D. Syndercombe Court

Investigation of the STR locus HUMTH01 using PCR and two electrophoresis formats: UK and Galician Caucasian population surveys and usefulness in paternity investigations

Two hundred unrelated Caucasians from the UK and 210 from Galicia (NW Spain) have been genotyped for the HUMTH01 locus using polymerase chain reaction (PCR) amplification followed by high sieving agarose electrophoresis (UK samples) and polyacrylamide electrophoresis (Galician samples). Allele and genotype frequencies obtained from both populations were in close agreement to those seen by other workers and chi 2 tests of the two populations showed that both were in Hardy-Weinberg equilibrium. The potential usefulness of HUMTH01 in paternity investigations was analysed by constructing, within each population, false family trios, each with a non-father, in order to estimate the actual exclusion rate. The observed rate of exclusion was in close agreement with the expected rate for both populations. Examination of the mother-child pairs in the false families showed a common allele in every case and no evidence of mutation or non-Mendelian inheritance was observed. The HUMTH01 locus shows an informative polymorphism and the production and analysis of population databases is easily achieved. The use of PCR followed by electrophoresis in either gel format appears to provide a quick and straightforward method for investigating the HUMTH01 locus.

Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel.

Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNPforID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNPforID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan, SNaPshot and Sequenom iPLEX SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler kit.

Forensic typing of autosomal SNPs with a 29 SNP-multiplex—Results of a collaborative EDNAP exercise

We report the results of an inter-laboratory exercise on typing of autosomal single nucleotide polymorphisms (SNP) for forensic genetic investigations in crime cases. The European DNA Profiling Group (EDNAP), a working group under the International Society for Forensic Genetics (ISFG), organised the exercise. A total of 11 European and one US forensic genetic laboratories tested a subset of a 52 SNP-multiplex PCR kit developed by the SNPforID consortium. The 52 SNP-multiplex kit amplifies 52 DNA fragments with 52 autosomal SNP loci in one multiplex PCR. The 52 SNPs are detected in two separate single base extension (SBE) multiplex reactions with 29 and 23 SNPs, respectively, using SNaPshot kit, capillary electrophoresis and multicolour fluorescence detection. For practical reasons, only the 29 SBE multiplex reaction was carried out by the participating laboratories. A total of 11 bloodstains on FTA cards including a sample of poor quality and a negative control were sent to the laboratories together with the essential reagents for the initial multiplex PCR and the multiplex SBE reaction. The total SNP locus dropout rate was 2.8% and more than 50% of the dropouts were observed with the poor quality sample. The overall rate of discrepant SNP allele assignments was 2.0%. Two laboratories reported 60% of all the discrepancies. Two laboratories reported all 29 SNP alleles in all 10 positive samples correctly. The results of the collaborative exercise were surprisingly good and demonstrate that SNP typing with SBE, capillary electrophoresis and multicolour detection methods can be developed for forensic genetics.

RNA/DNA co-analysis from human saliva and semen stains--results of a third collaborative EDNAP exercise.

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 μl saliva, 5-0.01 μl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 μl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies.

The recombination landscape around forensic STRs: Accurate measurement of genetic distances between syntenic STR pairs using HapMap high density SNP data

Family studies can be used to measure the genetic distance between same-chromosome (syntenic) STRs in order to detect physical linkage or linkage disequilibrium. However, family studies are expensive and time consuming, in many cases uninformative, and lack a reliable means to infer the phase of the diplotypes obtained. HapMap provides a more comprehensive and fine-scale estimation of recombination rates using high density multi-point SNP data (average inter-SNP distance: 900 nucleotides). Data at this fine scale detects sub-kilobase genetic distances across the whole recombining human genome. We have used the most recent HapMap SNP data release 22 to measure and compare genetic distances, and by inference fine-scale recombination rates, between 29 syntenic STR pairs identified from 39 validated STRs currently available for forensic use. The 39 STRs comprise 23 core loci: SE33, Penta D & E, 13 CODIS and 7 non-CODIS European Standard Set STRs, plus supplementary STRs in the recently released Promega CS-7™ and Qiagen Investigator HDplex™ kits. Also included were D9S1120, a marker we developed for forensic use unique to chromosome 9, and the novel D6S1043 component STR of SinoFiler™ (Applied Biosystems). The data collated provides reliable estimates of recombination rates between each STR pair, that can then be placed into haplotype frequency calculators for short pedigrees with multiple meiotic inputs and which just requires the addition of allele frequencies. This allows all current STR sets or their combinations to be used in supplemented paternity analyses without the need for further adjustment for physical linkage. The detailed analysis of recombination rates made for autosomal forensic STRs was extended to the more than 50 X chromosome STRs established or in development for complex kinship analyses.

RNA/DNA co-analysis from human menstrual blood and vaginal secretion stains: Results of a fourth and fifth collaborative EDNAP exercise

The European DNA Profiling Group (EDNAP) organized a fourth and fifth collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling. The task was to identify dried menstrual blood and vaginal secretion stains using specific RNA biomarkers, and additionally test 3 housekeeping genes for their suitability as reference genes. Six menstrual blood and six vaginal secretion stains, two dilution series (1/4-1/64 pieces of a menstrual blood/vaginal swab) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 24 participating laboratories, using RNA extraction or RNA/DNA co-extraction methods. Two novel menstrual blood mRNA multiplexes were used: MMP triplex (MMP7, MMP10, MMP11) and MB triplex (MSX1, LEFTY2, SFRP4) in conjunction with a housekeeping gene triplex (B2M, UBC, UCE). Two novel mRNA multiplexes and a HBD1 singleplex were used for the identification of vaginal secretion: Vag triplex (MYOZ1, CYP2B7P1 and MUC4) and a Lactobacillus-specific Lacto triplex (Ljen, Lcris, Lgas). The laboratories used different chemistries and instrumentation and all were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA allowed for positive identification of the tissue/fluid source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling, also from old and compromised casework samples. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid identification method that can easily be combined with current STR typing technology.

Reproducibility of mtDNA analysis between laboratories: a report of the European DNA profiling group (EDNAP)

The aim of this collaborative exercise was to determine whether uniformity of mtDNA sequencing results could be achieved among different EDNAP laboratories. Laboratories were asked to sequence mtDNAHV1 region (16024-16365) from three bloodstains, proceeding in accordance with the protocol and strategies currently used in each individual laboratory. Cycle sequencing was used by 11 laboratories and solid phase single stranded sequencing was used by one laboratory. Different PCR strategies and PCR conditions were used by the different laboratories. Three laboratories used semi-nested PCR, two nested PCR, three direct amplification of HV1 and four amplification of overlapping fragments covering the HV1 region. Despite the diversity of methodologies used, all the laboratories reported the same results. The successful result of this exercise shows that PCR based mtDNA typing by automated sequencing is a valid, robust and reliable means of forensic identification despite the different strategies and methodologies used by the different laboratories.

Reactivation of 2 Genetically Distinct Varicella-Zoster Viruses in the Same Individual

Varicella-zoster viruses recovered from 2 episodes of herpes zoster in an immunocompetent man were found to be different genotypes. The fact that the 2 isolates came from the same individual was confirmed by DNA fingerprinting. Immunity following chickenpox may not always protect against systemic reinfection. This finding raises questions about varicella-zoster virus pathogenesis and may have an impact on public health policy.

RNA/DNA co-analysis from human skin and contact traces – results of a sixth collaborative EDNAP exercise

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology.

An investigation of the HUMVWA31A locus in British Caucasians

A number of short tandem repeat (STR) loci are currently being examined for their usefulness as markers of identity; HUMVWA31A is one such locus. We used a high-sieving agarose technique to type 200 British Caucasians for this locus. Comparison of the resultant allele frequencies with other published databases showed that their distributions were similar. Observed heterozygosity was similar to that reported in other population studies but significantly lower than expected. A goodness of fit analysis of observed and expected genotypes was highly significant, suggesting a deviation from Hardy-Weinberg equilibrium, although reports from other populations appeared not to show such differences. The possibility that mistyping had led to an erroneous statistic was investigated by detailed examination of samples within our own laboratory and in two other laboratories, involving verification of the agarose typing by both automated fluorescent detection techniques, and sequencing. Other reported deviations from Hardy-Weinberg equilibrium in STR systems are discussed.