Investigation of the impact of biogeographic ancestry on DNA methylation based age predictions comparing a Middle East and a Central European population.

DNA methylation based age prediction is a new method in the toolbox of forensic genetics. Typically, the method is applied in the course of police investigation e.g. to predict the age of an unknown person that has left a biological trace at a crime scene. The method can also be used to answer other forensic questions, for example to estimate the age of unknown human bodies in the course of the identification process. In the present study, we tested for a potential impact of biogeographic ancestry (BGA) on age predictions using five age dependent methylated CpG sites within the genetic regions of ELOVL2, MIR29B2CHG, FHL2, KLF14 and TRIM59. We collected 102 blood samples each from donors living in Iraq, Middle East (ME) and Germany, Central Europe (EU). Both sample sets were matched in sex and age ranging from 18 to 68 years with exactly one male and female sample per year of age. All samples were analyzed by bisulfite pyrosequencing applying a multiplex pre-amplification strategy based on a single input of 35 ng converted DNA in the PCR. For the CpGs in MIR29B2CHG, FHL2 and KLF14, we observed significantly different methylation levels between the two populations. While we were able to train two highly accurate prediction models for the respective population with mean absolute deviations between predicted and actual ages (MAD) of 3.34 years for the ME model, and 2.72 years for the EU model, we found an absolute prediction difference between the two population specific models of more than 4 years. A combined model for both populations compensated the methylation difference between the two populations, providing MADs of prediction of only 3.81 years for ME and 3.31 years for EU samples. In total, the results of the present study strongly support the benefit of BGA information for more reliable methylation based age predictions.

Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine.

BACKGROUND: Mesenchymal stromal cells (MSCs) are promising cell therapy candidates. Clinical application is considered safe. However, minor side effects have included thromboembolism and instant blood-mediated inflammatory reactions suggesting an effect of MSC infusion on hemostasis. Previous studies focusing on plasmatic coagulation as a secondary hemostasis step detected both procoagulatory and anticoagulatory activities of MSCs. We now focus on primary hemostasis and analyzed whether MSCs can promote or inhibit platelet activation. METHODS: Effects of MSCs and MSC supernatant on platelet activation and function were studied using flow cytometry and further platelet function analyses. MSCs from bone marrow (BM), lipoaspirate (LA) and cord blood (CB) were compared to human umbilical vein endothelial cells or HeLa tumor cells as inhibitory or activating cells, respectively. RESULTS: BM-MSCs and LA-MSCs inhibited activation and aggregation of stimulated platelets independent of the agonist used. This inhibitory effect was confirmed in diagnostic point-of-care platelet function analyses in platelet-rich plasma and whole blood. Using inhibitors of the CD39-CD73-adenosine axis, we showed that adenosine produced by CD73 ectonucleotidase activity was largely responsible for the LA-MSC and BM-MSC platelet inhibitory action. With CB-MSCs, batch-dependent responses were obvious, with some batches exerting inhibition and others lacking this effect. CONCLUSIONS: Studies focusing on plasmatic coagulation suggested both procoagulatory and anticoagulatory activities of MSCs. We now show that MSCs can, dependent on their tissue origin, inhibit platelet activation involving adenosine converted from adenosine monophosphate by CD73 ectonucleotidase activity. These data may have strong implications for safety and risk/benefit assessment regarding MSCs from different tissue sources and may help to explain the tissue protective mode of action of MSCs. The adenosinergic pathway emerges as a key mechanism by which MSCs exert hemostatic and immunomodulatory functions.

Human platelet antigen polymorphisms (HPA-1, -2, -3, -4, -5 and -15) in major ethnic groups of Pakistan.

OBJECTIVES: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA-typed platelet donors in a given community. BACKGROUND: However, the pattern of HPA in Pakistani population is not known. AIM: The aim of present study was to determine the gene frequencies of HPA (HPA-1 to -5 and -15) in individuals belonging to major ethnic groups and castes of Pakistani population. MATERIALS AND METHODS: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction-sequence specific primers with detection on polyacrylamide electrophoresis. RESULTS: The gene frequencies of the 'a' and 'b' alleles of HPA-1 to -5 and -15 in Pakistanis were as follows: HPA-1a/b, 0.885/0.115; HPA-2a/b, 0.92/0.08; HPA-3a/b, 0.69/0.31; HPA-4a/b, 1/0; HPA-5a/b, 0.9/0.1; HPA-15a/b, 0.59/0.41. Except for significant difference regarding gene frequency of HPA-3 between Pathans and Sindhis, there was no significant difference of HPA-1 to -5 and -15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1-5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti-HPA-1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8-12 of 1000 in case of anti-HPA-5b. Homozygosity of HPA-1b, -2b and -5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA-3b and -15b was 11 and 18%. CONCLUSIONS: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.

[Clinical and laboratory aspects of the Aspirin-like defect as hereditary thrombocytopathy]. / Klinische und labordiagnostische Aspekte des Aspirin-like Defekts als hereditäre Thrombozytopathie.

UNLABELLED: The Aspirin-like defect (ALD) is caused by defects in the intraplatelet arachidonic acid (AA)-metabolism. We here present the characteristics of a larger cohort in a single centre. PATIENTS, METHODS: Based on 17 ALD index patients bleeding symptoms, agonist-induced platelet aggregation and closure times in the PFA-100 test were analysed in a family cohort of altogether 52 individuals from 17 families. Absent aggregation to AA (maximal aggregation or=1 bleeding symptoms. CONCLUSION: In case of a bleeding tendency diagnostic procedures should rule out primary haemostatic defects. Hereditary platelet function defects including ALD are an important differential diagnosis. Family studies are reasonable.

Molecular basis of the Rh antigen RH48 (JAL).

BACKGROUND AND OBJECTIVES: RH48 (JAL) is a low-incidence Rh antigen of unknown molecular background associated with weakened expression of RhCE antigens. The objective of this study was to establish the molecular basis of JAL. MATERIALS AND METHODS: Seventeen JAL+ samples, from seven black (one of them a Brazilian of mixed race: black/Caucasian), nine European Caucasians and one Asian individuals, were typed with anti-D, -C, -c, -E and -e. Some samples were also tested for V/VS and ce (f). Titration studies and flow cytometry were used to analyse the expression of the JAL antigen and genomic DNA sequencing of all RHCE exons was conducted on all samples. Routine genotyping for RHCE was carried out on all samples. Screening of RHD exons 1-10, which included detection of the DAU allele, was carried out on all except one of the black samples. The Caucasian samples and remaining black sample were screened for the DAU mutation 1136C>T (T379M). RESULTS: Six black individuals had the Dce haplotype with RHCE mutations 340C>T (R114W) and 733C>G (L245V) [V/VS] and the RHD mutation T379M [DAU]. One mixed race individual had the Dce haplotype with the RHCE mutation 340C>T (R114W) but without the V/VS or DAU mutation. Eight Caucasians had the DCe haplotype with the 340C>T mutation. One Caucasian and one Asian had the Dce haplotype with a different mutation in an adjacent nucleotide, 341G>A (R114Q). All Caucasian individuals were negative for the DAU mutation 1136C>T (T379M). Previously described weakness of CE-related Rh antigens when present in single dose on JAL+ samples of DCe and Dce haplotypes was observed. Weak expression of V/VS was observed in the three black samples tested and weakness of JAL was observed in the black samples compared to the Caucasian samples. CONCLUSION: The same mutation (340C>T, R114W) in two different haplotypes (DCe and Dce) and another mutation (341G>A, R114Q) in one of these haplotypes (Dce) are associated with expression of the JAL antigen. One of the RHCE mutations detected in our samples (340C>T) has been previously described but not in association with the JAL antigen. Our results indicate that the previously described RhCeMA and ce(s)(340) alleles encode the JAL antigen. Expression of V/VS antigen is weakened in the presence of JAL and expression of JAL is usually weaker when associated with the Dce haplotype compared to DCe.

Introduction of a validation concept for a PCR-based Mycoplasma detection assay.

BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

Effect of R-(+)-alpha-lipoic acid on experimental diabetic retinopathy.

AIMS/HYPOTHESIS: Hyperglycaemia-induced mitochondrial overproduction of reactive oxygen species (ROS) is central to the pathogenesis of endothelial damage in diabetes. R-(+)-alpha-lipoic acid has advantages over classic antioxidants, as it distributes to the mitochondria, is regenerated by glycolytic flux, and has a low redox potential. METHODS: To assess the effect of R-(+)-alpha-lipoic acid on experimental diabetic retinopathy, three groups of male Wistar rats were studied: non-diabetic controls, untreated diabetic controls, and diabetic rats treated with 60 mg/kg bodyweight R-(+)-alpha-lipoic acid i.p. for 30 weeks. Quantitative retinal morphometry included acellular occluded capillaries and pericyte numbers. The effects of R-(+)-alpha-lipoic acid on parameters of oxidative and nitrative stress, AGE and its receptor and nuclear factor kappa B (NFkappaB) were assessed by immunoblotting, and NFkappaB activation by electrophoretic mobility shift assay. Angiopoietin-2 and vascular endothelial growth factors were also determined by immunoblotting. RESULTS: After 30 weeks of diabetes, the number of acellular capillaries was significantly elevated in diabetic rats (57.1+/-10.6 acellular capillary segments [ac]/mm(2) of retinal area) compared with non-diabetic (19.8+/-5.1 ac/mm(2); p<0.001). Treatment with 60 mg/kg R-(+)-alpha-lipoic acid reduced the numbers by 88% (p<0.001 vs diabetic). Pericyte loss was also significantly inhibited in diabetic rats treated with R-(+)-alpha-lipoic acid (non-diabetic: 1,940+/-137 pericytes/mm(2)capillary area; untreated diabetic: 1,294+/-94 pericytes/mm(2)capillary area vs treated diabetic: 1,656+/-134 pericytes/mm(2); p<0.01). R-(+)-alpha-lipoic acid treatment reduced oxidative stress, normalised NFkappaB activation and angiopoietin-2 expression, and reduced vascular endothelial growth factor in the diabetic retina by 43% (p<0.0001). CONCLUSIONS/INTERPRETATION: R-(+)-alpha-lipoic acid prevents microvascular damage through normalised pathways downstream of mitochondrial overproduction of ROS, and preserves pericyte coverage of retinal capillaries, which may provide additional endothelial protection.

[A comparison of the gene expression patterns of human chondrocytes and chondrogen differentiated mesenchymal stem cells for tissue engineering]. / Vergleich der Genexpressionsmuster humaner Chondrozyten und chondrogen differenzierter mesenchymaler Stammzellen für das Tissue-Engineering.

BACKGROUND: Tissue engineering is a promising method for the generation of chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. METHODS: In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) harvested during septoplasty and human mesenchymal stem cells (hMSC) from cartilage biopsies in cell culture using the microarray technique. RESULTS: The genes for collagen 1alpha1, 2alpha1, 3alpha1, 4alpha1, 11alpha1, biglycan, fibromodulin and lumican were activated during the dedifferentiation of the HCs, collagen 9alpha2, 9alpha3, 10alpha1 and chondroadherin were inactivated. During chondrogenic differentiation of hMSCs, the genes for collagen 3alpha1, 9alpha2, 9alpha3, 10alpha1, 11alpha1 were activated, collagen 4alpha1 and fibromodulin inactivated and the genes for Col 1alpha1, biglycan und chondroadherin constantly expressed. CONCLUSION: The genetic profile for the investigated markers in human chondrocytes generated from hMSCs resembles the profile in differentiated chondrocytes. Collagen 2alpha1, 9alpha2, 9alpha3, 10alpha1 could represent markers for the differentiation of chondrocytes, Col 1alpha1, 3alpha1 und 4alpha1, biglycan, fibromodulin and lumican markers for the dedifferentiation into a more fibroblastoid cell type.

Effect of the psoralen-based photochemical pathogen inactivation on mitochondrial DNA in platelets.

Photochemical treatment (PCT) of platelet concentrates, using amotosalen HCl and UVA-light, inactivates pathogens by forming adducts between amotosalen and nucleic acids. The impact of the photochemical treatment on pathogens and leukocytes has been studied extensively. Yet little is known about the effect of PCT on nucleic acids in platelets. Platelets contain viable mitochondria and mitochondrial DNA (mtDNA) and this study aimed at evaluating the amotosalen modifications on platelet mtDNA. We applied two independent but complementary molecular assays to investigate qualitative as well as quantitative aspects of the psoralen-mediated DNA modifications in platelet mtDNA. The amotosalen-DNA modification density was measured using (14)C-labeled amotosalen. Amotosalen (150 microM) yielded 4.0 +/- 1.2 psoralen adducts per 1,000 bp in mtDNA after irradiation with 3 J/cm(2) UVA. Furthermore, we tested if the PCT-induced DNA modifications could be detected by a PCR assay. On the basis of PCR inhibition due to amotosalen-DNA adducts, mtDNA-specific PCR assays were developed and tested for their specificity and sensitivity. Our data revealed that mtDNA in platelets is substantially modified by PCT and that these modifications can be documented by a PCR inhibition system.

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.

Is prenatal HLA typing of uncultured amniocytes before the collection of related allogenic cord blood helpful?

The collection of related allogenic cord blood is gaining increasing importance in families with one child affected by haematopoietic disease. Within a family, there is only a 25% chance of a full HLA match between siblings. 50% of all collected cord blood samples cannot be used because of poor quality. Because of this, the determination of HLA type is useful for planning the collection of related allogenic cord blood transplants. We studied whether HLA typing is possible during late pregnancy if amniocentesis has not been performed during the first trimester. HLA -A, -B and -DRB loci were detected in amniotic fluid, as well as in corresponding cord blood and maternal blood using PCR-SSP. For the first time, HLA typing was performed from uncultured amniocytes. Unambiguous results were obtained from all samples. Fetal HLA-genotype in amniotic fluid was confirmed by typing results from corresponding cord blood. HLA typing of uncultured amniocytes during late pregnancy is a reliable and fast method. For the first time, prenatal HLA typing by amniocentesis after week 38 of gestation is possible in less than 8 h and without fetal risk.

Use of recombinant human deoxyribonuclease (DNase) for processing of a thawed umbilical cord blood transplant in a patient with relapsed acute lymphoblastic leukemia.

This case report describes for the first time the use of a recombinant human enzyme deoxyribonuclease (rhDNase) containing solution for the processing of a thawed umbilical cord blood (UCB) unit prior to successful transplantation to avoid cell losses by clotting phenomena. A 6-year-old boy received an unrelated 2/6 HLA antigen mismatched UCB transplant for high-risk Burkitt type acute lymphoblastic leukemia. The UCB unit was provided as a volume-reduced sample after buffy coat separation with a final volume of 36 ml. To avoid the loss of nucleated cells due to cell clumping during thawing procedure cells were washed with a solution containing the rhDNase. No visible clotting of the resuspended unit occurred, and the patient was transplanted with 2.9x10(7) nucleated cells/kg body weight without any acute or chronic side effects due to rhDNase. On day +35, PCR analysis of bone marrow aspirate showed complete chimerism, and the child engrafted with an absolute neutrophil count greater than 0.5x10(9)/l on day +47. Platelet transfusion independence was achieved on day +120. In conclusion, the supplementation of rhDNase to the washing and resuspension solutions of a thawed UCB unit is effective to prevent cell losses prior to transplantation. However, further investigations must be performed to confirm the safety of this procedure.

Prenatal HLA typing of uncultured amniocytes prior to the collection of related allogeneic cord blood.

DNA samples isolated from corresponding uncultured amniotic fluid, cord blood and maternal blood (n=5) were subjected to low resolution typing of the HLA-A, -B and -DRB loci by the polymerase chain reaction using sequence-specific primers (PCR-SSP). Furthermore, the effect of ethylene diamine tetraacetate disodium salt (EDTA) on the quality of genomic DNA isolated from amniotic fluid samples after long-term storage was evaluated. Unambiguous results of HLA typing could be achieved from all amniotic fluid samples stabilized with EDTA. PCR-SSP typing failed in DNA samples from amniotic fluid without the addition of EDTA. In all cases the fetal HLA type could be confirmed by the result from the corresponding cord blood typing. Contamination with maternal DNA led to additional weak PCR-SSP bands in one case, but data interpretation was still unambiguous. Reliable fetal HLA typing can be achieved directly from amniotic fluid and culturing of amniocytes is not required.

Cloning a calcium channel alpha2delta-3 subunit gene from a putative tumor suppressor gene region at chromosome 3p21.1 in conventional renal cell carcinoma.

We have identified loss of heterozygosity (LOH) of approx. 1 cM region around locus D3S1289 at chromosome 3p21.1 in a conventional renal cell carcinoma (RCC). During construction of a YAC/BAC contig for this region and shotgun sequencing of BACs 277p5, 55m24 and 428i24, we detected four new microsatellites. We narrowed down the target region by analysing these new loci to less than 100 kb within the BAC 55m24 and subsequently cloned a human calcium channel alpha2delta-3 subunit gene. This gene is widely expressed in fetal tissues and different types of adult tumors. The exons of the alpha2delta-3 subunit gene are distributed along approx. 500 kb DNA sequences. As the LOH involved exclusively intronic sequences and sequencing the entire coding region did not reveal any mutation, the alpha2delta-3 subunit gene is probably not a tumor suppressor gene.

Characterization of a novel O(1) variant allele at the ABO blood group locus.

We describe the identification and molecular characterization of a novel variant O(1) allele of the ABO blood group locus. The allele was found in a young child and by analyzing the maternal DNA we were able to show that a meiotic recombination event between the maternal O(1v-3) and B(1-1) alleles recreated a O(1)/B hybrid allele. Further characterization of intron 6 sequences delineated the putative recombination breakpoint between nucleotide position 42 and 163 of the intron. We propose that the novel O(1variant) allele should be named O(1v-7) and is a combination of exon 6 from a O(1v-3) allele and exon 7 from a B(1-1) allele.

The tcf17 gene at chromosome 5q is not involved in the development of conventional renal cell carcinoma.

The human Kid-1 homolog Tcf17 has been cloned and assigned to chromosome 5q35.3. Since the chromosome 5q22-qter region is duplicated in approximately 50% of conventional renal-cell carcinomas, it was suggested that Tcf17 is involved in the development of renal tumors. We have analyzed Tcf17 mRNA in normal kidneys and genetically distinct types of renal-cell tumor and found it expressed in nearly all normal kidney and tumor samples. There was no correlation between allelic duplication and expression of Tcf17. We did not find mutations within the coding sequences but did detect deletions within the zinc finger domain in a small proportion of RNA molecules in both normal and tumor tissues. We found ubiquitous expression of human Tcf17 as well as rat Kid-1 in different types of human and rat tissue, indicating that the putative transcription-regulating activity of this zinc finger gene, in contrast to the published data, is not restricted to the kidney. The results of expression and mutation analyses suggest that the Tcf17 gene is not involved in the development of renal-cell tumors.