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Endovascular treatment of femoropopliteal artery disease has shifted toward drug-coated balloons (DCB). However, limited data are available regarding the safety and efficacy of DCB vs bare-metal stents (BMS).To compare DCB vs BMS outcomes in a propensity-adjusted, pooled analysis of 4 prospective, multicenter trials.Patient-level data were pooled from 4 prospective, multicenter studies: the IN.PACT SFA I/II and IN.PACT SFA Japan randomized controlled DCB trials and the Complete SE and DURABILITY II single-arm BMS studies. Outcomes were compared using inverse probability of treatment weighting (IPTW). Clinical endpoints were 12-month primary patency, freedom from 36-month clinically driven target lesion revascularization, and cumulative 36-month major adverse events (MAE).The primary analysis included 771 patients (288 DCB, 483 BMS). IPTW-adjusted demographic, baseline lesion, and procedural characteristics were matched between groups. The adjusted mean lesion length was 8.1 ± 4.7 cm DCB and 7.9 ± 4.5 cm BMS. The IPTW-adjusted Kaplan-Meier estimates of 12-month primary patency (90.4% DCB, 80.9% BMS, P = 0.007), freedom from 36-month clinically driven target lesion revascularization (85.6% DCB, 73.7% BMS, P = 0.001), and cumulative incidence of 36-month MAE (25.3% DCB, 38.8% BMS, P0.001) favored DCB. There were no statistically significant differences observed in all-cause mortality, target limb major amputation, or thrombosis through 36 months.In a patient-level, IPTW-adjusted pooled analysis of prospective, multicenter pivotal studies, DCB demonstrated significantly higher patency, lower revascularization and MAE rates, and no statistically significant differences in mortality, amputation, or thrombosis versus BMS. This analysis supports DCB use vs BMS in moderately complex femoropopliteal lesions amenable to both treatments.

Strategies of balloon dilation during transfemoral carotid artery stenting include prestent dilation only (PRE), post-stent dilation only (POST), or both predilation and postdilation (PRE+POST). Concerns over higher neurological risk have been raised with POST and PRE+POST during transfemoral carotid artery stenting. Whether these concerns are applicable to transcarotid artery revascularization (TCAR), which uses proximal clamping and cerebral blood flow reversal during stent deployment and balloon angioplasty remains unknown. Our aim is to analyze outcomes of PRE, POST, or PRE+POST balloon dilation strategies during TCAR.We analyzed the prospectively collected data from the ROADSTER1 (pivotal), ROADSTER2 (US Food and Drug Administration indicated postmarket), and ROADSTER Extended Access TCAR trials. All trial patients had a high risk anatomic or clinical factors for carotid endarterectomy and were included, unless they did not undergo stent deployment or balloon dilation. For trial inclusion, asymptomatic patients had a carotid stenosis of more than 80%, and symptomatic patients had stenosis of more than 50%. Primary outcome measures were stroke, death, and myocardial infarction (MI) at 30 days. Data were statistically analyzed with χThere were 851 patients (566 male) who underwent dilation by PRE (n = 216), POST (n = 249), or PRE+POST (n = 386). Patients had carotid stenosis of greater than 70% (n = 828, 97%), and 207 (24%) were symptomatic. Flow reversal times were longer in the PRE+POST group (PRE 10.2 minutes, POST 9.8 minutes, and PRE+POST 13.3 minutes; P .001). The 30-day stroke rate for the whole cohort was 1.9%, mortality was 0.5%, and MI rate was 0.94%. Stroke rates for the PRE cohort (1.9%), POST cohort (2.0%), and PRE+POST cohort (1.8%; P = .98) were similar. Also, death rates at 30 days, and composite stroke, death, and MI rates were similar in the three cohorts. No significant differences in adverse outcomes were noted among the various dilation strategies for both symptomatic and asymptomatic patients.Based on these prospective trial data, there is no difference in neurological complications owing to balloon dilation strategy during TCAR. The balloon dilation technique best suited to the patient's specific lesion morphology should be used. Further studies are needed to evaluate the relationship of these dilation strategies to long-term outcomes, including stent patency, restenosis, and reintervention.

The SVS Wound, Ischemia, foot Infection (WIfI) limb staging system was established to estimate risk of major amputation in chronic limb-threatening ischemia (CLTI) and better stratify outcomes comparisons. There is little data on treatment outcomes beyond one year based on presenting WIfI stage.This is a single-institution retrospective study of 413 patients who underwent infrainguinal revascularization for CLTI (2011-2021) with data available for WIfI staging. Patient characteristics and outcomes were gathered from the electronic medical record. Data were analyzed based on presenting WIfI stage and initial treatment received at our center.Presenting WIfI stages were 1-2 (23%), 3 (27%) and 4 (50%). Index revascularization approach was endoluminal (59%), autogenous vein bypass (29%), or non-autogenous bypass (13%). Operative mortality within 30 days was 2.9% and was not associated with WIfI stage or revascularization approach. Median limb follow-up time was 502 days (IQR 112-1256), and median survival follow-up time was 932 days (IQR 343-1770). Major amputation or death occurred in 19% and 46% of patients at median times of 119 days (IQR 28-314) and 739 days (IQR 204-1475), respectively. WIfI stage was independently associated with major amputation (p=0.001), as was initial revascularization approach (p=0.01). In a Cox proportional hazards model, factors independently associated with major amputation were male sex [HR 1.4 (1.04-2.0), p=0.03], diabetes [HR1.8 (1.3-2.5), p=0.001], WIfI stage 4 [HR 2.3 (1.5-3.5), p0.001], and non-autogenous bypass [HR 2.9 (2.1-4.2) p0.001]. In a Cox proportional hazards model for mortality, independently associated factors were age [HR 1.04 (1.02-1.05), p0.001], ESRD [HR 2.8 (1.9-4.0), p0.001], CHF [HR 1.9 (1.4-2.5), p0.001], COPD [HR 1.5 (1.1-2.1, p=0.02), and WIfI stage 4 [HR 1.6 (1.04-2.2), p=0.03]. Among those presenting with WIfI stage 4 limbs, Kaplan-Meier estimated rates of freedom from major amputation or death at 2 years were 71% ± 3.7% and 68% ± 3.5%, respectively. In an inverse propensity weighted Cox proportional hazards model, non-white race [HR 1.5 (1.01-2.2), p=0.047], diabetes [HR 2.0 (1.2-3.3), p=0.008], GLASS IP grade [HR 1.2 (1.05-1.3), p=0.005], non-autogenous bypass [HR 3.2 (1.9-5.3), p0.001], and endoluminal revascularization [HR 2.6 (1.6-4.3), p0.001] were independently associated with major amputation in the WIfI stage 4 subgroup.Presenting WIfI stage is strongly associated with long-term risks of major amputation and death following infrainguinal revascularization for CLTI and should be used to stratify outcomes comparisons. Effective revascularization is critical in WIfI stage 4 disease, and autogenous vein bypass provides durable long-term limb preservation.

Although the benefits of carotid endarterectomy (CEA) for treating symptomatic carotid stenosis are well known, the optimal timing of intervention after acute stroke and whether the optimal timing will vary with preoperative stroke severity has remained unclear. Therefore, we assessed the effect of stroke severity and timing of the intervention on the postoperative outcomes for patients who had undergone CEA for stroke.We identified all patients in the Vascular Quality Initiative who had undergone CEA from 2012 to 2020 for prior stroke. The patients were stratified using the preoperative modified Rankin scale score (mRS score, 0-5) and time to CEA after stroke onset (≤2 days, 3-14 days, 15-90 days, 91-180 days). After univariate comparisons, the patients were stratified into the following mRS cohorts for further analysis: 0 to 1, 2, 3 to 4, and 5. The primary outcome was in-hospital stroke/death.We identified 15,601 patients, of whom 30% had had an mRS score of 0, 34% an mRS score of 1, 17% an mRS score of 2, 11% an mRS score of 3, 8% an mRS score of 4, and 1% an mRS score of 5. Overall, 9.3% of the patients had undergone CEA within ≤2 days, 46% within 3 to 14 days, 36% in 15 to 90 days, and 8.4% within 90 to 180 days. A decreasing mRS score and an increasing time to CEA were associated with lower rates of perioperative stroke/death (PPatients with minimal disability after stroke (mRS score, 0-1) seemed to benefit from CEA within 3 to 14 days. However, those with severe disability (mRS score 5) have a very high risk from CEA at any time point given the poor outcomes. In contrast to the current guidelines, patients with mild disability (mRS score 2) could benefit from delaying CEA to 15 to 90 days, and those with moderate disability (mRS score 3-4) might benefit from CEA within 3 to 90 days given the acceptable in-hospital outcomes. These data should be considered within the context of the clinical situation in the weeks after index event to determine the net benefit of delayed CEA.

Transcarotid artery revascularization (TCAR) is a carotid stenting technique that utilizes reversal of cerebral arterial flow to confer cerebral protection. Although carotid endarterectomy (CEA) remains the standard for treatment of symptomatic and asymptomatic carotid stenosis, the search for the optimal minimally invasive option for the high-risk surgical patient continues. The goal of the current study is to evaluate the 1-year safety and efficacy of TCAR in a prospective clinical trial.ROADSTER 2 is a prospective, open-label, single-arm, multicenter, post-approval registry for patients undergoing TCAR. All patients were considered high risk for CEA and underwent independent neurological assessments preoperatively, postoperatively, and had long-term clinical follow-up. The primary end point was incidence of ipsilateral stroke after treatment with the ENROUTE Transcarotid Stent System. Secondary end points included individual/composite rates of stroke, death, and perioperative myocardial infarction.Between June 2016 and November 2018, 155 patients at 21 centers in the United States and one in the European Union were enrolled and represented a subset of the overall trial. Asymptomatic (n = 119; 77%) and symptomatic patients (n = 36; 23%) with high-risk anatomic (ie, high lesion, restenosis, radiation injury; 43%), physiologic (32%), or combined factors (25%) were enrolled. No patient suffered a perioperative myocardial infarction or stroke. Over the year, no patient had an ipsilateral stroke, but four patients died (2.6%), all from non-neurological causes. Additionally, a technical success rate of 98.7% with a low cranial nerve deficit rate of 1.3% was achieved.In patients with high risk factors, TCAR yields high technical success with a low stroke and death rate at 1 year. Further comparative studies with CEA are warranted.

© 2019 The Authors Current forensic ancestry-informative panels are limited in their ability to differentiate populations in the Asia-Pacific region. MAPlex (Multiplex for the Asia-Pacific), a massively parallel sequencing (MPS) assay, was developed to improve differentiation of East Asian, South Asian and Near Oceanian populations found in the extensive cross-continental Asian region that shows complex patterns of admixture at its margins. This study reports the development of MAPlex; the selection of SNPs in combination with microhaplotype markers; assay design considerations for reducing the lengths of microhaplotypes while preserving their ancestry-informativeness; adoption of new population-informative multiple-allele SNPs; compilation of South Asian-informative SNPs suitable for forensic AIMs panels; and the compilation of extensive reference and test population genotypes from online whole-genome-sequence data for MAPlex markers. STRUCTURE genetic clustering software was used to gauge the ability of MAPlex to differentiate a broad set of populations from South and East Asia, the West Pacific regions of Near Oceania, as well as the other globally distributed population groups. Preliminary assessment of MAPlex indicates enhanced South Asian differentiation with increased divergence between West Eurasian, South Asian and East Asian populations, compared to previous forensic SNP panels of comparable scale. In addition, MAPlex shows efficient differentiation of Middle Eastern individuals from Europeans. MAPlex is the first forensic AIM assay to combine binary and multiple-allele SNPs with microhaplotypes, adding the potential to detect and analyze mixed source forensic DNA.

Patients undergoing revascularization for chronic limb-threatening ischemia (CLTI) are at elevated risk for both mortality and limb loss. To facilitate therapeutic decision-making, a mortality prediction model derived from the Vascular Quality Initiative (VQI) database has stratified patients into low, medium, and high risk, defined by 30-day mortality estimates of ≤3%, 3%-5%, or5% and 2-year mortality estimates of ≤30%, 30%-50%, or ≥50%, respectively. The purpose of this study was to compare expected mortality risk derived from this model with observed outcomes in a tertiary center.Consecutive patients treated at a single center between 2016 and 2019 were analyzed. Baseline demographics, approach, and mortality events were reviewed. Observed mortality was obtained using life-table methods and compared using a log-rank test with the expected mortality risk that was calculated using the VQI model.This study cohort consisted of 195 revascularization procedures in 169 unique patients stratified into 128 (66%) low-, 50 (26%) medium-, and 17 (8%) high-risk cases based on the VQI model. Ninety percent of revascularizations were performed for tissue loss. Compared with the VQI population, comorbidities were prevalent and included unstable angina or myocardial infarction within 6 months (6% vs 2.4% in VQI; P .001), congestive heart failure (30% vs 23%; P .001), and dialysis dependence (14% vs 0.9%; P .001). Patients were also older (31% vs 21% ≥80 years old; P .001) and more likely to be frail (45% vs 64% independent; P .001). High-risk patients were more prevalent in the endovascular group (11% of 132 endovascular interventions vs 3% of 63 bypasses; P = .056). Thirty-day observed mortality exceeded expected VQI prediction model mortality in all groups, although was not statistically significant. The VQI model adequately stratified the studied population into risk groups (P .001). Low-risk patients with CLTI (65% of the overall cohort) experienced 2-year mortality of 18.9%. However, observed mortality rates for medium- and high-risk VQI strata were similar. After a median follow-up of 28 months, medium-risk patients incurred a significantly higher mortality than predicted (53.5% ± 2.1% vs 36.8% ± 1.1%; P = .016).The VQI mortality prediction model discriminates mortality risk after limb revascularization in CLTI, accurately identifying a majority subgroup of patients who are suitable for either open or endovascular intervention. However, it may underestimate mortality in a tertiary referral population with high comorbidity burden and was not well calibrated for the medium-risk group. It may be more appropriate to dichotomize patients with CLTI who are candidates for limb salvage into an average-risk and high-risk group.

The interpretation of evidence continues to be one of the biggest challenges facing the forensic community. This is the first of two papers intended to provide advice on difficult aspects of evaluation and in particular on the formulation of propositions. The scientist has a dual role: investigator (crime-focused), where often there is no suspect available and a database search may be required; evaluator (suspect-focused), where the strength of evidence is assessed in the context of the case. In investigative mode, generally the aim is to produce leads regarding the source of the DNA. Sub-source level propositions will be adequate to help identify potential suspects who can be further investigated by the authorities. Once in evaluative mode, given the defence version of events of the person of interest, it may become necessary to consider alternatives that go beyond the source of the DNA (i.e., to consider activity level propositions). In the evaluation phase, it is crucial that formulation of propositions allows the assessment of all the results that will help with the issue at hand. Propositions should therefore be precise (indication of the number of contributors, information on the relevant population etc.), be about causes, not effects (e.g. a ‘matching’ DNA profile) and to avoid bias, must not be findings-led. This means that ideally, propositions should be decided based on the case information and before the results of the comparisons are known. This paper primarily reflects upon what has been coined as “sub-source level propositions“. These are restricted to the evaluation of the DNA profiles themselves, and help answer the issue regarding the source of the DNA. It is to be emphasised that likelihood ratios given sub-source level propositions cannot be carried over to a different level – for example, activity level propositions, where the DNA evidence is put into the context of the alleged activities. This would be highly misleading and could give rise to miscarriages of justice; this will be discussed in a second paper. The value of forensic results depends not only on propositions, but also on the type of results (e.g. allelic designations, peak heights, replicates) and upon the model used: it is therefore important to discuss these aspects. Finally, since communication is key to help understanding by courts, we will explore how to convey the value of the results and explain the importance of avoiding the practice of transposing the conditional.

In recent years, forensic mRNA profiling has increasingly been used to identify the origin of human body fluids. By now, several laboratories have implemented mRNA profiling and also use it in criminal casework. In 2018 the FoRNAP (Forensic RNA Profiling) group was established among a number of these laboratories with the aim of sharing experiences, discussing optimization potential, identifying challenges and suggesting solutions with regards to mRNA profiling and casework. To compare mRNA profiling methods and results a collaborative exercise was organized within the FoRNAP group. Seven laboratories from four countries received 16 stains, comprising six pure body fluid / tissue stains and ten mock casework samples. The laboratories were asked to analyze the provided stains with their in-house method (PCR/CE or MPS) and markers of choice. Five laboratories used a DNA/RNA co-extraction strategy. Overall, up to 11 mRNA markers per body fluid were analyzed. We found that mRNA profiling using different extraction and analysis methods as well as different multiplexes can be applied to casework-like samples. In general, high input samples were typed with high accuracy by all laboratories, regardless of the method used. Irrespective of the analysis strategy, samples of low input or mixed stains were more challenging to analyze and interpret since, alike to DNA profiling, a higher number of markers dropped out and/or additional unexpected markers not consistent with the cell type in question were detected. It could be shown that a plethora of different but valid analysis and interpretation strategies exist and are successfully applied in the Forensic Genetics community. Nevertheless, efforts aiming at optimizing and harmonizing interpretation approaches in order to achieve a higher consistency between laboratories might be desirable in the future. The simultaneous extraction of DNA alongside RNA showed to be an effective approach to identify not only the body fluid present but also to identify the donor(s) of the stain. This allows investigators to gain valuable information about the origin of crime scene samples and the course of events in a crime case.

Forensic Science International: Genetics and Forensic Science International: Reports communicate research on a variety of biological materials using genetics and genomic methods. Numerous guidelines have been produced to secure standardization and quality of results of scientific investigations. Yet, no specific guidelines have been produced for the ethical acquisition of such data. These guidelines summarize universally adopted principles for conducting ethical research on biological materials, and provide details of the general procedures for conducting ethical research on materials of human, animal, plant and environmental origin. Finally, the minimal ethics requirements for submission of research material are presented.

The use of biostatistical software programs to assist in data interpretation and calculate likelihood ratios is essential to forensic geneticists and part of the daily case work flow for both kinship and DNA identification laboratories. Previous recommendations issued by the DNA Commission of the International Society for Forensic Genetics (ISFG) covered the application of bio-statistical evaluations for STR typing results in identification and kinship cases, and this is now being expanded to provide best practices regarding validation and verification of the software required for these calculations. With larger multiplexes, more complex mixtures, and increasing requests for extended family testing, laboratories are relying more than ever on specific software solutions and sufficient validation, training and extensive documentation are of upmost importance. Here, we present recommendations for the minimum requirements to validate bio-statistical software to be used in forensic genetics. We distinguish between developmental validation and the responsibilities of the software developer or provider, and the internal validation studies to be performed by the end user. Recommendations for the software provider address, for example, the documentation of the underlying models used by the software, validation data expectations, version control, implementation and training support, as well as continuity and user notifications. For the internal validations the recommendations include: creating a validation plan, requirements for the range of samples to be tested, Standard Operating Procedure development, and internal laboratory training and education. To ensure that all laboratories have access to a wide range of samples for validation and training purposes the ISFG DNA commission encourages collaborative studies and public repositories of STR typing results. Published by Elsevier Ireland Ltd.

The statistical evaluation of autosomal Short Tandem Repeat (STR) genotypes is based on allele frequencies. These are empirically determined from sets of randomly selected human samples, compiled into STR databases that have been established in the course of population genetic studies. There is currently no agreed procedure of performing quality control of STR allele frequency databases, and the reliability and accuracy of the data are largely based on the responsibility of the individual contributing research groups. It has been demonstrated with databases of haploid markers (EMPOP for mitochondrial mtDNA, and YHRD for Y-chromosomal loci) that centralized quality control and data curation is essential to minimize error. The concepts employed for quality control involve software-aided likelihood-of-genotype, phylogenetic, and population genetic checks that allow the researchers to compare novel data to established datasets and, thus, maintain the high quality required in forensic genetics. Here, we present STRidER (http://strider.online), a publicly available, centrally curated online allele frequency database and quality control platform for autosomal STRs. STRidER expands on the previously established ENFSI DNA WG STRbASE and applies standard concepts established for haploid and autosomal markers as well as novel tools to reduce error and increase the quality of autosomal STR data. The platform constitutes a significant improvement and innovation for the scientific community, offering autosomal STR data quality control and reliable STR genotype estimates.

Africa is the cradle of all human beings, and although it has been the focus of a number of genetic studies, there are many questions that remain unresolved. We have performed one of the largest and most comprehensive meta-analyses of mitochondrial DNA (mtDNA) lineages carried out in the African continent to date. We generated high-throughput mtDNA single nucleotide polymorphism (SNP) data (230 SNPs) from 2024 Africans, where more than 500 of them were additionally genotyped for the control region. These data were analyzed together with over 12,700 control region profiles collected from the literature, representing more than 300 population samples from Africa. Insights into the African homeland of humans are discussed. Phylogeographic patterns for the African continent are shown at a high phylogeographic resolution as well as at the population and regional levels. The deepest branch of the mtDNA tree, haplogroup L0, shows the highest sub-haplogroup diversity in Southeast and East Africa, suggesting this region as the homeland for modern humans. Several demographic estimates point to the coast as a facilitator of human migration in Africa, but the data indicate complex patterns, perhaps mirroring the effect of recent continental-scaled demographic events in re-shaping African mtDNA variability.

A great amount of population and forensic genetic data are available for X-STRs supporting the need for having a common and accurate nomenclature among laboratories allowing for better communication, data exchange, and data comparison. DXS10148, DXS10074 and DXS10134 are commonly used X-STRs particularly due to their inclusion in the commercial kit Investigator Argus X-12 (Qiagen). Samples from West Africa and Iraq were sequenced for all three X-STRs allowing the detection of new DNA sequence variants. At DXS10148, variation was detected at four bases downstream from the flanking region from the repeat motif. The sequence AAGG-AAAG has been detected for the first time as a varying (AAGG-AAAG)1-3 motif, in the present work. One additional string when compared to the common one (AAGG-AAAG)2 adds eight bases to the fragment size of the tetranucleotide STR. This means that 2 repeats are added in these cases to the fragment size of the allele, while the presence of only one copy will reduce the expected allele size by 2 repeats. At DXS10074 two varying stretches consisting of AC and AG dinucleotide repeats were observed in the upstream flanking region, six bases from the main repeat core that also influence the expected allele size. DXS10134 revealed a simpler nomenclature in the Guinea-Bissau sample set when compared to the previously described allele nomenclature. This detected new hidden variation also has impact on the actual allele nomenclature at this locus as it contributes to a new class of short alleles so far undetected in other studies.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified. (C) 2015 Elsevier Ireland Ltd. All rights reserved.

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.

The pattern of human variation in Africa is the result of a complex series of demographic events. There are few databases related to the peoples of Africa, especially in western and central regions. In this study, 17 Y-STR and 31 Y-SNP loci were genotyped in a sample from Western Africa, which was compared with other representative populations of other African regions. The haplotype diversity observed in this region was 1.0000 (±0.0018) and that is similar to those values obtained in other African samples already studied. The most frequent haplogroup found in our sample belongs to E, showing the M2 derived allele. The haplogroup diversity was 0.6895 (±0.0200), very similar to that observed in countries where the Bantu groups are predominant (0.681 in Angola; 0.753 in Equatorial Guinea). Concerning the Western Africa, significant differences were observed between Guinea–Bissau and the remaining populations of this region. In addition, there was no significant difference between our sample and samples from Eastern and Central Africa. The genetic composition of the population studied was significantly different from that of Guinea–Bissau, although a genetic homogeneity was found among most countries belonging to West Africa.

Two statistical models have been developed to interpret locus drop-out resulting from degraded DNA samples. These models are based on an experimental dataset of 600 genotyping experiments. The first model presents the probability of locus drop-out against the average peak height with a lower and upper confidence interval. For a given peak height average, the probability of drop-out can be quite wide. This probability decreases rapidly when the profile quality increases. For the second model, we have fitted the probability of locus drop-out against the number of base pairs with a lower and upper confidence interval. The probability of drop-out is increasing along with the number of base pairs. Despite DNA degradation effects, the drop-out probability does not get higher than 0.5.

For the analysis of cell type-specific miRNA expression patterns qPCR is currently the method of choice owing to its high accuracy. However, to obtain reliable results, a proper normalization strategy is an absolute prerequisite, which is often underestimated. To demonstrate the importance of using a set of suitable reference genes, we tested two normalization strategies by comparing gene expressions of tissue-specific miRNA targets normalized against: (1) previously validated endogenous controls (miR92 and miR374) and (2) a commonly used miRNA reference (U6B).

The DNA Commission of the International Society of Forensic Genetics (ISFG) regularly publishes guidelines and recommendations concerning the application of DNA polymorphisms to the question of human identification. Previous recommendations published in 2000 addressed the analysis and interpretation of mitochondrial DNA (mtDNA) in forensic casework. While the foundations set forth in the earlier recommendations still apply, new approaches to the quality control, alignment and nomenclature of mitochondrial sequences, as well as the establishment of mtDNA reference population databases, have been developed. Here, we describe these developments and discuss their application to both mtDNA casework and mtDNA reference population databasing applications. While the generation of mtDNA for forensic casework has always been guided by specific standards, it is now well-established that data of the same quality are required for the mtDNA reference population data used to assess the statistical weight of the evidence. As a result, we introduce guidelines regarding sequence generation, as well as quality control measures based on the known worldwide mtDNA phylogeny, that can be applied to ensure the highest quality population data possible. For both casework and reference population databasing applications, the alignment and nomenclature of haplotypes is revised here and the phylogenetic alignment proffered as acceptable standard. In addition, the interpretation of heteroplasmy in the forensic context is updated, and the utility of alignment-free database searches for unbiased probability estimates is highlighted. Finally, we discuss statistical issues and define minimal standards for mtDNA database searches.

Several forensic age predictors based on DNA methylation have been developed, reporting a considerable number of CpG loci highly correlated with chronological age. Most of the published age prediction models so far were based on European samples, and therefore there is a need to explore additional worldwide populations. We performed a comprehensive analysis of methylation profiles from buccal mucosa samples of three independent population groups from Middle East, West Africa and Central Europe. For each population approx. 50 samples with an equal distribution of donor ages were collected. Using bisulfite pyrosequencing we analyzed the age dependent methylation changes at five CpG sites in the genes of ASPA , ITGA2B , PDE4C and ELOVL2 and predicted the donor ages using two previously reported age prediction models. Our results display significantly lower dispersions of methylations and thus errors of predicted ages in the Middle East population compared to the Central Europe and West African populations. Interestingly, the overall methylation variation was highly similar between the three populations at all investigated CpGs.

Insertion/deletion polymorphisms have recently received increased interest in the forensic genetics community. This class of markers combines the advantageous genetic properties of single nucleotide polymorphisms (i.e., low mutation rate, genetic stability, and short amplicon size) with the technical advantage of short tandem repeat markers (simple detection by fluorescence-labelled PCR and capillary electrophoresis). For a large number of indel markers significant differences in allele frequencies between the major populations have been reported, making this class of markers suitable for the analysis of biogeographic ancestry. We have developed a multiplex PCR assay designed to establish the biogeographic ancestry of forensic DNA samples based on insertion/deletion polymorphisms. A panel of 21 short indels with allele frequency differences between three major population groups (European, African and Asian) was selected to be incorporated into a single-tube multiplex PCR assay. The assay is highly sensitive, requiring less than 0.5 ng of genomic DNA for successful typing. Due to the short fragment lengths below 200 bp, the assay is ideally suited for the typing of challenging forensic genetic case work samples. A population genetic study has been performed proving the performance of the assay in inferring the ancestral population of individuals. The chosen 21 markers are sufficient to distinguish between three major global population groups. Furthermore, the assay design leaves room for an extension in order to cover additional population groups.

There is growing evidence that the histone-DNA complexes found in nucleosomes offer protection from DNA degradation processes, including apoptotic events in addition to bacterial and environmental degradation. We sought to locate human nucleosome regions and build a catalogue of SNPs sited near the middle of these genomic segments that could be combined into a single PCR multiplex specifically for use with extremely degraded human genomic DNA samples. Using recently optimized bio-informatics tools for the reliable identification of nucleosome sites based on sequence motifs and their positions relative to known promoters, 1395 candidate loci were collected to construct an 18-plex single base extension assay. Genotyping performance of the nucleosome SNPs was tested using artificially degraded DNA and 24 casework samples where the likely state of degradation of DNA was established by comparison to profile completeness in four other forensic assays: a standard 15-plex STR identification test, a miniaturized STR multiplex and two autosomal SNP multiplexes. The nucleosome SNP assay gave genotyping success rates 6% higher than the best existing forensic SNP assay: the SNPforID Auto-2 29-plex and significantly higher than the mini-STR assay. The nucleosome SNPs we located and combined therefore provide a new type of marker set that can be used to supplement existing approaches when the analysed DNA is likely to be extremely degraded and may fail to give sufficient STR genotypes for a reliable identification. © 2011 Elsevier Ireland Ltd. All rights reserved.

West Africa is characterized by a migration history spanning more than 150,000 years. Climate changes but also political circumstances were responsible for several early but also recent population movements that shaped the West African mitochondrial landscape. The aim of the study was to establish a Ghanaian mtDNA dataset for forensic purposes and to investigate the diversity of the Ghanaian population sample with respect to surrounding populations. We sequenced full mitochondrial control regions of 193 Akan people from Ghana and excluded two apparently close maternally related individuals due to preceding kinship testing. The remaining dataset comprising 191 sequences was applied as etalon for quasi-median network analysis and was subsequently combined with 99 additional control region sequences from surrounding West African countries. All sequences were incorporated into the EMPOP database enriching the severely underrepresented African mtDNA pool. For phylogeographic considerations, the Ghanaian haplotypes were compared to those of 19 neighboring populations comprising a total number of 6198 HVS1 haplotypes. We found extensive genetic admixture between the Ghanaian lineages and those from adjacent populations diminishing with geographical distance. The extent of genetic admixture reflects the long but also recent history of migration waves within West Africa mainly caused by changing environmental conditions. Also, evidence for potential socio-economical influences such as trade routes is provided by the occurrence of U6b and U6d sequences found in Dubai but also in Tunisia leading to the African West Coast via Mauritania and Senegal but also via Niger, Nigeria to Cameroon. © 2011 Elsevier Ireland Ltd. All rights reserved.

We present data from a multi-laboratory validation study of the Genplex typing system [C. Phillips, et al., Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel, Forensic Sci. Int.: Genet. 1 (2) (2007) 180–185.] (Applied Biosystems), which interrogates a subset of 48 SNPs selected from the panel of 52 previously developed for forensic analysis by the SNP for ID consortium [J.J. Sanchez, et al., A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (9) (2006) 1713–1724.], plus amelogenin. The Genplex technology was developed through modification of the SNPlex™ system (also Applied Biosystems) and utilises oligo-ligation of PCR products followed by probe hybridisation to generate dye-labeled, allele-specific oligonucleotides that are detected with capillary electrophoresis. We compare the success rate of Genplex in typing 55 samples in three laboratories with results obtained from STR typing of the same samples using Powerplex ® 16 (Promega) and SGMPlus ® (Applied Biosystems). The sample set was chosen to mimic extracts encountered in forensic situations and includes low concentration and degraded material as well as mixtures. We demonstrate that the Genplex technique provides a significantly higher success rate than STR-based methods when typing degraded DNA and is also capable of mixture detection up to a level of 1 in 10.

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. # 2007 Elsevier Ireland Ltd. All rights reserved.

Resume Objectif L'hypophosphatasie est une maladie metabolique hereditaire (MIM #241510) due a une mutation du gene de la phosphatase alcaline sans specificite tissulaire (TNSALP). Il n'existe pas de donnees sur l'evolution au long cours de la masse osseuse dans cette maladie. Methodes Nous avons suivi l'evolution de la densite minerale osseuse (DMO) de facon prospective pendant quatre ans chez six malades. La DMO a ete mesuree par absorptiometrie et tomodensitometrie quantitative peripherique (TDMqp). Resultats Au moment du diagnostic d'hypophosphatasie, une mineralisation accrue de l'os spongieux des metaphyses des os longs a ete notee. Pendant les quatre annees du suivi, la mineralisation de la metaphyse radiale a diminue de facon significative. En revanche, la DMO mesuree par absorptiometrie au rachis lombaire et au squelette total ne s'est pas modifiee par comparaison aux temoins bien portants. Les concentrations de prostaglandines circulantes etaient elevees chez la majorite des malades. La mesure des prostaglandines urinaires a permis de surveiller l'effet du traitement anti-inflammatoire non steroidien. Conclusions La mineralisation accrue des metaphyses, qui compenserait la resistance mecanique insuffisante de l'os, a diminue au cours du suivi. L'elevation durable des prostaglandines circulantes pourrait contribuer a la physiopathologie de ce phenomene.

The ISFG membership consists of scientists and medical professionals specialized in using genetic testing for kinship analysis and the individualization of biological material. This expertise makes the forensic geneticist a resource of advice to international and national organizations dealing with human identifications and causes many DNA laboratories to get involved in DVI tasks. The present recommendations are meant to educate more forensic geneticists about their potential involvement in mass fatality preparedness and possible DVI efforts, as well as to provide practical guidance for each of the laboratories’ individual tasks. The idea to work on DNA-specific recommendations was born after a round table discussion dealing with the 2004 Tsunami disaster in south east Asia during the 21st congress of the International Society for Forensic Genetics on the Azores, Portugal, in September 2005. The ensuing discussion between scientists and pathologists that had been involved in the International Center in Khao Lak, Thailand, revealed the need for the scientific community to be better prepared to answer the local authorities’ questions by formulating generally acceptable scientific standards for the most efficient use of DNA-based victim identification methods. These recommendations, as well as the many cited references, are intended to provide guidance on establishing preparedness for the forensic genetics laboratory, on collecting and storing ante-mortem and post-mortem samples suitable for DNA analysis, on DNA extraction and genetic typing strategies, on data management, and on issues related to the biostatistical interpretation and reporting of results. # 2006 Elsevier Ireland Ltd. All rights reserved.

Forensic molecular genetics has evolved from a rapidly developing field with changing technologies into a highly recognized and generally accepted forensic science, leading to the establishment of national DNA databases with DNA profiles from suspects and convicted offenders. DNA evidence has taken a central role by carrying a significant weight for convictions, as well as by excluding innocent suspects early on in a criminal investigation. Due to this impact on the criminal justice system, guidelines for research in forensic genetics have been introduced already since many years. The most important issues regarding the selection and definition of typing systems both for paternity testing and for forensic identification, the criteria for technical and biostatistical validation, as well as the use of mitochondrial DNA analysis are summarized and discussed.

Quadruplex and pentaplex systems for polymerase chain reaction amplification of X-chromosomal short tandem repeats DXS101, HPRTB, DXS8377, DXS981 (STRX1) and DXS6789 were developed for automated profiling of liquid and membrane-bound DNA samples. Chinese, Japanese and Thai populations were typed using a quadruplex system, while German and Philippine populations were analyzed using a five-locus system. Out of 88 meioses studied in Philippine family samples at each locus, a possible one repeat deletion (allele 51 to 50) at DXS8377 was observed in a father-daughter pair. Exact tests performed on genotype data from females in the Philippine, German and Thai populations indicated that these groups conform to Hardy-Weinberg equilibrium. Exact tests for population differentiation indicate significant variations in allele distributions, particularly at loci DXS101, DXS981 and DXS6789. Considered individually, DXS8377 was the most polymorphic and HPRTB the least polymorphic locus in these five populations. When the forensic efficiency of the quadruplex system was calculated, the combined power of discrimination among males (PD(M)) was no lower than 0.998, while among females the combined PD(F) was at least 0.9999 in all populations. The combined power of paternity exclusion was a minimum of 0.998 in trio cases and 0.98 in motherless cases. The addition of locus DXS6789 to the German and Philippine population databases using a pentaplex increased the forensic efficiency of the analysis system.

DNA typing through analysis of short tandem repeats (STRs) and mitochondrial DNA (mtDNA) by means of the polymerase chain reaction (PCR) and sequencing are the common methods for the forensic identification of persons and reconstruction of kinship, especially when skeletal human remains have to be analyzed. Furthermore, samples typically found at crime scenes may be both quantitatively and qualitatively inadequate since they may contain very scarce and often degraded DNA due to exposure to heat, light, humidity, and microorganisms. In order to improve the performance of STR typing technology in those cases where DNA availability is limited, it would be desirable to have a source of degraded DNA with known properties. For this purpose, we have developed a method to prepare artificially degraded DNA under controlled conditions. By treatment of genomic DNA with sonication and DNAse I we have produced DNA fragments within a defined range of lengths. STR typing of this degraded DNA with a commercially available multiplex kit could only produce partial profiles as indicated by the absence of STR alleles with sizes >200 bp. This artificially degraded DNA can be used for the improvement and standardization of STR typing protocols when only highly degraded DNA is available for analysis.

A systematic study was conducted to investigate whether DNA can be successfully extracted from latent fingerprints deposited on ordinary paper and analysed using short tandem repeat profiling and mitochondrial DNA sequencing. In order to evaluate the performance of latent fingerprint analysis in a criminal case, experiments with varying conditions were carried out to improve our understanding of low copy number (LCN) DNA typing. After optimising the extraction methods to achieve increased sensitivity, the examination of touched paper can routinely yield the STR profile of the individual who has touched it. A fingerprint can therefore be considered as a potential source of DNA for genetic identification. Nevertheless, the findings of our "after enhancement experiment" (using chemically or physically pre-treated fingerprints), and our "mixture experiment" (using fingerprints from three to four people on the same sheet of paper) help to define the limitations of the low copy number PCR technique in forensic casework.

1. IntroductionEngraftment of donor stem cells after allogeneic bone marrow transplantation can begenetically monitored by PCR typing of DNA polymorphisms [1]. Successful engraftmentwith complete chimerism and presence of the donor’s genotype in the bone marrow has tobe demonstrated, and the presence of the patient’s alleles has to be excluded. Detection ofthe patient’s alleles provides evidence for an incomplete chimerism or for a relapse ofmalignant disease. STRs have been used successfully for this type of genetic monitoring[2]. For the present study, we have developed an approach to quantify the ratio of donorchimerism using mock mixture experiments. The usefulness of our approach is demon-strated in typical cases where the donor chimerism could be monitored over periods ofmore than 1 year after transplantation (Tx).2. MethodsThe degree of donor–recipient chimerism was monitored by a semi-quantitativeanalysis of the mixed STR genotypes. DNA was extracted from bone marrow samplesas well as from FACS-separated mononuclear cells, CD3+ T cells and CD3 mono-nuclear cells. Furthermore, buccal swabs were collected from donor and recipient toestablish their genotypes prior to transplantation. The DNA was subjected to PCR typing