Body fluid identification by integrated analysis of DNA methylation and body fluid-specific microbial DNA.

Identification of body fluids found at crime scenes provides important information that can support a link between sample donors and actual criminal acts. Previous studies have reported that DNA methylation analysis at several tissue-specific differentially methylated regions (tDMRs) enables successful identification of semen, and the detection of certain bacterial DNA can allow for identification of saliva and vaginal fluid. In the present study, a method for detecting bacterial DNA was integrated into a previously reported multiplex methylation-sensitive restriction enzyme-polymerase chain reaction. The developed multiplex PCR was modified by the addition of a new semen-specific marker and by including amplicons for the 16S ribosomal RNA gene of saliva- and vaginal fluid-specific bacteria to improve the efficacy to detect a specific type of body fluid. Using the developed multiplex system, semen was distinguishable by unmethylation at the USP49, DACT1, and PFN3 tDMRs and by hypermethylation at L81528, and saliva could be identified by detection of saliva-specific bacteria, Veillonella atypica and/or Streptococcus salivarius. Additionally, vaginal fluid and menstrual blood were differentiated from other body fluids by hypomethylation at the PFN3 tDMR and the presence of vaginal fluid-specific bacteria, Lactobacillus crispatus and/or Lactobacillus gasseri. Because the developed multiplex system uses the same biological source of DNA for individual identification profiling and simultaneously analyses various types of body fluid in one PCR reaction, this method will facilitate more efficient body fluid identification in forensic casework.

DNA methylation-specific multiplex assays for body fluid identification.

Recent advances in whole-genome epigenetic analysis indicate that chromosome segments called tissue-specific differentially methylated regions (tDMRs) show different DNA methylation profiles according to cell or tissue type. Therefore, body fluid-specific differential DNA methylation is a promising indicator for body fluid identification. However, DNA methylation patterns are susceptible to change in response to environmental factors and aging. Therefore, we investigated age-related methylation changes in semen-specific tDMRs using body fluids from young and elderly men. After confirming the stability of the body fluid-specific DNA methylation profile over time, two different multiplex PCR systems were constructed using methylation-sensitive restriction enzyme PCR and methylation SNaPshot, in order to analyze the methylation status of specific CpG sites from the USP49, DACT1, PRMT2, and PFN3 tDMRs. Both multiplex systems could successfully identify semen with spermatozoa and could differentiate menstrual blood and vaginal fluids from blood and saliva. Although including more markers for body fluid identification might be necessary, this study adds to the support that body fluid identification by DNA methylation profiles could be a valuable tool for forensic analysis of body fluids.

Potential forensic application of DNA methylation profiling to body fluid identification.

DNA analysis of various body fluid stains at crime scenes facilitates the identification of individuals but does not currently determine the type and origin of the biological material. Recent advances in whole genome epigenetic analysis indicate that chromosome pieces called tDMRs (tissue-specific differentially methylated regions) show different DNA methylation profiles according to the type of cell or tissue. We examined the potential of tissue-specific differential DNA methylation for body fluid identification. Five tDMRs for the genes DACT1, USP49, HOXA4, PFN3, and PRMT2 were selected, and DNA methylation profiles for these tDMRs were produced by bisulfite sequencing using pooled DNA from blood, saliva, semen, menstrual blood, and vaginal fluid. The tDMRs for DACT1 and USP49 showed semen-specific hypomethylation, and the tDMRs for HOXA4, PFN3, and PRMT2 displayed varying degrees of methylation according to the type of body fluid. Preliminary tests using methylation-specific PCR for the DACT1 and USP49 tDMRs showed that these two markers could be used successfully to identify semen samples including sperm cells. Body fluid-specific differential DNA methylation may be a promising indicator for body fluid identification. Because DNA methylation profiling uses the same biological source of DNA for individual identification profiling, the determination of more body fluid-specific tDMRs and the development of convenient tDMR analysis methods will facilitate the broad implementation of body fluid identification in forensic casework.

Clinicopathologic report of uveal melanoma with persistent exudative retinal detachment after gamma knife radiosurgery.

AIMS: Our purpose was to report the clinical and pathological findings from uveal melanoma patients with persistent exudative retinal detachment (RD) after Gamma Knife radiosurgery (GKR). METHODS: A retrospective review was performed. RESULTS: GKR was performed on 19 uveal melanoma patients from 2004 to 2006, and 5 of them developed persistent exudative RD. The mean initial largest basal tumor diameter in these 5 patients was 14.4 +/- 1.9 mm, and the mean tumor height was 9.2 +/- 1.0 mm. Marginal doses of 40-50 Gy radiation were administered. RD developed or had become aggravated an average of 3 (1-10) months after GKR. Two patients underwent enucleation, while the remaining 3 underwent tumor removal by endoresection with retinal reattachment surgery by vitrectomy and silicone oil tamponade. These procedures were undertaken an average of 6.3 (1.5-14) months after radiation and 3.3 (0.5-5) months after the onset or aggravation of RD. Histologically 4 tumors showed 50-100% necrosis without any mitotic activity. Thus local tumor control after GKR appeared sufficient regardless of RD. CONCLUSION: Aggravation or development of exudative RD after GKR does not necessarily entail treatment failure. Therefore, retinal reattachment surgery in persistent exudative RD may be an option to preserve the eye.

DNA methylation-based age prediction from saliva: High age predictability by combination of 7 CpG markers.

DNA methylation is currently one of the most promising age-predictive biomarkers. Many studies have reported DNA methylation-based age predictive models, but most of these are based on DNA methylation patterns from blood. Only a few studies have examined age-predictive DNA patterns in saliva, which is one of the most frequently-encountered body fluids at crime scenes. In this study, we generated genome-wide DNA methylation profiles of saliva from 54 individuals and identified CpG markers that showed a high correlation between methylation and age. Because the age-associated marker candidates from saliva differed from those of blood, we investigated DNA methylation patterns of 6 age-associated CpG marker candidates (cg00481951, cg19671120, cg14361627, cg08928145, cg12757011, and cg07547549 of the SST, CNGA3, KLF14, TSSK6, TBR1, and SLC12A5 genes, respectively) in addition to a cell type-specific CpG marker (cg18384097 of the PTPN7 gene) in an independent set of saliva samples obtained from 226 individuals aged 18 to 65 years. Multiplex methylation SNaPshot reactions were used to generate the data. We then generated a linear regression model with age information and the methylation profile from the 113 training samples. The model exhibited a 94.5% correlation between predicted and chronological age with a mean absolute deviation (MAD) from chronological age of 3.13 years. In subsequent validation using 113 test samples, we also observed a high correlation between predicted and chronological age (Spearman's rho=0.952, MAD from chronological age=3.15years). The model composed of 7 selected CpG sites enabled age prediction in saliva with high accuracy, which will be useful in saliva analysis for investigative leads.

DNA methylation profiling for a confirmatory test for blood, saliva, semen, vaginal fluid and menstrual blood.

The ability to predict the type of tissues or cells from molecular profiles of crime scene samples has important practical implications in forensics. A previously reported multiplex assay using DNA methylation markers could only discriminate between 4 types of body fluids: blood, saliva, semen, and the body fluid which originates from female reproductive organ. In the present study, we selected 15 menstrual blood-specific CpG marker candidates based on analysis of 12 genome-wide DNA methylation profiles of vaginal fluid and menstrual blood. The menstrual blood-specificity of the candidate markers was confirmed by comparison with HumanMethylation450 BeadChip array data obtained for 58 samples including 12 blood, 12 saliva, 12 semen, 3 vaginal fluid, and 19 skin epidermis samples. Among 15CpG marker candidates, 3 were located in the promoter region of the SLC26A10 gene, and 2 of them (cg09696411 and cg18069290) showed high menstrual blood specificity. DNA methylation at the 2CpG markers was further tested by targeted bisulfite sequencing of 461 additional samples including 49 blood, 52 saliva, 34 semen, 125 vaginal fluid, and 201 menstrual blood. Because the 2 markers showed menstrual blood-specific methylation patterns, we modified our previous multiplex methylation SNaPshot reaction to include these 2 markers. In addition, a blood marker cg01543184 with cross reactivity to semen was replaced with cg08792630, and a semen-specific unmethylation marker cg17621389 was removed. The resultant multiplex methylation SNaPshot allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood using the 9CpG markers which show a methylation signal only in the target body fluids. Because of the complexity in cell composition, menstrual bloods produced DNA methylation profiles that vary with menstrual cycle and sample collection methods, which are expected to provide more insight into forensic menstrual blood test. Moreover, because the developed multiplex methylation SNaPshot reaction includes the 4CpG markers of which specificities have been confirmed by multiple studies, it will facilitate confirmatory tests for body fluids that are frequently observed in forensic casework.

Chondrogenic differentiation of human mesenchymal stem cells using a thermosensitive poly(N-isopropylacrylamide) and water-soluble chitosan copolymer.

Poly(N-isopropylacrylamide) (PNIPAAm) is known to be thermally responsive material and has a lower critical solution temperature (LCST, 32 degrees C) at which a macromolecular transition from a hydrophilic to a hydrophobic structure occurs. Chitosan is a useful natural polymeric biomaterial due to its biocompatibility and biodegradable properties. It has good characteristics for cell attachment, proliferation and viability. The aim of this study was to assess the ability to differentiate from mesenchymal stem cells (MSCs) to chondrocytes and mass formation using a newly developed injectable material, a thermosensitive (water-soluble chitosan-g-PNIPAAm) gel, and evaluate cartilage formation in vivo after injecting a cell-thermosensitive gel complex. The MSCs were cultured in the chitosan-PNIPAAm in vitro. Fluorescence-activated cell sort analysis, viability test, collagen type I, II, X formation and the aggrecan levels were examined. These cultured cells can be easily recovered from a copolymer gel by simply lowering the temperature. An animal study was performed to assess cartilage formation in the submucosal layer of the bladder of rabbits. The cartilage formation could be detected. This can be used to treat vesicoureteral reflux or reflux esophagitis by the effective mass effect. This is a simple method (sol-gel technique in LCST), and good cartilage formation occurs in the bladder tissue.

Rapid direct PCR for ABO blood typing.

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.

Histological and biomechanical properties of regenerated articular cartilage using chondrogenic bone marrow stromal cells with a PLGA scaffold in vivo.

The properties of regenerated cartilage using bone marrow-derived mesenchymal stem cells (MSCs) and poly lactic-co-glycolic acid (PLGA) scaffold composites pretreated with TGF-beta3 were investigated and compared to the non-TGF-beta3 treated MSCs/PLGA composites in a rabbit model. We prepared MSCs/PLGA scaffold composites and pretreated it with TGF-beta3 for 3 weeks prior to transplantation. Then, composites were transplanted to the osteochondral defect in the rabbit knee. After 12 weeks of transplantation, 10 of the 12 rabbits in which TGF-beta3 pretreated MSCs/PLGA scaffold composites were transplanted showed cartilaginous regeneration. In gross morphology, regenerated cartilage showed smooth, flush, and transparent features. In indentation test, this had about 80% of Young's modulus of normal articular cartilage. Histological examination demonstrated hyaline like cartilage structures with glycosaminoglycan and type II collagen expression. Histological scores were not statistically different to the normal articular cartilage. These results showed improvement of cartilage regeneration compared to the non-TGF-beta3 pretreated MSCs/PLGA scaffold composite transplanted group. Thus, we have successfully regenerated improved hyaline-like cartilage and determined the feasibility of treating damaged articular cartilage using MSCs/PLGA scaffold composite pretreated with TGF-beta3. Also, we suggest this treatment modality as another concept of cartilage tissue engineering.