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.

Y chromosomal deletion pattern in Koreans inhabiting Jeju Island.

ABSTRACT: Mutations occur in Y chromosome genes similar to autosomal genes. However, unlike autosomal genes, Y chromosome genes do not undergo recombination, which produce distinctive characteristics and distribution patterns in different geographic regions. Therefore, detailed analysis of mutations of Y chromosome genes might provide information for personal identification or analysis of phylogenetic history. In Y-STR (short tandem repeat) analysis tests on 668 habitants of Jeju Island, the largest island in the Korean peninsula located apart from the mainland, a deletion at DYS448 was found in 10 samples. The length of deletion was estimated by confirming specific Sequence Tagged Site (STS) markers ranging from G66018 to sY1201. Patterns found were similar to those of the Kalmyks, a tribe that has had strong social and genetic influences in Jeju Island in the past. Historically from 1273 on, Jeju Island was governed by Mongolian for about one hundred years. The results of this study suggest such historical aspects affected the genetic composition of people living in Jeju Island. Furthermore, previous reports showed that Y chromosomal deletions and region specific Y chromosomal mutations depended on regional differences. This study may be useful for a better understanding of the genetic structure of Jeju habitants as well as Korean population for the purpose of forensic practice and population genetics.

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.

Set up of cutoff thresholds for kinship determination using SNP loci.

The usefulness of single nucleotide polymorphism (SNP) loci for kinship testing has been demonstrated in many case works, and suggested as a promising marker for relationship identification. For interpreting results based on the calculation of the likelihood ratio (LR) in kinship testing, it is important to prepare cutoffs for respective relatives which are dependent on genetic relatedness. For this, analysis using true pedigree data is significant and reliable as it reflects the actual frequencies of markers in the population. In this study, the kinship index was explored through 1209 parent-child pairs, 1373 full sibling pairs, and 247 uncle-nephew pairs using 136 SNP loci. The cutoffs for LR were set up using different numbers of SNP loci with accuracy, sensitivity, and specificity. It is expected that this study can support the application of SNP loci-based kinship testing for various relationships.

Influence of immunologic status on age prediction using signal joint T cell receptor excision circles.

Age estimation based on quantifying signal joint T cell receptor excision circle (sjTREC) in T cells has been established to be a promising approach in forensic practice and demonstrated in different ethnic groups. Considering that the homeostasis of T cells carrying sjTRECs is closely related to the immunologic status of a person, it is important to investigate the influence of various immunologic statuses on the age estimation model. In this study, quantification of sjTREC contents was performed for groups of people with various immune system statuses, and the result showed less correlation with chronological age (r 2 = 0.424) than in the healthy group (r 2 = 0.648). The simulation model indicated that this influence could increase the range of prediction in the age estimation model, and the mean absolute deviation (MAD) between chronological age and predicted age. Through this study, it was demonstrated that immunologic status is a factor that affects the accuracy of age prediction using sjTREC quantification.

Kinship Testing Based on SNPs Using Microarray System.

BACKGROUND: Kinship testing using biallelic SNP markers has been demonstrated to be a promising approach as a supplement to standard STR typing, and several systems, such as pyrosequencing and microarray, have been introduced and utilized in real forensic cases. The Affymetrix microarray containing 169 autosomal SNPs developed for forensic application was applied to our practical case for kinship analysis that had remained inconclusive due to partial STR profiles of degraded DNA and possibility of inbreeding within the population. CASE REPORT: 169 autosomal SNPs were typed on array with severely degraded DNA of two bone samples, and the kinship compared to genotypes in a reference database of their putative family members. RESULTS: Two bone samples remained unidentified through traditional STR typing with partial profiles of 10 or 14 of 16 alleles. Because these samples originated from a geographically isolated population, a cautious approach was required when analyzing and declaring true paternity only based on PI values. In a supplementary SNP typing, 106 and 78 SNPs were obtained, and the match candidates were found in each case with improved PI values than using only STRs and with no discrepant SNPs in comparison. CONCLUSION: Our case showed that the utility of multiple SNPs on array is expected in practical forensic caseworks with an establishment of reference database.

7-acetoxycoumarin dimer-incorporated and folate-decorated liposomes: photoresponsive release and in vitro targeting and efficacy.

Photoresponsive and cancer cell (KB cell)-targetable liposome was developed by incorporating 7-acetoxycoumarin dimer (ACD) in the liposomal membrane and modifying the surface of liposome with folate. The liposomes were prepared from the dry mixed thin film of egg phosphatidylcholine (EPC), ACD, and folate conjugate (DSPE-PEG2000-folate) by a film hydration method, where the molar ratios of EPC/ACD/DSPE-PEG2000-folate were 10/0/0, 9/1/0, 9/1/0.05, and 9/0/0.05. The liposomal membranes were multilamellar and the diameter was on the order of hundreds of nanometers. The release degrees in 60 min of 5(6)-carboxyfluorescein (CF) from EPC/ACD/DSPE-PEG2000-folate liposome were less than 4% without the irradiation of UV light (λ = 254 nm, 6 W), but more than 20% under the irradiation of UV light (λ = 254 nm, 6W), possibly due to the phototriggered de-dimerization of ACD. Under confocal laser scanning microscopy, KB cells treated with EPC/ACD/DSPE-PEG2000-folate liposomes exhibited CF fluorescence of liposomes at the positions where 4',6-diamidino-2-phenylindole (DAPI) fluorescence of the cell nucleus was shown, indicating the liposomes targeted the cancer cells. In flow cytometric analysis, the cancer cells treated with EPC/ACD/DSPE-PEG2000-folate liposomes exhibited much higher fluorescence than the untreated cells did, indicating that there was a specific interaction between the liposome and the cancer cell. With the irradiation of UV light, EPC/ACD/DSPE-PEG2000-folate liposomes markedly promoted the in vitro anti-cancer efficacy of DOX without causing acute in vitro toxicity.

Liposomes composed of dioleoylphosphatidylethanolamine and 2-(hexadecyloxy)cinnamic acid: effects of UV irradiation and pH value on release.

Novel liposomes composed of dioleoylphosphatidylethanolamine (DOPE) and 2-(hexadecyloxy) cinnamic acid (HOCA) were prepared by a detergent removal method. When the molar ratio of DOPE to HOCA was 4:1, 3:2, and 2:3, the florescence quenching degree of 5(6)-carboxylic fluorescein (CF, a fluorescence marker) loaded in the assemblies was 62.4%, 68.9%, and 72.5%, respectively. The quenching degree was fairly high, indicating the assemblies were closed bilayer vesicles (liposomes). The release degree in 60 min of CF from the DOPE/HOCA liposomes was 9.6% to 15.7%, when the liposomes were subjected to the irradiation of a UV light (e.g., lambda = 254 nm). The photo-dimerization of the cinnamic acid residue of HOCA may destabilize the liposomal membrane, leading to a significant release. In addition, DOPE/HOCA (4/1) liposome exhibited a pH-sensitive release. It showed almost 100% of release degree in a few seconds under an acidic condition (e.g., pH 5.0), but it was stable at neutral and alkali conditions.

Light-sensitive liposomes containing coumarin-proteinoid conjugate.

Photo-sensitive liposomes were prepared by modifying the surface with coumarin derivative-proteinoid conjugate. Epoxypropoxy coumarin (EPC) was conjugated to proteinoid composed of Asp, Ser and Leu (PASL) using the reaction mixture of which EPC/PASL ratio was 8/1 (w/w). The molar ratio of Asp residue/Ser residue/Leu residue/EPC residue of the resulting conjugate was calculated to be 71.1:18.6/6.3/4.0 on 1H NMR spectrum. Egg phosphatidylcholine (egg PC) liposomes bearing PASL-EPC conjugate was prepared by hydrating egg PC dry film with a buffer solution containing the conjugate. Egg PC/PASL-EPC conjugate ratios (w/w) in the preparation were 100:1 to 100:20, and the specific loadings of EPC residue in the liposomes were 0.0002 mg/mg to 0.0018 mg/mg. The release from the liposomes prepared using the egg PC/PASL-EPC conjugate ratios of 100:5 began to markedly increase 30 min after the irradiation of lambda = 254 nm and the release degree in 180 min was about 30%. Without the UV irradiation, no appreciable release was observed.

Characteristics and photo-responsive release property of liposome containing 7-acetoxy coumarin.

A photo-responsive liposome was developed by loading 7-acetoxy coumarin (ATC) in egg phosphatidylcholine (EPC) liposome. ATC was derivetized from 7-hydroxy coumarin using sodium acetate. The ATC-loaded liposomes were prepared by suspending the dry mixture film of ATC/EPC in distilled water and sonicating the suspension. When the ATC to EPC ratio was less than 1:8, homogeneous liposomal suspensions were obtained without any precipitate. On 1H NMR spectra, the unsaturated chains of liposomal EPCs were somewhat deteriorated by a subsequent irradiation, the irradiation of lambda = 365 nm and then the irradiation of lambda = 254 nm. On TEM photos, the size of liposomes incorporating ATC markedly increased by the subsequent irradiation. Photo-dimerization (under lambda = 365 nm) and de-dimerization (under lambda = 254 nm) of ATC took a place even in EPC liposomes. Upon the irradiation of lambda = 254 nm, liposome containing ATC dimers exhibited an enhanced release of 5(6)-carboxyfluorescein compared with liposome containing ATC monomer and liposome free of ATC. The de-dimerization of ATC dimers is believed to fluidize the liposomal membrane and promote the release.