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N Morling

Publications and source records attributed to N Morling.

At least 37 records · Page 2Linked to original sources
PubMed1999

Detection of fetal-specific DNA after enrichment for trophoblasts using the monoclonal antibody LK26 in model systems but failure to demonstrate fetal DNA in maternal peripheral blood.

Trophoblast cells can be detected in maternal blood during normal human pregnancy and DNA from these cells may be used for non-invasive prenatal diagnosis of inherited diseases. The possibility of enriching trophoblast cells from maternal blood samples using a monoclonal antibody (LK26) against a folate-binding protein, which recognizes trophoblast in normal tissues, in conjunction with immunomagnetic cell sorting was investigated. Verification of the presence of fetal DNA in the sorted samples was done by detection of fetal/paternal-specific short tandem repeat (STR) alleles using polymerase chain reaction (PCR) and automated fluorescence-based genotyping. After successful initial experiments using retroplacental blood samples with a high number of trophoblast cells or an artificial mixture of trophoblast cells and blood, several versions of the enrichment method were attempted on peripheral maternal blood samples. However, it was not possible to detect fetal DNA sequences in these samples, most probably due to the extremely low number of trophoblast cells. Positive identification and retrieval of trophoblast cells in suspension or trophoblast nuclear material prepared on microscope slides after cell sorting procedures can be a solution to this problem.

Antibodies, Monoclonal↗
PubMed1999

Polymorphism in the regulatory region located more than 1.1 kilobases 5' to the start site of transcription, the promoter region, and exon 1 of the HLA-G gene.

The non-classic Human Leucocyte Antigen class Ib molecule, HLA-G, is expressed on the invasive, extra-villous cytotrophoblast in human placenta. HLA-G protects against natural killer (NK)-cell-mediated lysis and may modulate the secretion of cytokines. Aberrant expression of HLA-G has been reported in certain disorders of pregnancy. We have studied the DNA sequences of the putative regulatory region located more than 1.1 kilobases 5' from the start site of transcription (a 244 bp HindIII/EcoRI fragment) of the HLA-G gene and of the promoter region to detect any possible polymorphism. We detected one nucleotide substitution in the HindIII/ EcoRI region and one in the promoter region in the alleles G*01012, G*01013, G*0104, and G*0105N compared to G*01011. Several nucleotide substitutions were detected in the region between the 1.1 kb 5' regulatory region and the promoter region. Alleles can be divided into two groups based on the detected polymorphism. The nucleotide substitutions may have implications for the binding of nuclear factors to the regulatory regions. To our knowledge this is the first study of any polymorphism in the 5'-flanking sequences to the HLA-G gene. Further studies are needed to elucidate the exact impact of the detected polymorphism on levels or developmental regulation of HLA-G expression.

Base Sequence↗
PubMed1998

Report of the European DNA Profiling Group (EDNAP)--an investigation of the hypervariable STR loci ACTBP2, APOAI1 and D11S554 and the compound loci D12S391 and D1S1656.

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were asked to test 7 blood stains, one of which was a known control, and to report the results to the co-ordinating laboratory. The exercise demonstrated that ACTBP2 showed good reproducibility between laboratories, whereas further testing would be needed to validate APOAI1 and D11S554 for interlaboratory comparisons. In separate exercises, the simple loci D12S391 and D1S1656 were tested; both of these showed excellent reproducibility between laboratories.

Alleles↗
PubMed1998

Co-dominant expression of the HLA-G gene and various forms of alternatively spliced HLA-G mRNA in human first trimester trophoblast.

Genes may be silenced at the transcriptional level by 'genomic imprinting' in such a way that only one of the parental alleles is expressed. Imprinting may be tissue-specific and in some cases it seems also to be time-dependent during development. The phenomenon has been studied in pre- and post-implantation developmental processes. Animal studies of genomic imprinting of major histocompatibility complex (MHC) antigens in the placenta have shown discordant results. To address this issue in the human placenta, we examined the expression of the non-classical human leukocyte antigen (HLA) class I gene, HLA-G. Genomic imprinting of the HLA-G locus could have implications for the interaction in the feto-maternal relationship. Restriction Fragment Length Polymorphism (RFLP), allele-specific amplification and Single Strand Conformation Polymorphism (SSCP) analysis followed by DNA sequencing were performed on Reverse Transcription (RT) Polymerase Chain Reaction (PCR) products of HLA-G mRNA to examine the expression of maternal and paternal alleles. Our results demonstrate that HLA-G is co-dominantly expressed in first trimester trophoblast cells. A "new" non-synonymous base substitution in exon 4 was detected. We also investigated the different alternatively spliced forms of HLA-G mRNA in first trimester trophoblast and found the full-length transcript to be the far most abundant.

Alleles↗
PubMed1997

A report of the 1995 and 1996 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Haemogenetics.

We report the results of the 1995 and 1996 Paternity Testing Workshops of the English Speaking Working Group of the International Society for Forensic Haemogenetics. In 1995, 18 laboratories participated and in 1996, 21 laboratories participated. Each year, blood samples from three persons (child, mother and alleged father) were sent to participating laboratories which performed paternity testing according to their usual protocols. The results and answers to questionnaires concerning methods were compiled and are presented in this report. From the questionnaires, a general tendency to a more frequent use of polymerase chain reaction (PCR) based methods was seen. In 1996, 62% of the laboratories used PCR based methods. Ten per cent of the laboratories used only PCR based methods. The remaining 90% of the laboratories performed restriction fragment length polymorphism (RFLP) investigations of variable numbers of tandem repeat (VNTR) loci with single locus probes (SLPs) either alone or in combination with PCR based typing, multi locus probing, classical systems (ABO etc.), or serological HLA typing. In 1996, typing with classical systems was used in 29% of the laboratories. The majority of the laboratories performed RFLP typing of VNTR loci using very similar methods. The results and the inter-laboratory variations of the measured lengths of the DNA-fragments of the VNTR regions D2S44, D7S21, D7S22, and D12S11 of the trios were analysed. The overall coefficient of variation was 2.15% in 1995 and 1.43% in 1996. During the period 1991-1996, the inter-laboratory variation has decreased, most probably due to the fact that the methods have now been optimised and the majority of the participating laboratories have adopted the standardised method for RFLP typing with SLPs which was agreed upon for investigations in crime cases by the European DNA Profiling Group. In 1996, eight laboratories reported the results of PCR based typing of the short tandem repeat (STR) locus HumTH01, six laboratories reported results of HumVWA31A typing, and five laboratories reported the results of typing of the STR locus HumF13A1 and the VNTR locus D1S80. The results were concordant although the nomenclature was slightly inconsistent concerning the classification of an irregular repeat of the HumTH01 system.

Blood Grouping and Crossmatching↗
PubMed1997

Polymorphism of exon 3 of the HLA-G gene.

HLA-G is a non-classical MHC class I gene with a limited tissue distribution. The most pronounced expression is detected in the cytotrophoblast of first trimester placenta. It is possible to detect mRNA for HLA-G in preimplantation blastocysts where expression is correlated with a high cleavage rate of embryos. HLA-G seems to play an important role in the feto-maternal relationship. The polymorphism of the HLA-G locus is not fully clarified. One study has shown extensive nucleotide sequence variation in the exon 3 (alpha-2 domain) in healthy African Americans. A few studies in other populations have only revealed a limited polymorphism. We investigated the polymorphism of the exon 3 of HLA-G by means of Polymerase Chain Reaction (PCR)-Single Strand Conformation Polymorphism (SSCP)- and DNA sequencing analysis in a Danish population. We detected four single-base substitutions in exon 3 compared to the sequence of HLA-6.0 (G*01011); one of these has not been reported before. We also found a deletion of the first base of codon 130 or the third of codon 129 in a heterozygous individual. This study, together with previous results, suggests that the polymorphism of exon 3 of the HLA-G gene in Caucasians is limited, in contrast to that observed in Americans originating from Africa. Implications of this discrepancy and the detected deletion in relation to certain disorders of pregnancy are discussed.

Alleles↗
PubMed1997

HLA typing in acute optic neuritis. Relation to multiple sclerosis and magnetic resonance imaging findings.

OBJECTIVE: To study the association of brain magnetic resonance imaging (MRI) findings and HLA findings to clarify the relationship between monosymptomatic optic neuritis (ON) and ON as part of clinically definite multiple sclerosis (CDMS). DESIGN: Population-based cohort of patients with ON referred prospectively during 6 years by neurologists and ophthalmologists within 4 weeks of onset of ON. SETTING: Referral center in the general community of greater Copenhagen (Denmark) (population, 1.5 million). PATIENTS: A consecutive sample of 199 patients aged 12 to 59 years with ON (133 with idiopathic ON, 66 with ON + CDMS), ethnically matched with 192 healthy volunteers. MAIN OUTCOME MEASURES: Relation between the HLA-DR15, -DR17, -DQA-1B, and -DQB-1B polymorphisms as defined by restriction fragment length polymorphism analysis, and presence of plaques on T2-weighted brain MRI. RESULTS: The frequency of HLA-DR15 was significantly increased in patients with ON + CDMS (52%) and ON (47%) compared with control subjects (31%). The frequency of HLA-DR17 was almost equal in the ON + CDMS (18%), ON (23%), and control (23%) groups. The frequencies of HLA-DQA-1B (55% in ON + CDMS, 58% in ON) and HLA-DQB-1B (49% in ON + CDMS, 59% in ON) were significantly increased compared with control subjects (41%, HLA-DQA-1B; 37%, HLA-DQB-1B). Brain MRI was abnormal in 48 of 56 examined patients with ON + CDMS and in 64 of 120 examined patients with ON (P < .001). In contrast, the frequencies of HLA alleles did not differ between patients with and without demyelinating lesions. However, patients with ON and normal MRI findings did not show association with HLA-DR15. CONCLUSIONS: The frequencies of alleles were similar in patients with ON and ON + CDMS, confirming that they are not 2 immunogenetically distinct disease entities. The heterogeneity within the group of patients with ON suggests that the HLA-DR15 molecule is involved in susceptibility to initial demyelinating lesion formation.

Acute Disease↗
PubMed1997

Detection and quantitative characterization of artificial extra peaks following polymerase chain reaction amplification of 14 short tandem repeat systems used in forensic investigations.

Detection on automated DNA sequencers of polymerase chain reaction (PCR) products of tetra- and penta-nucleotide short tandem repeat (STR) loci frequently reveals one or more extra peaks along with the true, major allele peak. The most frequent extra peak pattern is a single smaller peak which is one repeat unit shorter than the true allele peak. The existence of such artificial peaks is of special importance when the methods are used for forensic investigations because the artificial extra peaks may simulate true alleles when samples containing mixtures of DNA from different individuals are analyzed. We have investigated the relative levels of formation of extra peaks in 14 STR marker systems. We found that not only the parameters of the PCR but also factors determining the stringency during the post-PCR and pre-electrophoresis handling of samples were of importance for the formation of extra peaks. In our hands, the amounts of extra peaks were reduced (i) if the samples were effectively denatured immediately before loading, (ii) if they contained substantial amounts of formamide (i.e. > or = 50%), and (iii) if the temperature of the electrophoresis gel was above a certain level (i.e. > or = 43 degrees C). The results suggest that extra peaks may in part be due to re-annealing of the PCR product under suboptimal conditions. When efforts had been made to reduce the post-PCR formation of extra peaks, the relative peak areas of the extra peaks ranged from 1% to 17% of those of the true alleles. Similar results were obtained when the PCR products were analyzed under native conditions. Low-copy genome analysis excluded that somatic heterogeneity of the STR regions caused the extra peaks. The systems HumVWA31A, HumFibra/FGA, and D21S11 were especially affected by low-stringency conditions, while Hum-TH01, HumCD4, and D12S391 were virtually unaffected by low-stringency conditions. Replacement of the Taq DNA polymerase with DNA polymerases with lower processivity resulted in higher levels of extra peaks. Our results support the hypothesis that extra peaks are produced due to slipped-strand mispairing.

DNA↗
PubMed1997

Platelet alloimmunization after transfusion. A prospective study in 117 heart surgery patients.

BACKGROUND AND OBJECTIVES: The frequency of platelet-specific antibodies after one series of blood transfusions has not been reported, and in multiply transfused patients is controversial. MATERIALS AND METHODS: We studied the frequency of alloimmunization against platelet antigens in 117 patients who received a single series of blood transfusions. They received mostly saline-adenine-glucose+mannitol red blood cell components (poor in leukocytes and platelets) in connection with cardiac surgery. Platelet-specific antibodies were detected with the platelet ELISA and the monoclonal-antibody-specific immobilization of platelet antigen assay. HLA antibodies were detected by the standard lymphocyte cytotoxicity techniques. RESULTS: We found platelet-specific anti-HPA-5b (anti-Bra) in 2 cases (1.7%). One antibody was the result of de novo immunization. We detected lymphocytotoxic HLA antibodies in 21 patients (17.9%), of whom 18 (15.4%) had had no detectable antibodies before transfusion. There was a positive correlation between the transfused load of immunogenic materials and the frequency of alloimmunization against HLA antigens. In one third of the immunized patients, there was no history of previous immunization. CONCLUSION: There was a low incidence of platelet-specific antibodies after one series of blood transfusions in this group of patients. This is similar to the results of some previous studies in multiply transfused patients, but not with those of others who found a higher incidence.

Adolescent↗
PubMed1996

PCR typing of DNA fragments of the two short tandem repeat (STR) systems upstream of the human myelin basic protein (MBP) gene in Danes and Greenland Eskimos.

DNA from the double short tandem repeat (STR) system MBP (locus 18q23-pter) was amplified by the polymerase chain reaction (PCR) and the two polymorphic repeat systems were separated by cutting with the restriction enzyme NlaIII. The lengths of the DNA fragments of the two MBP STR systems MBP-A and MBP-B were analyzed by vertical electrophoresis in polyacrylamide gels followed by silver staining. DNA samples from 112 unrelated Danes, 140 unrelated Greenland Eskimos, and 88 Danish mother/child pairs were analyzed. The distributions of MBP phenotypes were in Hardy-Weinberg equilibrium in both the Eskimo and Danish populations. Significant differences were observed between the distribution of fragments ('alleles') in Greenland Eskimos and in Danes. The allele MBP-A7 was considerably more frequent in Eskimos (0.2214) than in Danes (0.0775) and also the allele MBP-B9 was considerably more frequent in Eskimos (0.225) than in Danes (0.06). Strong gametic associations were found between fragments from MBP-A and MBP-B series in both Danes and Eskimos. Some of the associations were different in Danes and Eskimos. In the 88 Danish mother/child pairs, the segregation of the MBP genotypes were in accordance with a genetic model of co-dominant inheritance and no mutation was found. Two MBP STR regions with irregular structures were sequenced. One fragment had a single base G to A transition at position 124 in the primer binding region between the MBP-A and MBP-B regions. In the other fragment, a deletion starting at position 117 and including the primer binding region between MBP-A and MBP-B regions was found.

Alleles↗