C.R. Thacker

Forensic validation of the SNPforID 52-plex assay

The advantages of single nucleotide polymorphism (SNP) typing in forensic genetics are well known and include a wider choice of high-throughput typing platforms, lower mutation rates, and improved analysis of degraded samples. However, if SNPs are to become a realistic supplement to current short tandem repeat (STR) typing methods, they must be shown to successfully and reliably analyse the challenging samples commonly encountered in casework situations. The European SNPforID consortium, supported by the EU GROWTH programme, has developed a multiplex of 52 SNPs for forensic analysis, with the amplification of all 52 loci in a single reaction followed by two single base extension (SBE) reactions which are detected with capillary electrophoresis. In order to validate this assay, a variety of DNA extracts were chosen to represent problems such as low copy number and degradation that are commonly seen in forensic casework. A total of 40 extracts were used in the study, each of which was sent to two of the five participating laboratories for typing in duplicate or triplicate. Laboratories were instructed to carry out their analyses as if they were dealing with normal casework samples. Results were reported back to the coordinating laboratory and compared with those obtained from traditional STR typing of the same extracts using Powerplex 16 (Promega). These results indicate that, although the ability to successfully type good quality, low copy number extracts is lower, the 52-plex SNP assay performed better than STR typing on degraded samples, and also on samples that were both degraded and of limited quantity, suggesting that SNP analysis can provide advantages over STR analysis in forensically relevant circumstances. However, there were also additional problems arising from contamination and primer quality issues and these are discussed.

An Israeli Arab patient with a de novo TNFRSF1A mutation causing tumor necrosis factor receptor-associated periodic syndrome.

OBJECTIVE To investigate genetic susceptibility to recurrent fevers, generalized severe myalgia, and migratory erythema in an Israeli Arab child with no family history of similar disease. METHODS DNA sequencing of exons 1-6 of the TNFRSF1A gene (formerly TNFR1) was performed in the patient and his parents to determine the presence of the autosomal-dominant tumor necrosis factor receptor-associated periodic syndrome (TRAPS); informative markers spanning the TNFRSF1A locus were used to genotype all available members of the patients family. The TNFRSF1A gene was subsequently screened in 69 healthy Arab controls and 96 Caucasian controls. Formal forensic paternity testing was performed on the child. RESULTS We found a de novo missense mutation in exon 3 of the TNFRSF1A gene, involving a novel C-->T transition encoding a Cys70Arg (C70R) variant, in the Israeli Arab patient. Eight of the common familial Mediterranean fever (FMF) gene MEFV mutations were excluded. This mutation was not present in the parents or siblings, or among the 69 healthy Arab controls. However, another TNFRSF1A variant, Pro46Lys (P46L), was present in 1 of the Arab controls. CONCLUSION We have identified a TNFRSF1A mutation associated with periodic fever in an Arab patient, and a TNFRSF1A variant, which is variably pathogenic in Caucasians, in an Arab control. This is the first report of a de novo mutation in periodic fevers in general, and also of TRAPS in the Arab population. These findings demonstrate the need to include TRAPS in the differential diagnosis of recurrent fevers in this population.

Autosomal SNP typing of forensic samples with the GenPlex TM HID System: Results of a collaborative study

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.

Case study: paternity testing—when 21 loci are not enough

Abstract A seemingly routine paternity case which involved the testing of a mother, child and man led to inconclusive results as an exclusion involving only a single repeat was found at one of the 14 loci tested. This led to the testing of further loci. Only the single exclusion was found after profiling a total of 21 loci, with the addition of a further single locus providing a second exclusion. Even with both mutation events incorporated into the calculation a paternity index (PI) of 4957 was calculated, still a significant figure. However, when a second likelihood ratio was calculated assessing the likelihood of the results if the biological father of the child was the tested man or the tested mans brother, then the results were not significant, only 0.15. This analysis led to the profiling of the tested mans brother who matched at all 19 loci that were profiled and was concluded to be the biological father.

Comparison of Y-chromosome haplotypes in three racial groups and the possibility of predicting ethnic origin

Abstract Currently, most STRs found on the Y-chromosome exhibit much lower levels of polymorphism when compared to autosomal STRs. However, unlike autosomal STRs, they often show marked differences in allele frequency distributions between racial groups. Two hundred unrelated males from each of the three principal ethnic groupings within the UK were typed for 11 loci and used to build a predictive model for those classifications. An additional 50 individuals from each group were used in a blind trial to validate the model and the utility of the assignments assessed by calculating likelihood ratios. Use of a haplotype consisting of only three Y-chromosome STRs correctly identified 81%, 96% and 70% of individuals who defined themselves as white, black or South Asian.

Haplotype discrimination amongst three UK population groups using three multiplexes to type eleven Y chromosome STRs

Abstract In certain circumstances, analysis with Y chromosome markers provides potential benefits over autosomal STRs, for example, paternity cases in which the putative father is deceased and in rape cases. The polymorphism level of Y chromosome STRs, however, is generally quite low and discrimination is further reduced as a result of linkage. Consequently, profiles need to be analysed as haplotypes rather than independent loci. To improve the current level of discrimination, we extended the range of Y-chromosome STRs most commonly used in forensic analysis, with the addition of three recently reported STR markers: DYS437, 438, and 439 to give a total of 11 loci. Two PCR triplex reactions were developed and optimised, combining the three new loci with established markers. Six hundred UK individuals who separately described themselves as white, black or South Asian (from the Indian subcontinent) were typed and intermediate alleles and duplications further characterised. Addition of the new loci significantly increased haplotype diversity.