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 high-throughput 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.

Application of array-based age prediction models to post-mortem tissue samples.

Since DNA methylation at specific CpG sites exhibits a strong age association, researchers have developed numerous age prediction models based on the methylation BeadChip array. These models harness epigenetic clocks that hold the potential to narrow down the search range for unknown suspects and unidentified victims. This study collected 180 post-mortem tissue samples comprising nine tissue types (blood, brain, heart, lung, liver, kidney, muscle, epidermis, and dermis) from autopsies of 20 Koreans aged 18-78. Subsequently, DNA methylation profiling was conducted using the Infinium MethylationEPIC array. We tested several array-based age prediction models using the data obtained from various tissues. The pan-tissue clock exhibited a moderately accurate prediction across all nine tissue types (MAE = 8.7 years, r = 0.88). Notably, the DNAm ages of the Hannum clock, the skin & blood clock, and the Zhang clock strongly correlated with the actual age in blood samples (MAE < approximately 5 years, r > 0.9). PhenoAge yielded an MAE of 10.1 years and an r-value of 0.92. The muscle-specific epigenetic clock, the MEAT package, demonstrated high prediction accuracy in muscle samples (MAE = 4.7 years, r = 0.93). Those previously reported array-based age prediction models were mainly constructed in Europeans but performed well in Koreans. In addition, tests involving various quantities of DNA and fragmented DNA have shown that DNA quantity and quality affected methylation measurements and age prediction results. However, robust age prediction models exist under low amounts of DNA and fragmented DNA conditions.

A novel approach of kinship determination based on the physical length of genetically shared regions of chromosomes.

BACKGROUND: Determination of genetic relatedness between individuals plays a crucial role in resolving numerous civil cases involving familial relationships and in forensic investigation concerning missing persons. Short tandem repeats (STRs), known for their high degree of DNA polymorphism, have traditionally been the primary choice of DNA markers in genetic testing, but their application for kinships testing is limited to cases involving close kinship. SNPs have emerged as promising supplementary markers for kinship determination. Nevertheless, the challenging remains in discriminating between third-degree or more distant relatives, such as first cousins, using SNPs. OBJECTIVE: To investigate a kinship analysis method for distant degree of familial relationships using high-density SNP data. METHODS: A high-density SNP data from 337 individuals of Korean families using Affymetrix Axiom KORV1.0-96 Array was obtained for this study. SNPs were aligned by chromosomal positions, and identity-by-state (IBS) was determined, and then shared regions as consecutive SNPs with IBS of 1 or 2 were investigated. The physical lengths of these IBS segments were measured and summed them to create an Index, as a measure of kinship. RESULTS: The kinship was determined by the physical length of shared chromosomal regions that are distinguished by each kinship. Using this method, the relationship was able be distinguished up to the fourth degree of kinship, and non-relatives were clearly distinguished from true relatives. We also found a potential for this approach to be used universally, regardless of microarray platforms for SNP genotyping and populations. CONCLUSION: This method has a potential to determine the different degree of kinship between individuals and to distinguish non-relatives from true relatives, which can be of great help for practical applications in kinship determination.

DNA methylation-based organ tissue identification: Marker identification, SNaPshot multiplex assay development, and interlaboratory comparison.

Identifying body fluids and organ tissues is highly significant as they can offer crucial evidence in criminal investigations and aid the court in making informed decisions, primarily through evaluating the biological source and possibly at the activity level up to death or fatal damage. In this study, organ tissue-specific CpG markers were identified from Illumina's methylation EPIC array data of nine organ tissues, including epidermis, dermis, heart, skeletal muscle, blood, kidney, brain, lung, and liver, from autopsies of 10 Koreans. Through the validation test using 43 samples, 18 hypomethylation markers, with two markers for each organ tissue type, were selected to construct a SNaPshot assay. Two multiplex assays involving forward and reverse SBE primers were designed to help investigators accurately determine the organ origin of the analyzed tissue samples through repeated analysis of the same PCR products for markers. The developed multiplex demonstrated high accuracy, achieving 100.0 % correct detection of the presence of nine organ tissue types in 88 samples from autopsies of 10 Asians. However, two lung samples showed additional positive indications of the presence of blood. An interlaboratory comparison using 80 autopsy samples (heart, skeletal muscle, blood, kidney cortex, kidney medulla, brain, lung, and liver) from 10 individuals in Germany revealed overall comparable results with correct detection of the presence of eight organ tissue types in 92.5 % samples (74 of 80 samples). In the case of six samples, it was impossible to determine the correct tissue successfully due to drop-outs of unmethylation signals at target tissue marker loci. One of these lung samples revealed only non-intended off-target signals for blood. The observed differences might be due to differences in sample collection during routine autopsy, technical differences due to the PCR cycler, and the threshold used for signal calling. Indicating the presence of additional tissue type and off-target unmethylation signals seems alleviated by applying more stringent hypomethylation thresholds. Therefore, the developed SNaPshot multiplex assays will be valuable for forensic investigators dealing with organ tissue identification, as well as for prosecutors and defense aiming to establish the circumstances that occurred at the crime scene.

Paleoparasitological report on Ascaris aDNA from an ancient East Asian sample

In this study, Ascaris DNA was extracted and sequenced from a medieval archaeological sample in Korea. While Ascaris eggs were confirmed to be of human origin by archaeological evidence, it was not possible to pinpoint the exact species due to close genetic relationships among them. Despite this shortcoming, this is the first Ascaris ancient DNA (aDNA) report from a medieval Asian country and thus will expand the scope of Ascaris aDNA research.