Assessment of the effectiveness of the EUROFORGEN NAME and Precision ID Ancestry panel markers for ancestry investigations.

The EUROFORGEN NAME panel is a regional ancestry panel designed to differentiate individuals from the Middle East, North Africa, and Europe. The first version of the panel was developed for the MassARRAY system and included 111 SNPs. Here, a custom AmpliSeq EUROFORGEN NAME panel with 102 of the original 111 loci was used to sequence 1098 individuals from 14 populations from Europe, the Middle East, North Africa, North-East Africa, and South-Central Asia. These samples were also sequenced with a global ancestry panel, the Precision ID Ancestry Panel. The GenoGeographer software was used to assign the AIM profiles to reference populations and calculate the weight of the evidence as likelihood ratios. The combination of the EUROFORGEN NAME and Precision ID Ancestry panels led to fewer ambiguous assignments, especially for individuals from the Middle East and South-Central Asia. The likelihood ratios showed that North African individuals could be separated from European and Middle Eastern individuals using the Precision ID Ancestry Panel. The separation improved with the addition of the EUROFORGEN NAME panel. The analyses also showed that the separation of Middle Eastern populations from European and South-Central Asian populations was challenging even when both panels were applied.

NGMSElect™ and Investigator(®) Argus X-12 analysis in population samples from Albania, Iraq, Lithuania, Slovenia, and Turkey.

The analysis of STRs is the main tool when studying genetic diversity in populations or when addressing individual identification in forensic casework. Population data are needed to establish reference databases that can be used in the forensic context. To that end, this work investigated five population samples from Albania, Iraq, Lithuania, Slovenia, and Turkey. Individuals were typed for 16 autosomal STRs and 12 X-chromosomal STRs using the NGMSElect™ and Investigator(®) Argus X-12 kits, respectively. The aim of the study was to characterize the diversity of both STR kits in these population samples and to expand our forensic database. The results showed that all markers were polymorphic in the five populations studied. No haplotype was shared between the males analysed for X-STRs. No statistically significant deviations from Hardy-Weinberg equilibrium were observed for any of the genetic markers included in both the kits. Pairwise LD was only detected in X-STRs between markers located in the same linkage group. Power of discrimination values for males and females and the probability of exclusion in duos and trios were high for the populations in this study.

Concordance study and population frequencies for 16 autosomal STRs analyzed with PowerPlex® ESI 17 and AmpFℓSTR® NGM SElect™ in Somalis, Danes and Greenlanders.

A concordance study of the results of PowerPlex(®) ESI 17 and AmpFℓSTR(®) NGM SElect™ kits obtained from 591 individuals from Somalia (N=198), Denmark (N=199) and Greenland (N=194) was performed. Among 9456 STR types, seven discordant results were found with the two kits: one observed in the D19S433 system in an individual from Denmark and six in the SE33 system in six individuals from Somalia. Sequencing of SE33 in the six samples with discordant results showed G>A transition 15bp downstream of the repeat unit in three of the individuals, and G>A transition 68bp downstream of the repeat unit in the other three individuals. Population data for 16 autosomal STR systems analyzed in 989 individuals from Somalia, Denmark and Greenland are also presented. The highest mean heterozygosity was observed in Danes (82.5%). With the exception of D8S1179 in Danes, no significant deviations from Hardy-Weinberg expectations were observed. Only one pair of systems (D12S391 and D18S51) showed significant allelic association in Greenlanders (after Holm-Sidák correction). A MDS plot drawn from pairwise FST values calculated between 21 populations showed a clear displacement of the Greenlandic population versus the other ones included in the analyses. The highest combined chance of exclusion and power of discrimination was observed for Danes reaching values of 99.9999987% and 1 in 1.8×10(21), respectively.

Non-uniform phenotyping of D12S391 resolved by second generation sequencing.

Non-uniform phenotyping of five case work samples were observed in the D12S391 locus. The samples were typed at least twice with the AmpFℓSTR NGM SElect PCR Amplification Kit and different alleles were called with GeneMapper ID-X in the different experiments. Detailed analyses of the electropherograms suggested that the individuals were heterozygous with two alleles that differed in size by one nucleotide. This was confirmed by amplifying the samples with the PowerPlex ESX 17 system. D12S391 is a complex STR with variable numbers of AGAT and AGAC repeats. Second generation sequencing revealed that separation of two alleles differing by one nucleotide in length was poor if the number of AGAT repeats in the short allele was higher than in the long allele. A total of 45 individuals with microvariants or off-ladder alleles in D12S391 were sequenced. Thirty different alleles were detected and sixteen of these were not previously reported.

Autosomal SNP typing of forensic samples with the GenPlex™ 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.

Analysis of artificially degraded DNA using STRs and SNPs--results of a collaborative European (EDNAP) exercise.

Recently, there has been much debate about what kinds of genetic markers should be implemented as new core loci that constitute national DNA databases. The choices lie between conventional STRs, ranging in size from 100 to 450 bp; mini-STRs, with amplicon sizes less than 200 bp; and single nucleotide polymorphisms (SNPs). There is general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new markers is to increase the chance of amplifying highly degraded DNA rather than to increase the discriminating power of the current techniques. A collaborative study between nine European and US laboratories was organised under the auspices of EDNAP. Each laboratory was supplied with a SNP multiplex kit (Foren-SNPs) provided by the Forensic Science Service, two mini-STR kits provided by the National Institute of Standards and Technology (NIST) and a set of degraded DNA stains (blood and saliva). Laboratories tested all three multiplex kits, along with their own existing DNA profiling technique, on the same sets of degraded samples. Results were collated and analysed and, in general, mini-STR systems were shown to be the most effective. Accordingly, the EDNAP and ENFSI working groups have recommended that existing STR loci are reengineered to provide smaller amplicons, and the adoption of three new European core loci has been agreed.

AMPA receptor subunit mRNAs and intracellular [Ca(2+)] in cultured mouse and rat cerebellar granule cells.

Cultured mouse cerebellar granule cells differ from their rat counterparts in that they survive well when grown in non-depolarising medium (5 mM K(+)). However, when chronically stimulated by added glutamate agonists, including (RS)alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), rat cerebellar granule cells also survive well in non-depolarising medium. We hypothesised that the relatively good survival of mouse cerebellar granule cells in the absence of added glutamate agonists might reflect AMPA receptors resistant to desensitisation. These receptors might be stimulated by endogenous glutamate. We tested this hypothesis by comparing cultured mouse and rat cerebellar granule cells grown in depolarising (25 mM K(+)) and non-depolarising (5 mM K(+)) medium. We studied the AMPA-induced increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), using the fluorescent Ca(2+) chelator, Fluo-3, and the relative concentrations of mRNAs for the four AMPA receptor subunits, GluR1-4. GluR1-4 mRNAs were measured by restriction enzyme analysis of a PCR product containing cDNA with a composition proportional to the four subunit mRNAs. We found that the [Ca(2+)](i)-response to AMPA receptor activation in cultured cerebellar granule cells is determined mainly by the desensitisation properties of the AMPA receptors rather than by their ion permeability. We also found that mouse cerebellar granule cells express AMPA receptors which are more resistant to desensitisation than the corresponding rat AMPA receptors. Thus, relatively slow AMPA receptor desensitisation kinetics may contribute to the survival of mouse cerebellar granule cells in non-depolarising medium.

Amyloid beta-peptide(25-35) changes [Ca2+] in hippocampal neurons.

Insoluble aggregates of the amyloid beta-peptide (A beta) is a major constituent of senile plaques found in brains of Alzheimer disease (AD) patients. The detrimental effects of aggregated A beta is associated with an increased intracellular Ca2+ concentration ([Ca2+]i). We examined the effects of A beta(25-35) on [Ca2+]i and intracellular H+ concentration ([H+]i) in single hippocampal neurons by real time fluorescence imaging using the Ca(2+)- and H(+)-specific ratio dyes, indo-1 and SNARF-1. Incubation of these cultures with A beta(25-35) for 3-12 days in vitro increased [Ca2+]i and [H+]i in large, NMDA-responsive neurons.

Weaver mutant mouse cerebellar granule cells respond normally to chronic depolarization.

We studied the effects of chronic K(+)-induced membrane depolarization and treatment with N-methyl-D-aspartate (NMDA) on cerebellar granule cells (CGCs) from weaver mutant mice and non-weaver litter-mates. The weaver mutation is a Gly-to-Ser substitution in a conserved region of the Girk2 G protein-coupled inward rectifying potassium channel [Patil N., Cox D. R., Bhat D., Faham M., Myers R. M. and Peterson A. S. (1995) Nature Genet. 11, 126-129] which induces early death of CGCs. The biochemical differentiation of CGCs was estimated as the rate of 2-deoxy-D-glucose accumulation and the expression of neural cell adhesion molecule (NCAM). High (25 mM) K+ ion concentration or treatment with NMDA greatly promoted the biochemical differentiation of both weaver mutant and non-weaver litter-mate mouse CGCs. In contrast to the marked effect on biochemical differentiation in both weaver and non-weaver mice CGSs, chronic high K+ treatment only had limited effect on survival. The survival of weaver mutant mouse CGCs in medium containing 5 mM K+ ions was very low, only 20% of the plated cells surviving at 7 days after plating, as opposed to the 50% for non-weaver CGCs. Chronic high K+ treatment improved the relative survival of weaver mutant mouse CGCs 1.6 2.2-fold and that of non-weaver CGCs 1.2-1.4-fold; the same number of CGCs (about 20% of the plated cells) were rescued by high K+ in both types of culture. The findings indicate that, in culture weaver mutant mouse, CGCs have a normal response to membrane depolarization and that the normal function of the Girk2 potassium channel is not critical for the survival of differentiated CGCs.

NMDAR1 mRNA expression and glutamate receptor stimulated increase in cytosolic calcium concentration in rat and mouse cerebellar granule cells.

We have previously reported that, unlike their rat counterparts, the survival of mouse cerebellar granule cells is independent of chronic stimulation whether owing to elevated K(+)-induced depolarization or NMDA (N-methyl-D-aspartate) receptor activation. One explanation could be that during the critical period mouse granule cells are very sensitive to NMDA receptor stimulation by endogenous glutamate released in the cultures. If so, this might be reflected by an increased expression of NMDA receptors or an increased response to their activation. We tested this hypothesis by measuring (a) the concentration of mRNA for the obligatory NMDA receptor subunit, NMDAR1, and (b) the glutamate/NMDA stimulated increase in cytosolic Ca(2+)-ion concentration in cultures at physiological or elevated K(+)-ion concentration. The expression of NMDAR1 mRNA was measured by competitive PCR of reversely transcribed mRNA and was normalized to that of the constitutively expressed H3.3 histone mRNA. The glutamate and NMDA stimulated increase in cytosolic Ca(2+)-ion concentration was measured using the fluorescent Ca(2+)-chelator Fluo3. In contrast to the hypothesis, we found NMDAR1 mRNA expression to be lower in mouse than in rat granule cells cultured for 4 days at physiological K(+)-ion concentration. However, the NMDA stimulated increase in cytosolic Ca(2+)-ion concentration did not differ in 4-day rat and mouse cultures. Although the glutamate-stimulated increase in cytosolic Ca(2+)-ion concentration in 2-day cultures was higher in mouse granule cells than in rat granule cells, the developmental profile of the glutamate-stimulated increase in cytosolic Ca(2+)-ion concentration was the same in both cases. In conclusion, we found no obvious evidence for increased NMDA receptor activity in mouse cerebellar granule cells cultured at physiological K(+)-ion concentration.

The survival of cultured mouse cerebellar granule cells is not dependent on elevated potassium-ion concentration.

The effects of K(+)-induced membrane depolarization were studied on the survival and biochemical parameters in mouse and rat cerebellar granule cells grown in micro-well cultures. Cell numbers were determined by estimating DNA content using the Hoechst 33258 fluorochrome binding assay. DNA from degenerated cells was removed by prior DNAase treatment. These DNA estimates of cell numbers were comparable with values obtained by direct counting of fluorescein diacetate-stained viable cells. In agreement with previous studies, the survival of rat granule cells was promoted by increasing the concentration of K+ in the medium from 5 to 25 mM throughout a 7-day culture period. In contrast, mouse granule cells survived in culture containing 'low' K+ (5 or 10 mM), as well as in the presence of 'high' K+ (25 mM). On the other hand, several biochemical parameters in mouse granule cells were markedly increased by cultivation in 'high' as compared with 'low' K(+)-containing media, demonstrated by increased fluorescein diacetate esterase activity, enhanced rate of NADPH-dependent tetrazolium reduction, augmented 2-deoxy-D-glucose accumulation and increased N-methyl-D-aspartate-evoked 45Ca2+ influx. It was concluded that although cultivation in 'high' K+ promotes biochemical differentiation in mouse cerebellar granule cells, these cells differ from their rat counterparts in that they do not develop a survival requirement for K(+)-induced membrane depolarization.