Highlights
- •We selected 15 CpGs as menstrual blood-specific marker candidates using HumanMethylation450 BeadChip array data.
- •cg09696411 and cg18069290 in the SLC26A10 gene demonstrated high menstrual blood-specificity.
- •A previously reported multiplex methylation SNaPshot was modified to detect 9 CpGs, which include cg09696411 and cg18069290.
- •The resultant multiplex allowed positive identification of blood, saliva, semen, vaginal fluid and menstrual blood.
- •Menstrual bloods produced methylation profiles that vary with menstrual cycle and sample collection methods.
Abstract
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 15 CpG 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 2 CpG 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 9 CpG 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 4 CpG 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.
Keywords
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Article info
Publication history
Published online: June 13, 2016
Accepted:
June 11,
2016
Received in revised form:
June 7,
2016
Received:
February 11,
2016
Identification
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