Carmen Entrala

Social benefits of non-criminal genetic databases: missing persons and human remains identification

Abstract A Missing Persons Genetic Identification Program (Phoenix Program) was implemented in Spain in order to try to identify cadavers and human remains that could not be identified using traditional forensic approaches; to our knowledge, this is the first database ever implemented and in function in the world. Two separate mitochondrial DNA (mtDNA) databases have been generated and comparisons can be made automatically to match identical or similar sequences contained in both databases. One database is called the Reference Database (RD), which contains mtDNA sequences from maternal relatives of missing persons that provide the samples voluntarily after informed consent. The other database is called the Questioned Database (QD) and is comprised of mtDNA data on unknown remains and cadavers that could not be unequivocally identified. The combined database is a civil database designed solely for human identification and because of the informed consent and voluntary donation of reference samples is different from other databases now used to solve criminal cases. It is timely and incumbent on other willing countries to begin an international collaboration so compatibility and full utility can be enjoyed with this kind of non-criminal database.

Dandruff as a Potential Source of DNA in Forensic Casework

Dandruff is a clinical alteration of the skin that consists histologically of orthokeratotic clumps with minute parakeratotic foci found in inflammatory pathologies such as seborrheic dermatitis and psoriasis. Therefore, some nucleated cells should be found in dandruff and hence there is a possibility that forensically typeable DNA could be extracted from dandruff. Because of a particular case in which we were involved, a study was carried out to determine whether or not DNA could be extracted from dandruff, and if the two most widely used extraction techniques (Chelex and organic) would be applicable. Results show that sufficient quantities of DNA (more than 30 to 40 ng) can be obtained from as little as 1.0 to 1.5 mg of dandruff. Both methods yield DNA, although the organic procedure seems to yield more (72.5 ng Chelex vs. 183.3 ng organic). All the DNA samples extracted were typed correctly for the loci HUMTH01 and HUMvWA. Therefore, dandruff can be considered a potential source of DNA for forensic identification.

Fluorescent multiplex analysis of nine STR loci: Spanish population data

A total of 171 Caucasians living in Andalucia (southern Spain) have been typed for nine short tandem repeat (STR) loci by multiplex PCR amplification using a commercially available kit (Profiler Plus; Perkin-Elmer, Norwalk, CT, USA) and semi-automatic electrophoresis (ABI Prism 377 DNA Sequencer, Applied Biosystems, Foster City, CA, USA). The kit enables typing of the STR loci D3S1358, VWA, FGA, D5S818, D13S317, D7S820, D8S1179, D21S11, and D18S51. All loci, except D7S820, meet Hardy-Weinberg equilibrium. Because of the large number of loci that can be analyzed, the power of discrimination (PD) is greater than 0.99999, and the probability of exclusion (PE) reaches 0.99991 in our population sample.

Noninvasive Fetal RhD Status Determination in Early Pregnancy

Introduction: The aim of this study was to examine if noninvasive fetal RhD genotyping from maternal blood cell-free fetal DNA performed in the first trimester of pregnancy is accurate enough to propose its routine application to replace usual immunoprophylaxis. Material and Methods: We carried out a prospective study analyzing fetal RhD genotype in 149 nonimmunized RhD-negative women with single pregnancies between 8 and 13 weeks of gestation. Fetal RhD genotype was detected by quantitative PCR targeting exons 5 and 7. The results were compared with postnatal cord blood phenotype, and discrepancy rates were calculated. Results: The concordance of fetal RhD genotypes in maternal plasma and newborn D phenotypes at delivery was 98.2%, including 1 false-positive and 1 false-negative result. The specificity and sensitivity of the assay were 97.5% (95% CI 87.1-99.9) and 98.6% (95% CI 92.7-99.9), respectively, and 6.5% of the results were inconclusive. The application of this test in early pregnancy would avoid unnecessary antenatal prophylaxis in about 27% (40/143) of nonsensitized RhD-negative women. Discussion: Determination of the fetal RhD status from cell-free fetal DNA in maternal plasma in the first trimester of pregnancy is feasible and highly accurate, thus allowing consideration of replacing general routine immunoprophylaxis in the cases of mothers with Rh-negative fetuses.

Paraguayan population data on the fifteen STR loci included in the PowerPlex 16 kit.

Blood samples were obtained by venipuncture from unrelated individuals (n = 168) living in Paraguay.

Population data on nine STR loci in an El Salvadoran (Central American) sample population.

Blood samples were obtained by venipuncture from unrelated individuals (n = 323) living in El Salvador. Approximately 1–3 ng of DNA were used in each PCR. The samples were amplified using the Profiler Plus™ kit (PE) and the alleles were separated and detected using an Applied Biosystems ABI310 genetic analyzer. The frequency of each allele for each locus was calculated from the numbers of each genotype in the sample set (i.e., the gene count method). Unbiased estimates of expected heterozygosity were computed as described by Edwards et al. (1). Possible divergence from Hardy-Weinberg expectations (HWE) was tested by calculating the unbiased estimate of the expected homozygote/heterozygote frequencies (1–4) and the exact test (5), based on 2000 shufflings experiments. An interclass correlation criterion (6) for two-locus associations was used for detecting disequilibrium between the STR loci. The program for this analysis was kindly provided by R. Chakraborty (University of Texas, School of Biomedical Sciences, Houston, TX).

Next generation sequencing: an application in forensic sciences?

Abstract Context: Over the last few decades, advances in sequencing have improved greatly. One of the most important achievements of Next Generation Sequencing (NGS) is to produce millions of sequence reads in a short period of time, and to produce large sequences of DNA in fragments of any size. Libraries can be generated from whole genomes or any DNA or RNA region of interest without the need to know its sequence beforehand. This allows for looking for variations and facilitating genetic identification. Objectives: A deep analysis of current NGS technologies and their application, especially in forensics, including a discussion about the pros and cons of these technologies in genetic identification. Methods: A systematic literature search in PubMed, Science Direct and Scopus electronic databases was performed for the period of December 2012 to June 2015. Results: In the forensic field, one of the main problems is the limited amount of sample available, as well as its degraded state. If the amount of DNA input required for preparing NGS libraries continues to decrease, nearly any sample could be sequenced; therefore, the maximum information from any biological remains could be obtained. Additionally, microbiome typification could be an interesting application to study for crime scene characterisation. Conclusions: NGS technologies are going to be crucial for DNA human typing in cases like mass disasters or other events where forensic specimens and samples are compromised and degraded. With the use of NGS it will be possible to achieve the simultaneous analysis of the standard autosomal DNA (STRs and SNPs), mitochondrial DNA, and X and Y chromosomal markers.

Population data of Ecuador for fifteen STR loci (POWERPLEX 16)

Blood samples were obtained by venipuncture from unrelated individuals (n = 150) living in Ecuador.

Spanish Population Data on the Loci D13S317, D7S820, and D16S539 Generated Using Silver Staining (SilverSTR III™ Multiplex)

A set of 212 samples from unrelated Spanish Caucasians living in Andalucia (southern Spain) were analyzed with a new commercially-available kit for multiplex amplification of 3 STR loci (D13S137, D7S820, and D16S539), manual denaturing polyacrylamide gel electrophoresis and silver staining. These three loci are of special interest for the forensic community since they are a part of the 13 CODIS-core STR loci. The results show that the loci D13S317 and D16S539 meet Hardy-Weinberg expectations (HWE), but the locus D7S820 did not meet HWE (p = 0.003). However, there was no detectable departures from independence (i.e., linkage disequilibrium) between any pair-wise combination of loci. The D7S820 data were further investigated. The excess homozygosity was due to an excess of D7S820 10, 10 homozygotes. To determine if the allele frequency data are meaningful and can be applied to forensic identity cases, the Spanish D7S820 allele frequency data were compared with four other Caucasian sample populations. The D7S820 allele frequencies were statistically similar; thus, the results support that the allele frequency data can be used reliably for estimating DNA profile frequencies.

Spanish population data for the 15 STRs loci included in Powerflex-16

Short tandem repeat (STR) loci are the most informative PCR-based genetic markers available to date for attempting to individualize biological material. The 16 STR loci: D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA, and the locus amelogenin can be amplified simultaneously using the the PowerPlex16 kit (Promega, Madison, WI, USA). This paper presents allele distribution data in the Spanish population. The data demonstrate that these loci can be useful for providing estimates of the frequency of a DNA profile in forensic identity testing and that a multiple locus profile is extremely rare in all the population. Whole blood was obtained in EDTA vacutainer tubes by venipuncture from unrelated individuals (N=323) residing in Andalucia (Spain). Extracted DNA samples were amplified at the 16 loci using the PowerPlex 16 kit (Promega). Samples were analyzed using the ABI Prismk 310 Genetic Analyzer (PE Biosystems, Foster City, CA) according to the manufacturer’s recommended protocol. All 15 loci are highly polymorphic in the Spanish sample population with the locus TPOX (66.8%) having the lowest observed heterozygosity, and the locus D18S51 (87.5%) displaying the highest heterozygosity. The most discriminating loci were D18S51 (PD=0.969) and PentaE (PD=0.968). The combined probability of exclusion for the 15 STR loci is 0.99999953. There was little evidence for departures from Hardy-Weinberg expectations (HWE) in this sample population. Based on the exact test, the locus that departed significantly from HWE was vWA ( p=0.0488). After employing the Bonferroni correction for the number of loci analyzed, these observations are not likely to be significant. An interclass correlation test analysis was performed to detect any correlations