Y-STR analysis for detection and objective confirmation of child sexual abuse

We evaluated 26 child sexual assault cases for the incorporation of Y-STR screening in the routine detection and objective confirmation of sexual contact between the child victim and the perpetrator. Various samples, e.g. vaginal or anal swabs from patients aged 2–17 years old (25 females, 1 male), were collected 6–72 h after the incident. Due to the limited amounts of DNA in these samples, total DNA was extracted using a one-step procedure and screened with autosomal STRs to detect signs of a victim-assailant DNA mixture and with Y-STRs for assailant DNA. Autosomal STRs failed to give signs of victim-assailant DNA mixtures while Y-STRs were detected in 24 of the 26 cases corresponding to a success rate of 92.3%. With the possible presence of both male sperm and/or male epithelial cells in forensic evidence, Y-STR DNA markers were detected regardless of external ejaculation, microscopic detection of sperm and with post-coital intervals of up to 72 h. While only partial profiles were generated owing to low quantities of male DNA present, Y-STR screening results can serve as objective evidence of sexual contact in child sexual abuse cases involving victims who do not have any previous sexual history. This type of evidence can corroborate child victim testimony and spare the child victim from further trauma caused by prolonged forensic investigations and court proceedings. Alternatively, Y-STR screening can provide objective proof of non-involvement of an accused with the victim.     

Y-STR analysis of digital and/or penile penetration cases with no detected spermatozoa

This forensic casework trial involved Yfiler^® testing samples from 47 digital and/or penile penetration cases where the medical examination had occurred within 48 h of the alleged incident and no spermatozoa had been detected following Sperm Elution^©. 30% of these cases yielded at least one Y-STR profile comprising three or more alleles per profile and 21% yielded at least one Y-STR profile of ten or more alleles per profile. This trial further investigated the persistence of male DNA in different case types, the location of samples submitted for testing and whether samples from different locations benefit from being combined prior to testing. The data supports the use of Y-STR profiling to provide scientific evidence to investigate whether the alleged sexual activity had occurred as well as to obtain probative evidence in spermatozoa negative penetration cases.     

Fetal male lineage determination by analysis of Y-chromosome STR haplotype in maternal plasma

The aim of this study is to determine the fetus Y-STR haplotype in maternal plasma during pregnancy and estimate, non-invasively, if the alleged father and fetus belong to the same male lineage. The study enrolled couples with singleton pregnancies and known paternity. All participants signed informed consent and the local ethics committee approved the study. Peripheral blood was collected in EDTA tubes (mother) and in FTA paper (father). Maternal plasma DNA was extracted by using NucliSens EasyMAG. Fetal gender was determined by qPCR targeting DYS-14 in maternal plasma and it was also confirmed after the delivery. From all included volunteers, the first consecutive 20 mothers bearing male fetuses and 10 mothers bearing female fetuses were selected for the Y-STR analysis. The median gestational age was 12 weeks (range 12–36). All DNA samples were subjected to PCR amplification by PowerPlex Y23, ampFLSTR Yfiler, and two in-house multiplexes, which together accounts for 27 different Y-STR. The PCR products were detected with 3500 Genetic Analyzer and they were analyzed using GeneMapper-IDX. Fetuses’ haplotypes (Yfiler format) were compared to other 5328 Brazilian haplotypes available on Y-chromosome haplotypes reference database (YHRD). As a result, between 22 and 27 loci were successfully amplified from maternal plasma in all 20 cases of male fetuses. None of the women bearing female fetuses had a falsely amplified Y-STR haplotype. The haplotype detected in maternal plasma completely matched the alleged father haplotype in 16 out of the 20 cases. Four cases showed single mismatches and they did not configure exclusions; 1 case showed a mutation in the DYS 458 locus due to the loss of one repeat unit and 3 cases showed one DYS 385I/II locus dropout. All mismatches were confirmed after the delivery. Seventeen fetuses’ haplotypes were not found in YHRD and one of them had a mutation, which corresponded to the paternity probability of 99.9812% and 95.7028%, respectively. Three fetuses’ haplotypes occurred twice in YHRD, which corresponded to paternity probability of 99.9437%. In conclusion, high discriminatory fetal Y-STR haplotype could be determined from maternal plasma during pregnancy starting at 12 weeks of gestation. All male fetuses could be attributed to the alleged father male lineage early in pregnancy. The high probability of paternity associated with each case suggests that the relationship is not random and this strategy can be use as an alternative for male fetal kinship analysis.     

Autosomal and Y-STR analysis of degraded DNA from the 120-year-old skeletal remains of Ezekiel Harper

The 120-year-old skeletal remains of Confederate Civil War soldier Captain Ezekiel “Zeke” Harper were exhumed by court order in January 2011 for DNA analysis. The goal of the DNA testing was to support or refute whether Captain Harper had fathered a son (Earl J. Maxwell) with his Native American maid prior to his murder in 1892. Bones with adequate structural integrity (left tibia, right tibia, right femur, mandible, four teeth) were retrieved from the burial site and sent to the Institute of Applied Genetics in Fort Worth, Texas for analysis. Given the age and condition of the remains, three different extraction methods were used to maximize the probability of DNA recovery. The majority of the DNA isolates from over fifty separate bone sections yielded partial autosomal STR genotypes and partial Y-STR haplotypes. After comparing the partial results for concordance, consensus profiles were generated for comparison to reference samples from alleged family members. Considering the genetic recombination that occurs in autosomal DNA over the generations within a family, Y-STR analysis was determined to be the most appropriate and informative approach for determining potential kinship. Two of Earl J. Maxwell's grandsons submitted buccal samples for comparison. The Y-STR haplotypes obtained from both of these reference samples were identical to each other and to the alleles in Ezekiel Harper's consensus profile at all 17 loci examined. This Y-STR haplotype was not found in either of two major Y-STR population databases (U.S. Y-STR database and YHRD). The fact that the Y-STR haplotype obtained from Ezekiel's skeletal remains and Earl's grandsons is not found in either population database demonstrates its rarity and further supports a paternal lineage relationship among them. Results of the genetic analyses are consistent with the hypothesis that Earl J. Maxwell is the son of Ezekiel Harper.     

A Case of Amelogenin Y-null: A simple primer binding site mutation or unusual genetic anomaly?

A thirteen year old boy was murdered by a gunshot wound to the head. In order to confirm identity of the boy, samples were sent to the Instituto de Ciencias Forenses de Puerto Rico (PR-ICF) DNA laboratory. Autosomal DNA results exhibited only an X at the Amelogenin locus, whereas the autopsy results reported the child to be anatomically male. The sample was amplified with four separate YSTR marker systems. While a full Y-STR profile for the father of the boy was obtained, the boy only amplified at STR markers on the p arm of the Y chromosome. Theories that could account for this large absence of Y-STR results include an X-Y translocation or Yp isochromosome.     

Weight of evidence of Y-STR matches computed with the discrete Laplace method: Impact of adding a suspect’s profile to a reference database

The discrete Laplace method is recommended by multiple parties (including the International Society for Forensic Genetics, ISFG) to estimate the weight of evidence in criminal cases when a suspect’s Y-STR profile matches the crime scene Y-STR profile. Unfortunately, modelling the distribution of Y-STR profiles in the population reference database is time-consuming and requires expert knowledge. When the suspect’s Y-STR profile is added to the database, as would be the protocol in many cases, the parameters of the discrete Laplace model must be re-estimated. We found that the likelihood ratios with and without adding the suspect’s Y-STR profile were almost identical with 1,000 or more Y-STR profiles in the database for Y-STR profiles with 8, 12, and 17 loci. Thus, likelihood ratio calculations can be performed in seconds if an established discrete Laplace model based on at least 1,000 Y-STR profiles is used. A match in a population reference database with 17 Y-STR loci from at least 1,000 male individuals results in a likelihood ratio above 10,000 in approximately 94% of the cases, and above 100,000 in approximately 82% of the cases. We offer free software accessible without restrictions to estimate a discrete Laplace model using a Y-STR reference database and subsequently to calculate likelihood ratios.     

Assessing the presence of female DNA on post-coital penile swabs: Relevance to the investigation of sexual assault

During the investigation of sexual assault, penile swabs from an alleged perpetrator are processed as part of the routine procedure. The results of the forensic DNA analysis may subsequently be presented in court. Documentation on the expectancy of finding female cells on post-coital penile swabs is scarce. Reviews from assault cases show that retrieval of female cells from penile swabs is generally reported for less than 50% of the cases. The perception may therefore be that female cells are not expected to be recovered on penile swabs, even few hours after vaginal penetration. However, these reviews do not provide certainty that penetration of body cavities had taken place. Furthermore, the alleged perpetrator may have washed himself before the examination. In the present study, eleven couples provided fourteen sets of post-coital penile swabs collected between 5 and 24 h after vaginal intercourse. At time intervals between 5 and 12 h, the full female DNA profiles were recovered in 90% of the samples. At the lowest, 67% of the full female profile was typed as an average of two swabs sampled at each time point. Samples collected from three couples at 20, 22 and 24 h post-coital, retrieved 100% (15 AMPFlSTR^®Identifiler markers, in addition to the amelogenin gene) of the female DNA profile from one couple, and 37% and 30% of the full female DNA profiles from the other two couples. For the majority of samples, male DNA was present in slightly greater abundance than female DNA. In this study, female DNA was recovered on all post-coital penile swabs taken at 5–24 h intervals.     

Identifying the most likely contributors to a Y-STR mixture using the discrete Laplace method

In some crime cases, the male part of the DNA in a stain can only be analysed using Y chromosomal markers, e.g. Y-STRs. This may be the case in e.g. rape cases, where the male components can only be detected as Y-STR profiles, because the fraction of male DNA is much smaller than that of female DNA, which can mask the male results when autosomal STRs are investigated. Sometimes, mixtures of Y-STRs are observed, e.g. in rape cases with multiple offenders. In such cases, Y-STR mixture analysis is required, e.g. by mixture deconvolution, to deduce the most likely DNA profiles from the contributors.We demonstrate how the discrete Laplace method can be used to separate a two person Y-STR mixture, where the Y-STR profiles of the true contributors are not present in the reference dataset, which is often the case for Y-STR profiles in real case work. We also briefly discuss how to calculate the weight of the evidence using the likelihood ratio principle when a suspect's Y-STR profile fits into a two person mixture. We used three datasets with between 7 and 21 Y-STR loci: Denmark (n = 181), Somalia (n = 201) and Germany (n = 3443). The Danish dataset with 21 loci was truncated to 15 and 10 loci to examine the effect of the number of loci. For each of these datasets, an out of sample simulation study was performed: A total of 550 mixtures were composed by randomly sampling two haplotypes, h₁ and h₂, from the dataset.We then used the discrete Laplace method on the remaining data (excluding h₁ and h₂) to rank the contributor pairs by the product of the contributors’ estimated haplotype frequencies. Successful separation of mixtures (defined by the observation that the true contributor pair was among the 10 most likely contributor pairs) was found in 42–52% of the cases for 21 loci, 69–75% for 15 loci and 92–99% for 10 loci or less depending on the dataset and how the discrete Laplace model was chosen. Y-STR mixtures with many loci are difficult to separate, but even haplotypes with 21 Y-STR loci can be separated.     

Analysis of clinical forensic examination reports on sexual assault

Medical-forensic examination of sexual assault victims and alleged offenders is a common task of many forensic institutes. In the current study, the results from samples taken at the Institute of Legal Medicine, Hanover Medical School, during a period from 2005 to 2007 were retrospectively evaluated. In total, 292 victims (283 females and nine males) and 88 suspects were examined. At the time of the assault, 41.8% of the victims and 43.2% of the alleged perpetrators were under the influence of alcohol. Injuries were found in 84.9% of the victims and 39.8% of the suspects. Thirty victims (10.3%) reported having been choked or strangled. Cytology was performed in 218 victims. In 81 cases (38.0%), sperm could be detected in vaginal swabs up to 3 days post-assault. In seven (18.9%) out of 37 anal samples, evidence of sperm could be found 24 h post-assault. None of 22 oral samples was positive for sperm. Out of 301 sexual assault cases, 171 could be proved by means of medical-forensic examination. In summary, our evaluation shows that an early medical-forensic examination of both victim and suspect can secure numerous medical findings. Furthermore, persons intoxicated by alcohol, handicapped persons and persons with psychiatric disorders are more vulnerable to become a sexual assault victim.     

Validation of a combined autosomal/Y-chromosomal STR approach for analyzing typical biological stains in sexual-assault cases

DNA testing is an established part of the investigation and prosecution of sexual assault. The primary purpose of DNA evidence is to identify a suspect and/or to demonstrate sexual contact. However, due to highly uneven proportions of female and male DNA in typical stains, routine autosomal analysis often fails to detect the DNA of the assailant. To evaluate the forensic efficiency of the combined application of autosomal and Y-chromosomal short tandem repeat (STR) markers, we present a large retrospective casework study of probative evidence collected in sexual-assault cases. We investigated up to 39 STR markers by testing combinations of the 16-locus NGMSElect kit with both the 23-locus PowerPlex Y23 and the 17-locus Yfiler kit. Using this dual approach we analyzed DNA extracts from 2077 biological stains collected in 287 cases over 30 months. To assess the outcome of the combined approach in comparison to stand-alone autosomal analysis we evaluated informative DNA profiles. Our investigation revealed that Y-STR analysis added up to 21% additional, highly informative (complete, single-source) profiles to the set of reportable autosomal STR profiles for typical stains collected in sexual-assault cases. Detection of multiple male contributors was approximately three times more likely with Y-chromosomal profiling than with autosomal STR profiling. In summary, 1/10 cases would have remained inconclusive (and could have been dismissed) if Y-STR analysis had been omitted from DNA profiling in sexual-assault cases.     

Method to predict the chance of developing a male profile out of mixtures of male and female DNA

In forensic examination it is a standard to take vaginal swabs from victims of sexual assault for further molecular genetic analysis. Laboratories then are usually confronted with mixtures of lots of female and only a small amount of male DNA. Nowadays it is possible to work with specific Y chromosomal markers after DNA extraction by differential lysis. The determined ratio of autosomal DNA and Y chromosomal DNA can be used to identify the possibility of generating a male profile in these samples.     

Prevalence and persistence of male DNA identified in mixed saliva samples after intense kissing

Identification of foreign biological material by genetic profiling is widely used in forensic DNA testing in different cases of sexual violence, sexual abuse or sexual harassment. In all these kinds of sexual assaults, the perpetrator could constrain the victim to kissing. The value of the victim's saliva taken after such an assault has not been investigated in the past with currently widely used molecular methods of extremely high sensitivity (e.g. qPCR) and specificity (e.g. multiplex Y-STR PCR).In our study, 12 voluntary pairs were tested at various intervals after intense kissing and saliva samples were taken from the women to assess the presence of male DNA. Sensitivity-focused assays based on the SRY (single-copy gene) and DYS (multi-copy gene) sequence motifs confirmed the presence of male DNA in female saliva after 10 and even 60 min after kissing, respectively. For specificity, standard multiplex Y-STR PCR profiling was performed and male DNA was found in female saliva samples, as the entire Y-STR profile, even after 30 min in one sample.Our study confirms that foreign DNA tends to persist for a restricted period of time in the victim's mouth, can be isolated from saliva after prompt collection and can be used as a valuable source of evidence.     

DNA transfer during laundering may yield complete genetic profiles

In a number of child sexual abuse cases, the alleged perpetrator is a member of the nuclear family. In those cases, there is a possibility that the suspect’s DNA was innocently deposited onto the child’s clothing without acts of sexual assault ever occurring, for example via secondary transfer within the washing machine. To assess the quantity and quality of DNA that may be transferred among clothing during laundering, we conducted three series of experiments. First, we evaluated the level of spermatozoa that may be transferred by washing pristine pairs of underwear with bed sheets containing a varying number of ejaculates. Secondly, we explored whether current genetic methods may also detect the transfer of DNA from vaginal secretions during a machine wash. Finally, we analyzed the background levels of DNA on children’s underwear collected from control families where sexual abuse never occurred. For both spermatozoa and vaginal secretions, we revealed that sufficient amounts of DNA may transfer onto laundered clothing to yield complete genetic profiles. Furthermore, DNA from relatives living within the same household was found in most cuttings taken from control children’s underwear. Based on these findings, we present a framework for the handling and interpretation of intrafamilial sexual abuse cases. These suggestions should help determine whether DNA was deposited directly onto a fabric or merely transferred during a wash.     

Improved specificity of Y-STR typing in DNA mixture samples

Y-STR loci are beneficial for the analysis of forensic samples especially in sexual assault cases or other situations where mixtures of male and female cells are present. However, the amplification of Y-chromosomal STRs is also known to result in the formation of artefactual amplification products, mainly due to insufficient PCR specificity. This is a major drawback of the method, as the sensitivity as well as the correct Y-STR interpretation are affected. In our study, the addition of a PCR enhancer to the reaction master-mix resulted in a significant increase of specificity of Y-STR typing. This was clearly demonstrated by a loss of artefactual signal with increasing enhancer concentration, while the peak heights of the Y-STR alleles were not significantly affected by the enhancer. Mixtures of up to 1:500 (200 pg male and 100 ng female DNA) gave correct Y-STR profiles when the PCR enhancer was added to the reaction, while artefactual amplification succeeded over Y-specific amplification when no PCR enhancer was present.     

Description of artefacts in the PowerPlex Y23® system associated with excessive quantities of background female DNA

Male on female sexual assault cases that involve azoospermic individuals, those where the male has penetrated but failed to ejaculate, those where there has been an extended interval between the sexual assault and sample collection or where there has been only digital penetration are often difficult to investigate by employing traditional autosomal STR testing. Such cases often involve minimal amounts of male DNA either being deposited initially or remaining after the passage of time. These cases are often further complicated by the presence of large amounts of female DNA compared to the relatively small amounts of male DNA on the intimate samples taken. Y-STR kits provide a solution that allows targeting of male DNA in a mixed male/female sample. However, large quantities of excess female DNA have the potential to generate non-specific artefact peaks. Here we characterise a number of previously reported artefacts observed in the PowerPlex^® Y23 system. We demonstrate that some of these artefacts can impact on profile interpretation and that they are highly dependent on the levels of female DNA present. These artefacts have been characterised to assist practitioners with the interpretation of such samples.     

Persistence of DNA from laundered semen stains: Implications for child sex trafficking cases

In sexual assault cases, particularly those involving internal child sex trafficking (ICST), victims often hide their semen-stained clothing. This can result in a lag time of several months before the items are laundered and subsequently seized during a criminal investigation. Although it has been demonstrated previously that DNA can be recovered from clothing washed immediately after semen deposition, laundered items of clothing are not routinely examined in ICST cases, due to the assumption that the time delay and washing would result in no detectable DNA. The aim of this study was to examine whether viable DNA profiles could be recovered from laundered semen stains where there has been a significant lag time between semen deposition from one or more individuals and one or more washes of the stained clothing.Items of UK school uniform (T-shirts, trousers, tights) were stained with fresh semen (either from a single donor or a 1:1 mixture from two donors) and stored in a wardrobe for eight months. Stained and unstained items (socks) were then washed at 30 °C or 60 °C and with non-biological or biological detergent. DNA samples extracted from the semen-stained sites and from the unstained socks were quantified and profiled.High quantities of DNA, (6–18 μg) matching the DNA profiles of the semen donors, were recovered from all semen-stained clothing that had been laundered once, irrespective of wash conditions. This quantity,and profile quality,did not decline significantly with multiple washes. The two donor semen samples yielded ∼10-fold more DNA from the T-shirts than from the trousers. This disparity resulted in the T-shirts yielding a ∼1:1 mixture of DNA from the two donors, whereas the trousers yielded a major DNA profile matching only that of the second donor. The quantities of DNA recovered from the unstained socks were an order of magnitude lower, with most of the DNA being attributable to the donor of the semen on the stained clothing within the same wash, demonstrating the transfer of semen-derived DNA among items of clothing in the washing machine.This study demonstrates that complete DNA profiles can be obtained from laundered semen stains on school uniform-type clothing, with an eight-month lag time between semen deposition and laundering, despite multiple washes and stains from two semen donors. These data emphasise the need to recover and examine the clothing of victims for semen and DNA evidence, even if the clothing has been stored for several months or washed multiple times since the sexual offence took place.     

Evaluation of incest cases of Turkey in terms of DNA profiling difficulties

We scanned suspicious 1200 paternity cases and 650 sexual abuse victims in Council of Forensic Medicine of Turkey between 2011 and 2014 and detected 50 incest cases and evaluated the forensic and genetic data of incest cases for source of DNA evidence, gender, age, SES (Socioeconomic status) and geographic location of victim, abusive person, extent of incest, pregnancy from incest and date of gestation termination and also aimed to discuss some DNA profiling difficulties.We detected incest from DNA evidences of curettage material (34%; Chorionic Villi (12%) and fetal tissue (22%)), alive baby after pregnancy (28%), sperm in vaginal swab (10%), sperm in anal swab (2%), sperm on clothing (24%) and in one case both sperm on clothing and in vaginal swab (2%). It was found that the most common incestuous relationship was elder-brother-sister incest (34%) and the second most common relationship was father-daughter incest (28%). The rarest incest was mother-son incest with only one reported case (2%). Forty-three victims (86%) were younger than 18 years old and 7 victims (14%) were older than 18 years old. Thirty-eight cases described full sexual intercourse and 31 of them culminated in pregnancy and 14 of them gave birth at the end of pregnancy.We had paternity rejection problem 3 (10%) of 31 incest cases between tested genetically related alleged fathers. Totally 20 STR loci did not discriminate the alleged fathers in two cases and we treated this problem increasing the number of STR loci and finally got the discrimination.In one case we detected same triallelic variant pattern at the same D3S1358 STR locus in both tested parents but child had not got STR variant; had only two alleles at this loci. We then evaluated the peak height values of STR variant alleles of tested persons and concluded a tetra-allelic baby without any STR incompatibility of 15 STR loci.Finally, forensic experts should aware of some DNA profiling difficulties while analyzing paternity incest cases due to increasing intra familial allelic share. We suggested that first try increasing the number of compared STR loci and secondly use alternative genetic markers and also be careful while evaluating triallelic STR variants.     

The proportion of false-positives in positive Seratec® prostate-specific antigen SemiQuant test results in postmortem screening for seminal fluid

Prostate-specific antigen (PSA) tests are used in forensics to conduct rapid screening for semen in vaginal swab samples from alleged victims of sexual abuse. Although PSA membrane tests have been applied to autopsy specimens, no study has evaluated predictors of false-positive test results in relation to factors such as age, cause of death, postmortem interval, drugs, and alcohol. This study describes the results obtained with the Seratec® PSA SemiQuant Kit test in 283 deceased women, with or without a history of sexual assault. Overall, 18.4% (52/283) of the vaginal swab samples tested positive for PSA. However, 63.5% (33/52) of the PSA-positive vaginal swab samples had no sperm detected. The proportion of false-positives in positive PSA results was 94.4% in those aged over 60 years. Multivariate logistic regression for PSA-positive samples showed that the proportion of false-positives in positive PSA results increased with the age of the deceased. However, the cause of death, postmortem interval, and presence of drugs or alcohol in the blood or urine of the deceased did not affect the PSA determination. These results show that PSA membrane tests are relatively unreliable and can be misleading, especially when derived from vaginal swab samples of older women, obtained at autopsy. In forensic cases, positive PSA screening test results may have an impact on subsequent legal actions and criminal charges brought against the accused. These findings are important for both forensic pathologists and the police to ensure accurate screening of older women in cases of suspected sex crimes.     

Positive prostate-specific antigen (PSA) reaction in post-mortem rectal swabs: A cautionary note

Prostate-specific antigen (PSA) tests are considered a valuable screening method for the forensic examination of semen in vaginal and rectal swabs of alleged victims of sexual abuse. Although these membrane tests have been also applied to autopsy specimens no study has assessed their reliability when performed on post-mortem (PM) rectal swabs from decomposed cadavers. The present study describes the results obtained with the Seratec^® PSA Semiquant Kit test on 39 male and 10 female adult cadavers with no history of sexual assault and with a PM interval up to 136 days. Overall 64% of the 39 male cadavers tested positive for the PSA, the positive PSA reaction being more frequent in the 20 males with advanced decomposition than in the 19 males with no putrefaction signs (70% vs. 58%). The Phosphatesmo KM Paper Test^® for detection of acid phosphatase (AP) gave a positive color reaction with 60% of the rectal swabs obtained from decomposed male cadavers. Both the PSA-test and the Phosphatesmo KM paper-test gave a negative result in each of the rectal samples from female cadavers. Y STR multiplex revealed no DNA other than that of the subject tested in the rectal swab positive for PSA. The results of the present study show that PSA membrane tests are unreliable and can be misleading when derived from male rectal samples obtained at autopsy.