Faculty Bibliography

AIM: To test the reliability, robustness, and reproducibility of short tandem repeat (STR) profiling of low template DNA (LT-DNA) when employing a defined set of testing and interpretation parameters. METHODS: DNA from known donors was measured with a quantitative real time polymerase chain reaction (PCR) assay that consistently detects less than 1 pg/microL of DNA within a factor of 0.3. Extracts were amplified in triplicate with AmpFlSTR Identifiler reagents under enhanced PCR conditions. Replicates were examined independently and alleles confirmed using a consensus approach. Considering observed stochastic effects inherent to LT-DNA samples, interpretation protocols were developed and their accuracy verified through examination of over 800 samples. RESULTS: Amplification of 100 pg or less of DNA generated reproducible results with anticipated stochastic effects. Down to 25 pg of DNA, 92% or more of the expected alleles were consistently detected while lower amounts yielded concordant partial profiles. Although spurious alleles were sometimes observed within sample replicates, they did not repeat. To account for allelic dropout, interpretation guidelines were made especially stringent for determining homozygous alleles. Due to increased heterozygote imbalance, stutter filters were set conservatively and minor components of mixtures could not be resolved. Applying the resultant interpretation protocols, 100% accurate allelic assignments for over 107 non-probative casework samples, and subsequently 319 forensic casework samples, were generated. CONCLUSION: Using the protocols and interpretation guidelines described here, LT-DNA testing is reliable and robust. Implementation of this method, or one that is suitably verified, in conjunction with an appropriate quality control program ensures that LT-DNA testing is suitable for forensic purposes

AIM: To evaluate the effect of genetic instability and degradation in archived histology samples from cancerous tumors and to investigate the validity of short tandem repeat (STR) typing of these samples and its potential effect on human identification. METHODS: Two hundred and twenty eight slides of archival pathology tissues from 13 different types of malignant tumors were compared with healthy tissues from the same individuals. DNA analysis was performed using standard techniques for forensic STR analysis, PowerPlex16 and Identifiler on 2 distinct sample sets. Genetic instability was assessed by comparing reference tissues with cancerous tissues derived from the same individual. Loss of heterozygosity, a > or =50% reduction in heterozygosity ratio between healthy and diseased samples, and microsatellite instability, the presence of an additional allele not present in reference tissue, were assessed. The quality of profiles obtained with respect to completeness among the archived samples and degradation using the 2 platforms were also compared. RESULTS: Profiles obtained using the Identifiler system were generally more complete, but showed 3-fold higher levels of instability (86%) than those obtained using PowerPlex 16 (27%). Instances of genetic instability were distributed throughout all loci in both multiplex STR systems. CONCLUSION: After having compared 2 widely used forensic chemistries, we suggest individual validation of each kit for use with samples likely to exhibit instability combined with fixation induced degradation or artifact. A 'one size fits all' approach for interpretation of these samples among commercially available multiplexes is not recommended

Qualitative information on the sequence composition of the allele and locus structure of the X-STRs DXS8378, DXS9898, DXS6789, GATA31E08, and GATA172D05 was generated in this study. Sequence data were obtained from chimpanzees (Pan troglodytes) and diverse human population groups including Africans, Caucasians, Asians, African-Americans, and Hispanics. Results revealed DXS8378 as the most stable locus. On the other hand, DXS9898 and GATA172D05 showed unstable regions identified through chimpanzee-human sequence comparison. At DXS6789, intra-allelic variation was found in all human populations, i.e., alleles with same fragment sizes showed structural differences only detected by sequencing. At the GATA31E08 locus, a previously unreported variation between humans and chimpanzees was identified in an adjacent region upstream from the repeat. This resulted in the addition of two repeat units and the proposal of a new allele nomenclature at this locus. Also, the sequence analyses did not detect ethnic differences between the studied population samples that would justify the use of these markers to help identify ethnic origin in an anthropological context

The Paternity Testing Commission (PTC) of the International Society for Forensic Genetics has taken up the task of establishing the biostatistical recommendations in accordance with the ISO 17025 standards and a previous set of ISFG recommendations specific to the genetic investigations in paternity cases. In the initial set, the PTC recommended that biostatistical evaluations of paternity are based on a likelihood ratio principle - yielding the paternity index, PI. Here, we have made five supplementary biostatistical recommendations. The first recommendation clarifies and defines basic concepts of genetic hypotheses and calculation concerns needed to produce valid PIs. The second and third recommendations address issues associated with population genetics (allele probabilities, Y-chromosome markers, mtDNA, and population substructuring) and special circumstances (deficiency/reconstruction and immigration cases), respectively. The fourth recommendation considers strategies regarding genetic evidence against paternity. The fifth recommendation covers necessary documentation, reporting details and assumptions underlying calculations. The PTC strongly suggests that these recommendations should be adopted by all laboratories involved in paternity testing as the basis for their biostatistical analysis

An X-chromosomal multiplex amplifying ten short tandem repeats (STRs) in one single PCR reaction was developed and optimized in this work. The X-STRs included were DXS8378, DXS9898, DXS8377, HPRTB, GATA172D05, DXS7423, DXS6809, DXS7132, DXS101, and DXS6789. Decaplex performance was tested on 377 male samples from three United States population groups, namely, 130 African Americans, 104 Asians, and 143 Hispanics. DXS8377 was the most polymorphic locus across all three populations, whereas DXS7423 was the least informative marker. Genetic distance analysis (R (ST) and F (ST)) performed for the three populations residing in New York showed significant genetic distances between population groups for most pairwise comparisons, except for HPRTB, DXS6809, and DXS7132. When testing linkage disequilibrium for all pairs of loci in the three groups, no significant association was found between any pair of the loci studied, after applying Bonferroni correction. The high values for the average probability of excluding a random man obtained in all three populations when both mother and daughter are tested or when father/daughter relationships are evaluated support the potential of this decaplex system in kinship analysis. Also, the overall high power of discrimination values for samples of female and male origin, confirms the usefulness of this decaplex system in identification analysis. As expected, results also support the use of independent databases comprising these ten X-linked loci for the three US populations evaluated

Detailed histopathologic examination remains to be the basis for the diagnosis of hydatidiform mole (HM). However, poor sampling, necrosis, and earlier uterine evacuation can lead to uncertainty in the diagnosis. Also, the criteria are subjective, resulting in considerable interobserver variability. The p57(KIP2) gene is paternally imprinted and maternally expressed, and the presence of its protein product serves as a surrogate marker for the nuclear maternal genome. Because a complete HM (CHM) is the only type of conceptus lacking a maternal contribution, p57(KIP2) immunostaining is correspondingly absent, whereas it is present in CHM mimics. Although analysis of DNA microsatellite polymorphisms is a reliable method for the diagnosis and classification of HM, it is not universally available. To assess the relative accuracy of p57(KIP2) immunostaining and molecular diagnosis by nuclear DNA microsatellite polymorphisms in discriminating CHM from its mimics, we analyzed archival tissue from 33 case patients (7 with a definitive diagnosis of CHM, 16 with a possible diagnosis of HM, and 10 with normal placentas) by both methods. Concordant results were obtained in all cases, and p57(KIP2) immunostaining accurately identified all cases of CHM from the groups with a definitive or possible diagnosis of HM. p57(KIP2) immunohistochemistry is a time- and cost-effective means of distinguishing CHM from its mimics in challenging cases

Using high sensitivity forensic STR polymerase chain reaction (PCR) typing procedures, we have found low concentrations of DNA contamination in plasticware and water assumed to be sterile, which is not detected by standard DNA procedures. One technique commonly used to eliminate the presence of DNA is ultraviolet (UV) irradiation; we optimized such a protocol used in the treatment of water, tubes, plates, and tips for low copy number DNA (LCN) amplification. UV light from a Stratalinker((R)) 2400 was administered to 0.2, 1.5 mL tubes, and PCR plates contaminated with up to 500 pg of DNA. They were subsequently quantified with an ALU-based real-time PCR method using the Rotorgene 3000. Overall, there was a decrease in concentration of DNA recovered as the duration of treatment increased. Nonetheless, following 45 min of irradiating a PCR plate with 500 pg of DNA, nearly 6 pg were still detected. However, when the plate was raised within an inch of the UV source, less than 0.2 pg of DNA was detected. Additionally, lining the area around the samples with aluminum foil further reduced the amount of time necessary for irradiation, as only 30 min eliminated the presence DNA in the raised PCR plate. Similar experiments were conducted using tubes filled with a solution of DNA and water in equivalent concentrations for 50, 15, and 1.5 mL tubes with comparative results. It is plausible that the aluminum foil increased the amount of reflection in the area thereby enhancing penetration of UV rays through the walls of the plasticware. This protocol was tested for the possibility of inhibitors produced from irradiation of plastic tubes. As our protocols require less irradiation time than previous studies, PCR sensitivity was not affected. Moreover, the lifespan of the UV lamps was extended. Our findings demonstrate that this method is useful as an additional precautionary measure to prevent amplification of extraneous DNA from plasticware and water without compromising the sensitivity of LCN DNA amplifications