can predict this particular propensity among Korean population and to assess the applicability of this finding to build a prediction model as forensic DNA phenotyping tool to operate in practical forensic cases. Five hundred seventy unrelated Koreans were genotyped using microfluidic technology with 24 possible candidate single nucleotide polymorphism (SNP) markers. Of the 24 candidate SNPs, four markers, rs671, rs2074356, rs4646776, and rs10849915, on chromosome 12 showed statistically significant association with P-values ranging from 1.39×10⁻¹⁴ to 0.004988 among our subjects. All four markers show relatively high specificity values, ranging from 0.804651 to 0.972093, presenting their capabilities as differential SNPs that can distinguish a person with or without alcohol-induced flushing syndrome. Maneuvering these candidate SNPs as well as finding additional potential markers through future studies will help building an appropriate prediction model for Koreans that can be used as supplementary tool for individual identification.

Single Nucleotide Polymorphisms Associated with Alcohol-Induced Flushing Syndrome in Korean Population / 대한법의학회지

Alcohol-induced flushing syndrome is one of the alcohol hypersensitivity reactions commonly found among Asian population. This study was designed to find markers that can predict this particular propensity among Korean population and to assess the applicability of this finding to build a prediction model as forensic DNA phenotyping tool to operate in practical forensic cases. Five hundred seventy unrelated Koreans were genotyped using microfluidic technology with 24 possible candidate single nucleotide polymorphism (SNP) markers. Of the 24 candidate SNPs, four markers, rs671, rs2074356, rs4646776, and rs10849915, on chromosome 12 showed statistically significant association with P-values ranging from 1.39×10⁻¹⁴ to 0.004988 among our subjects. All four markers show relatively high specificity values, ranging from 0.804651 to 0.972093, presenting their capabilities as differential SNPs that can distinguish a person with or without alcohol-induced flushing syndrome. Maneuvering these candidate SNPs as well as finding additional potential markers through future studies will help building an appropriate prediction model for Koreans that can be used as supplementary tool for individual identification.

Detection of Innate and Artificial Mitochondrial DNA Heteroplasmy by Massively Parallel Sequencing: Considerations for Analysis

BACKGROUND: Mitochondrial heteroplasmy, the co-existence of different mitochondrial polymorphisms within an individual, has various forensic and clinical implications. But there is still no guideline on the application of massively parallel sequencing (MPS) in heteroplasmy detection. We present here some critical issues that should be considered in heteroplasmy studies using MPS. METHODS: Among five samples with known innate heteroplasmies, two pairs of mixture were generated for artificial heteroplasmies with target minor allele frequencies (MAFs) ranging from 50% to 1%. Each sample was amplified by two-amplicon method and sequenced by Ion Torrent system. The outcomes of two different analysis tools, Torrent Suite Variant Caller (TVC) and mtDNA-Server (mDS), were compared. RESULTS: All the innate heteroplasmies were detected correctly by both analysis tools. Average MAFs of artificial heteroplasmies correlated well to the target values. The detection rates were almost 90% for high-level heteroplasmies, but decreased for low-level heteroplasmies. TVC generally showed lower detection rates than mDS, which seems to be due to their own computation algorithms which drop out some reference-dominant heteroplasmies. Meanwhile, mDS reported several unintended low-level heteroplasmies which were suggested as nuclear mitochondrial DNA sequences. The average coverage depth of each sample placed on the same chip showed considerable variation. The increase of coverage depth had no effect on the detection rates. CONCLUSION: In addition to the general accuracy of the MPS application on detecting heteroplasmy, our study indicates that the understanding of the nature of mitochondrial DNA and analysis algorithm would be crucial for appropriate interpretation of MPS results.

SNP-Based Fetal DNA Detection in Maternal Serum Using the HID-Ion AmpliSeq™ Identity Panel / 대한법의학회지

Fetal DNA (fDNA) detection in maternal serum is a challenge due to low copy number and the smaller size of fDNA fragments compared to DNA fragments derived from the mother. Massively parallel sequencing (MPS) is a useful technique for fetal genetic analysis that is able to detect and quantify small amounts of DNA. In this study, seven clinical samples of maternal serum potentially containing fDNA were analyzed with a commercial single nucleotide polymorphism (SNP) panel, the HID-Ion AmpliSeq™ Identity Panel, and the results were compared to those from previous studies. Reference profiles for mothers and fetuses were not available, but multiple Y chromosomal SNPs were detected in two samples, indicating that fDNA was present in the serum and thereby validating observations of autosomal SNPs. This suggests that SNP-based MPS can be valuable for fDNA detection, thereby offering an insight into fetal genetic status. This technology could also be used to detect small amounts of DNA in mixed DNA samples for forensic applications.

Asian Ethnic Group Classification Model Using Data Mining / 대한법의학회지

In addition to identifying genetic differences between target populations, it is also important to determine the impact of genetic differences with regard to the respective target populations. In recent years, there has been an increasing number of cases where this approach is needed, and thus various statistical methods must be considered. In this study, genetic data from populations of Southeast and Southwest Asia were collected, and several statistical approaches were evaluated on the Y-chromosome short tandem repeat data. In order to develop a more accurate and practical classification model, we applied gradient boosting and ensemble techniques. To infer between the Southeast and Southwest Asian populations, the overall performance of the classification models was better than that of the decision trees and regression models used in the past. In conclusion, this study suggests that additional statistical approaches, such as data mining techniques, could provide more useful interpretations for forensic analyses. These trials are expected to be the basis for further studies extending from target regions to the entire continent of Asia as well as the use of additional genes such as mitochondrial genes.

Forensic DNA Phenotyping: A Review in Korean Perspective / 대한법의학회지

Forensic DNA phenotyping (FDP) using human externally visible characteristics (EVCs) is an emerging new technique that allows for the prediction of phenotypic traits of a person of interest using relevant sets of genetic markers. This technique predicts not only physical appearances, but also the behavioral characteristics as well as biogeographical information, serving as a powerful supplementary tool to narrow down the investigative pool in various forensic cases. Over the past few years, many countries, Europe and America being at the forefront, have conducted significant research to identify related markers for predicting pigmentation traits such as eye, hair, and skin color. Furthermore, some commercial platforms are now available for practical use in forensic cases. Korea and other Asian countries have also dedicated remarkable research to identify relevant markers to utilize FDP in forensic investigations. However, a slightly different approach is needed because Asians have limited phenotypic variations than Western populations. Thus, medically irrelevant and simple propensity traits such as smoking and alcohol consumption could be used to compensate for the limited phenotypic variations. This article is intended to inform readers about the progress and worldwide trends in EVC research, as well as the whereabouts and future prospects of FDP-related research in Korea. Although various legal and ethical disputes must be resolved beforehand, employing an FDP system can certainly be a powerful complementary tool for providing additional clues in forensic investigations.

Entire Mitochondrial DNA Sequencing on Massively Parallel Sequencing for the Korean Population

Mitochondrial DNA (mtDNA) genome analysis has been a potent tool in forensic practice as well as in the understanding of human phylogeny in the maternal lineage. The traditional mtDNA analysis is focused on the control region, but the introduction of massive parallel sequencing (MPS) has made the typing of the entire mtDNA genome (mtGenome) more accessible for routine analysis. The complete mtDNA information can provide large amounts of novel genetic data for diverse populations as well as improved discrimination power for identification. The genetic diversity of the mtDNA sequence in different ethnic populations has been revealed through MPS analysis, but the Korean population not only has limited MPS data for the entire mtGenome, the existing data is mainly focused on the control region. In this study, the complete mtGenome data for 186 Koreans, obtained using Ion Torrent Personal Genome Machine (PGM) technology and retrieved from rather common mtDNA haplogroups based on the control region sequence, are described. The results showed that 24 haplogroups, determined with hypervariable regions only, branched into 47 subhaplogroups, and point heteroplasmy was more frequent in the coding regions. In addition, sequence variations in the coding regions observed in this study were compared with those presented in other reports on different populations, and there were similar features observed in the sequence variants for the predominant haplogroups among East Asian populations, such as Haplogroup D and macrohaplogroups M9, G, and D. This study is expected to be the trigger for the development of Korean specific mtGenome data followed by numerous future studies.

Factors Affecting DNA Yields from Serum and Plasma Samples Used for Personal Identification Testing / 대한법의학회지

Serum or plasma is free of cellular components. As DNA is in the nucleus or mitochondria of a cell, it can be presumed that serum/plasma is DNA free. However, there are cases wherein serum/plasma is the only resource available for identification analysis, yet no sufficient data are available regarding whether reliable DNA testing can be applied to such cases, and what the influencing factors are when testing is a valid course of action. The aim of this study is to illustrate the factors that can be used in the genetic testing of serum/plasma when identifying an individual. The results showed that the concentration of serum DNA significantly increased over time in 4℃ storage, and the DNA yields from samples stored in heparin tubes were overall higher than from samples stored in ethylenediaminetetraacetic acid tubes. We observed that the concentration of DNA in serum successfully matched 100% to the short tandem repeat data of blood DNA.

Looking Back at Our 5-Year Experience of Paternity Testing: A Summary / 대한법의학회지

We have been testing familial relationships based on short tandem repeats (STRs) in families who requested it either voluntarily or by order of the court. Here, we present a summary of our 5-year experience of autosomal STR-based paternity tests. A total of 1,431 individuals from 588 cases were tested, including 878 pairs of either of the parent, and a child. Among these 588 cases, genetic information about the other parent was available only for 135 cases. Five hundred eighteen pairs were concluded to be parent-child relations, for which the median paternity index (PI) was 72,826, and the median decimal logarithm was 4.860. Autosomal mutation was observed in nine pairs (1.74%), and the pairs harbored only one mismatched locus among the 15 standard loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA). The number of mismatched loci did not increase even after additional loci were included in the study. The observed mutation rates were D13S317 (0.193%), D18S51 (0.193%), D19S433 (0.193%), FGA (0.193%), vWA (0.386%), Penta D (0.387%), and Penta E (0.193%). There were 14 pairs with two mismatched loci, which we excluded through additional tests on either autosomal or X chromosomal STRs, and mitochondrial sequencing. Although PI is useful for determining parent-child relation, it provides indirect information; it is an interpretation of the test results that is based on probability. Additional genotyping on sex chromosome and mitochondrial DNA, or participation of other family members might be beneficial for a reliable conclusion.

Forensic Application of Age Estimation Using Signal Joint T-Cell Receptor Excision Circles in Human Lymphoid Tissues / 대한법의학회지

The declining tendency of signal joint T-cell receptor excision circles (sjTRECs) in peripheral blood is known to be age-dependent, and their quantification in blood or bloodstains has recently been introduced as a tool for age estimation. Lymphoid tissues such as the thymus and spleen represent potential candidates for age estimation because they undergo age-related structural and functional changes. In the present study, the correlation between age and sjTREC levels in human lymphoid tissues, namely the thymus, spleen, and blood, obtained from autopsy cases were investigated, with the goal of establishing a reliable age estimation model. Results showed negative regression curves with coefficient values of r=-0.410, r=-0.611, and r=-0.584 for thymus, spleen, and blood, respectively. In addition, this model was testing using thymus samples from the torsos of dismembered bodies from two real forensic cases, and results showed the predicted ages to be close to the actual ages of the victims. Further study will be required to improve accuracy and reduce estimation error, particularly within the lower age range. Nonetheless, these results suggest that quantification of sjTRECs in not only blood but also in other lymphoid tissues could be a useful tool for age estimation in forensic cases.

Mixture Patterned Short Tandem Repeat Profile in a Perimortem Transfused Patient / 대한법의학회지

Recently, it has been reported that transfused patients can generate admixture-like genetic profiles. As genetic material of the donor can survive for a reasonable time after transfusion, the recipient's genomic DNA is likely to have a mixture pattern. An autopsy case of a man transfused perimortem generated a mixture patterned short tandem repeat profile. Notably, the patient was transfused mostly with nuclear-deficient cells, limiting the donor genetic material available for the recipient. As a result, mixture-like patterns were observed consistently, regardless of change in input DNA content; the sample DNA content, which was serially diluted, ranged from 1 ng to 0.0625 ng. The distributions of foreign peaks appeared to be irreproducible, showing stochastic behaviors throughout the genotyped results. This study suggests that a cautious approach is required when genotyping of a patient who has undergone recent transfusion. One must consider the possibility of obtaining a mixture patterned profile in such patients, and therefore, choose parenchymal organs or tissues for reliable results.

Searching for Appropriate Statistical Parameters for Validation of Mitochondrial DNA Database / 대한법의학회지

Recently, studies on mitochondrial DNA (mtDNA) have increased rapidly. Conventional parameters, such as diversity index, pairwise comparison, are used to interpret and validate data on autosomal DNA; however, the use of these parameters to validate data from mitochondrial DNA databases (mtDNA DBs) needs to be verified because of the different transmission patterns of mtDNA. This study was done to verify the use of these conventional parameters and to test the "coverage concept" for a new parameter. The mtDNA DB is not very big; however, it is necessary to check how the change in parameters corresponds to the DB size. For this, we artificially rearranged a Korean DB into several small sub-DBs of variable sizes. The results show that the diversity in nucleotide variations and the different haplotype numbers do not vary as the size of DB increases. However, the "coverage" changed a lot. The coverage increased from 0.113 in a DB of 100 people to 0.260 in a DB of 653 people. Additionally, using the "coverage concept", we predicted how the total number of haplotypes changed with variations in the sub-DB size and compared the predicted result with final result. In conclusion, "coverage", in addition to conventional statistical parameters, can be used to check the usability of an mtDNA DB. Finally, we tried to predict the size of the whole mtDNA number in Korea using "saturation concept".

Limitation of Regular Autosomal STR Testing for Paternity within an Isolated Population / 대한법의학회지

In order to determine paternity by genetic testing, the Paternity Index (PI) and probability of paternity are calculated using likelihood ratio method. However, when it is necessary, additional testing can be performed to validate the genetic relationship. This research demonstrates autosomal short tandem repeat (STR) results of Jeju Island population in order to determine genetic relationship. Two notable cases showed that despite the acceptable PI value obtained from STR testing, average of 12 mismatches were found in total of 169 autosomal single nucleotide polymorphism typing. Such cases imply that cautious statistical approach is necessary when determining genetic relationship, especially within an isolated population group. Moreover, this would suggest that a further research and investigation are needed in order to understand the population structure of Korea.

Genetic Relationship in Bone Samples Using SNP-Based Human Identification DNA Chip / 대한법의학회지

DNA profiling with sets of short tandem repeat (STR) markers is the most popular method for identifying human DNA in forensics. Identification by STR typing might fail when DNA is degraded or is present in low amounts, such as in disaster victim identification (DVI) samples. In such cases, more information might be obtained by using additional markers such as single nucleotide polymorphisms (SNPs). Multiplex PCR and microarray are convenient techniques to analyze SNP markers. We used an AccuID(TM) Chip, SNP-based DNA chip manufactured by DNA Link Corporation, to confirm genetic relationship between two human bone samples that had been buried for more than 50 years and blood samples from the alleged descendants of the sources of the bone fragments. The chip combines an Affymetrix resequencing array with a multiplex PCR technology and can genotype hundreds of SNP markers in a single experiment. Genotyping the two bone samples yielded 90.5 and 77 SNP markers. The commonly genotyped markers (61 and 47 SNP loci) in each bone-family pair provided high paternity indices to support the genetic relationships in both cases.

DNA Profiling via Short Tandem Repeat Analysis by Using Serum Samples / 대한법의학회지

Serum is free of cellular components. Because DNA is located in the nuclei or mitochondria of cells, serum could be assumed DNA free. Few previously published case reports to date have used serum for DNA typing. Here, we report on human genotyping via short tandem repeat (STR) analysis using serum as a sample, and discuss problems involved in the process.