Transfer, persistence and recovery of DNA and mRNA vaginal mucosa markers after intimate and social contact with Bayesian network analysis for activity level reporting
Forensic Science International: Genetics, 2022
Authors
Journal
Forensic Science International: Genetics
Study Design
Addressed Question
1. How long after intimate contact is it possible to detect mRNA vaginal mucosa markers in fingernail or penile swabs. 2. if mRNA vaginal mucosa markers can be detected in fingernail swabs, penile swabs or boxershors if only social contact has occurred. 3. The associateion between the mRNA and DNA results.
Activity Context
Category
Specifications
Variables of Interest
Stringency of Control
Number of Individuals
24 (12 couples)
Replicates per Individual and Condition
1 sampling event per individual/location/time-point where available, no repeated experimental replicates reported.
Nucleic Acid
Bodily Origin
Depositor & Contact
Depositor Characteristics
Gender
Criteria for Shedder Status
n.a.
Previous Activities
n.a.
Contact Scenario
Intimate contact, defined as vaginal penetration by penis and by fingers from dominant hand, and no intimate contact by couples.
Primary Substrate
Primary Substrate Type
fingernails, penis and boxershorts.
Primary Substrate Material
Deposit
Natural deposition during intimate contact
Delay
n.a.
Secondary Substrate
Secondary Substrate Type
n.a.
Secondary Substrate Material
N/A
Secondary Substrate Contact
n.a.
Further Transfer
n.a.
Sampling
Background DNA on Sampled Surface
Sampling Time
for finger and penile samples: 0, 12, 18, 24 and 36 h post-intimate contact. Non-intimate-contact (at least 72 h since last intimate contact) samples were also collected. Boxer shorts was collected after it was worn for 5-6 hours after intimate contact.
Persistence
0-36 hours after intimate contact. Conditions were normal daily life.
Sampling Method
Wet cotton swab for fingernails and penile surface. mini-tape (Scenesafe FAST TM K545) for boxer shorts.
Sampling Area
The crotch area (ca. 18x20cm) in the boxer shorts. Area of swabs for underneath all fingernails of dominant hand and penile swaps from shaft of penis not specified.
Laboratory Analysis
Extraction
All experimental samples were co-extracted using QIAamp DNA mini kit (QIAGEN) and mirVANA TM miRNA isolation kit (invitrogen by Thermo Fisher Scientific)
DNA Quantification
DNA fractions and refrence samples were quantified with Power-Quant R System (Promega)
Input for Profiling
Amplified with Promegas Power-PlexRFusrion 6C System (25 uL reaction volume, 1 ng DNA input, 29 amplification cycles) as recommended by the manufacturer.
Profiling
Samples were injected onto the applied biosystems 3500xl Genetic Analyzer at 1.2 kV for 24s. Results were analysed using the GeneMapper R ID-X Software version 1.6 (Applied Biosystems TM) and the analytical threshold for the alleles was set to 100 RFU. No stochastic threshold was applied.
Reference Samples
A mouth swab was collected as a DNA reference sample from each of the 24 participants.
Profile Interpretation and Mixture Analysis
DNA results were evaluated using likelihood ratios, with LR ≥ 10,000 used to classify a result as supporting the proposition that the POI was the donor. The data were then incorporated into a Bayesian network for activity-level evaluation. A DNA mixture was assigned if there was three or more alleles at a locus. Sub-source likelihood ratios (LR) were calculated using EuroForMix v.3.2.0.
RNA Data Interpretation
The markers included were: blood markers HBB, ALAS2, CD93; saliva markers HTN3 and STATH; semen markers SEMG1, PRM1 and KLK3; vaginal mucosa markers MUC4, MYOZ1 and CYP2B7P1; menstrual secretion markers MMP7, MMP10 and MMP11; nasal mucosa markers BPIFA1; male marker RPS4Y1; female marker XIST; and housekeeping genes ACTB and 18S-rRNA. The results were analysed using the GeneMapper® ID-X Software version 1.6 (Applied Biosystems™) and the limit of detection (LOD) for the alleles was set to 50 RFU.
Results
DNA Quantity
All values in ng/ul. Penile 0h: 0,42-180,9. Penile 12h: 0,4-10,5. Penile 18h: 0,04-10,02. Penile 24h: 0,17-2,36. Penile 36h: 0,07-2,16. Penile non-intimate: 0,01 – 1,16. Fingernail 0h: 0,88-25,61. Fingernail 12h: 0,02– 2,66. Fingernail 18h: 0,02– 1,44. Fingernail 24h: 0,02– 2,41. Fingernail 36h: 0,03– 0,4. Fingernail non-intimate: 0,01-6,38. Boxer shorts 0h: 0,4 – 16,35. Boxer shorts non-intimate: 0,01-1,74.
Profile Quality
DNA results were sufficiently informative to support POI-donor propositions in 94% of intimate-contact samples and 50% of non-intimate-contact samples at LR ≥10,000
Parameter Used for Comparison
Detection of vaginal-mucosa mRNA. DNA quantity/mean RFU. DNA likelihood ratio for POI contribution. time since intimate contact. sampling location.
Summary of Results
Vaginal mucosa was detectable for 36 h post intimate contact, although detection declined with time. It was also detected in one non-intimate-contact sample, demonstrating that detection of a vaginal-mucosa marker was not uniquely associated with intimate contact. Full profiles of the POI were detected in some of the non-intimate contact penile and boxershorts samples, highlighting that the evaluation of DNA evidence must be carried out with care. DNA evidence supporting the POI as donor was obtained in 94% of intimate-contact samples and 50% of non-intimate-contact samples at LR ≥10,000. The Bayesian-network analysis showed that the evidential value was driven mainly by the relatively high DNA quantity/mean RFU, whereas detection of vaginal-mucosa mRNA had comparatively little influence on the resulting activity-level LR.
Raised Questions
The study addresses the need to replace MUC4 as a vaginal mucosa marker, as it did not perform well in this study.
Cautionary Remarks
N/A