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.

[Why people delay seeking care after a positive HIV test: a qualitative study in Burkina]. / La déperdition entre les postes de dépistage et de prise en charge des personnes vivant avec le VIH-sida au Burkina Faso : une approche qualitative.

UNLABELLED: Delayed care-seeking occurs when a person who received a positive HIV serology test result does not immediately seek medical treatment for this HIV infection. It has serious consequences for patient survival. This study aims to analyze the factors leading to delayed care-seeking in this circumstance. METHODOLOGY: Applying a qualitative approach, we conducted individual interviews and focus groups in 9 community-based organizations of people living with HIV and AIDS in Burkina Faso. In total, 112 people including 70 HIV-positive patients, 30 healthcare providers, and 12 people leaving the laboratory after an HIV test, were interviewed. A thematic content analysis identified the factors that delayed care-seeking. RESULTS: Several factors explain the delay in seeking care. The weight of the negative representations of HIV and AIDS, its impact on those diagnosed with them, and fear of stigmatization (especially by family members) are major factors in delayed care. The poor quality of pre- and post-test counseling is another factor. This study also shows that financial barriers remain important in this delay. CONCLUSION: These findings suggest that earlier HIV care may be possible through efforts to reduce stigma, removal of financial barriers, and improvement of the quality of pre- and post-test counseling in mobile-device strategies and during large-scale testing campaigns.

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.

Influence of chorionicity on perinatal outcome in a large cohort of Danish twin pregnancies.

OBJECTIVE: To assess outcome in twin pregnancies according to chorionicity. METHODS: A cohort was retrieved from local ultrasound databases at 14 obstetric departments in Denmark, comprising all twin pregnancies with two live fetuses scanned between weeks 11 and 14 in the period 1 January 2004 to 31 December 2006. Outcome data were retrieved from the National Board of Health. RESULTS: Among 2038 twin pregnancies, 1757 (86.2%) were dichorionic (DC) and 281 (13.8%) were monochorionic diamniotic (MC). In MC pregnancies, the rate of spontaneous fetal loss in both second and third trimesters was more than threefold higher than the comparable rate in DC pregnancies: 6.0% vs. 1.9% for at least one fetus in the second trimester (P < 0.001) and 2.1% vs. 0.7% in the third trimester (P = 0.03). In 98.4% of DC pregnancies and in 91.1% of MC pregnancies, at least one infant was liveborn. Amongst pregnancies with two live fetuses at 24 weeks, the proportion with two live infants at 28 days after delivery was 97.5% and 95.1%, respectively. CONCLUSIONS: The increased incidence of fetal loss in MC pregnancies compared with DC pregnancies predominantly occurs before 24 weeks' gestation. After this stage, although the risk of intrauterine fetal death is still higher in MC than in DC pregnancies, if both fetuses are alive at 24 weeks, the chance of a woman having two live infants 1 month after delivery is similar in MC and DC pregnancies.

Screening of subfertile men for testicular carcinoma in situ by an automated image analysis-based cytological test of the ejaculate.

Testicular cancer (TC) is usually diagnosed after manifestation of an overt tumour. Tumour formation is preceded by a pre-invasive and asymptomatic stage, carcinoma in situ (CIS) testis, except for very rare subtypes. The CIS cells are located within seminiferous tubules but can be exfoliated and detected in ejaculates with specific CIS markers. We have built a high throughput framework involving automated immunocytochemical staining, scanning microscopy and in silico image analysis allowing automated detection and grading of CIS-like stained objects in semen samples. In this study, 1175 ejaculates from 765 subfertile men were tested using this framework. In 5/765 (0.65%) cases, CIS-like cells were identified in the ejaculate. Three of these had bilateral testicular biopsies performed and CIS was histologically confirmed in two. In total, 63 bilateral testicular biopsy were performed in conjunction with analysis of the ejaculates because of infertility work-up. Histological analysis of the biopsies for the presence of CIS yielded a test sensitivity of 0.67 and a specificity of 0.98. In addition, ejaculates from 45 patients with clinical signs of an overt TC were investigated and yielded a slightly lower sensitivity (0.51), possibly because of obstruction. We conclude that this novel non-invasive test combining automated immunocytochemistry and advanced image analysis allows identification of TC at the CIS stage with a high specificity, but a negative test does not completely exclude CIS. On the basis of the results, we propose that the assay could be offered to subfertile men and other patients who are at increased risk of TC.

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.

Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis.

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7 day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.

Double fertilization in maize: the two male gametes from a pollen grain have the ability to fuse with egg cells.

In flowering plants, two male gametes from a single pollen grain fuse with two female gametes, the egg and central cells, to form the embryo and endosperm, respectively. The question then arises whether the two male gametes fuse randomly with the egg and central cells. We investigated this question using two nearly isogenic maize lines with supernumerary B chromosomes (TB10L18) or without (r-tester). B chromosomes regularly undergo non-disjunction at the second pollen mitosis, producing one sperm cell with zero B chromosomes and one with two. We first confirmed earlier studies showing an excess of transmission of the B chromosomes to the embryo rather than to the endosperm. We then tested the possibility of a directed fertilization. For TB10L18 pollen, we could demonstrate the existence of a size dimorphism between the two sperm cells, correlated to the content in B chromosomes, as detected by fluorescence in situ hybridization (FISH). However, no directed fusion of B chromosome containing sperm to egg cells could be detected when using in vitro fertilization. The absence of directed fusion in vitro could also be demonstrated for control lines. We conclude that both male gametes have the capacity to fuse with the egg cell in maize, although sexual reproduction results in a preferential transmission of supernumerary B chromosomes.

Fertility in Prader-Willi syndrome: a case report with Angelman syndrome in the offspring.

UNLABELLED: We report on a 32-y-old woman with Prader-Willi syndrome (PWS) and her daughter with Angelman syndrome (AS). PWS in the mother was confirmed as due to a deletion of 15q11-q13, and molecular analysis in the neonate indicated an inherited maternal deletion of the same region. Features of AS in early infancy, such as jerky movements, feeding problems and poor sleep, were observed. At 5 mo of age, a triphasic high voltage EEG pattern was reported. CONCLUSIONS: This case confirms the non-Mendelian inheritance of PWS and AS and, in addition to previous reports, provides evidence of fertility in PWS women. We recommend the provision of information regarding fertility in females with PWS to parents, guardians and individuals with PWS, and frequent EEG monitoring for early AS diagnosis. Given the different genetic aetiologies for PWS and AS, cytogenetic and molecular genetic analysis is strongly indicated for counselling and risk estimation.

False teeth and real suffering: the social course of 'germectomy' in eastern Uganda.

The removal of the incipient canine teeth ('germectomy') in small babies is a practice carried out in many parts of eastern Africa. This article describes how 'germectomy' among the Jop'Adhola in Eastern Uganda is an important idiom of distress, referred to as false teeth by English speaking people, and lakijo marach (bad teeth) or gira kwanya (that which is removed) in the local language Dhop'Adhola. Through an analysis of how the notion of false teeth is shaped by macro social forces of war and poverty as well as by negotiations within the local social world, the discussion is taken beyond the question of cultural belief. False teeth as a practice seems to have spread through vast geographical areas within a few decades, but as the example of the Jop'Adhola shows, it has taken a particular social course in eastern Uganda--as it is most likely to also have done everywhere else it has gained a footing. By analyzing its social course we may gain insight into important mediating social processes which may have as much to do with actual health outcome in a particular area as health care per se.

Occurrence of plastids in rye (Poaceae) sperm cells.

Studies using classic genetics as well as restriction fragment length polymorphism analysis have demonstrated that rye, unlike most flowering plants, has biparental inheritance of both plastids and mitochondria. Yet, a previous in-depth ultrastructural study found no plastids in rye sperm cells, and DNA-specific staining revealed no cytoplasmic DNA in the male gametes of this plant. In the present study, we examined serial ultrathin sections of eight rye sperm cells (four pairs) and found unambiguous examples of plastids in all cases. The number of plastids per sperm cell varies from two to 12. The sperm of a pair may vary with regard to plastid number; however, these differences are not consistent among the sperm pairs examined.

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.

B chromosome behavior in maize pollen as determined by a molecular probe.

The B chromosomes of maize typically undergo nondisjunction during the second microspore division (generative cell division). When the microspore nucleus contains only one B chromosome, two kinds of sperm result, one with two B chromosomes and one with no B chromosomes. The sperm with the B chromosomes preferentially fertilizes the egg cell. Previous studies of these phenomena have been limited to genetic analysis and chromosome spreads. In this study we show that a B chromosome-specific probe can be used with fluorescence in situ hybridization (FISH) analysis to detect the presence, location, and frequency of B chromosomes in intact interphase nuclei within mature pollen of maize. Using genetic line TB-10L18, our results indicate that nondisjunction of the B centromere occurs at an average frequency of 56.6%, based on four plants and 1306 pollen grains analyzed. This is consistent with the results of genetic studies using the same B-A translocation. In addition, our results suggest that B chromosome nondisjunction can occur during the first microspore division. Spatial distribution of the B chromosome-specific probe appears to be largely confined to one tip of the sperm nucleus, and a DNA fragment found outside the pollen nuclei often hybridizes to the B chromosome-specific probe.

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.

The narrative of AIDS among the Tonga of Zambia.

The Tonga of Southern Zambia usually refer to a traditional disease, kahungo, when talking about AIDS. Such an association of AIDS with a traditional disease could easily be interpreted as a cultural obstacle to an understanding of AIDS and thus to a change of behaviour. However, a close investigation shows that this association is not the result of categorical thinking, but rather of narrative logic. What people are actually articulating when they associate AIDS with kahungo is a narrative about order, disorder and respect for existing rules and values of the society. The paper investigates how the dynamic notion of narrative may help us to get a better understanding of how people work towards a shared understanding of a new disease, and the implications this may have for AIDS education. It is argued that such local versions of the "story about AIDS" should be taken seriously and that they may contain as much "truth" as the version of the "North" which is usually promoted in AIDS education. AIDS education should, rather than being a transfer of knowledge, be an exchange of narratives and an attempt to "set a story in motion", that hinders the spread of AIDS.

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.