Biomedical subjects

S Simsek

Publications and source records attributed to S Simsek.

30 records · Page 2Linked to original sources

PubMed1994

Human platelet antigen-1 (Zw) typing of fetuses by analysis of polymerase chain reaction-amplified genomic DNA from amniocytes.

Prenatal typing for the human platelet antigens-1 (HPA) permits identification of a fetus at risk for neonatal alloimmune thrombocytopenia (NAITP) in cases of HPA-1 incompatibility in which the father is heterozygous for the HPA-1a antigen. Diagnostic cordocentesis and phenotyping of the fetal platelets are used for this purpose. We applied allele-specific restriction enzyme analysis on polymerase chain reaction (PCR)-amplified DNA purified from amniocytes. This assays allows early second trimester typing for HPA-1 alleles. We were able to determine the genotype of three fetuses at risk. Iatrogenic fetal loss is lower with amniocentesis than with cordocentesis. Therefore, this technique is a welcome addition to the antenatal management of NAITP.

Alleles↗

PubMed1994

A new private platelet antigen, Groa, localized on glycoprotein IIIa, involved in neonatal alloimmune thrombocytopenia.

The serum of a Caucasian woman who gave birth to a child with neonatal alloimmune thrombocytopenia contained antibodies directed against a platelet antigen of the newborn. There was no incompatibility for the known platelet alloantigens HPA-1 to HPA-7 or for the private or low-frequency antigens Sra and Vaa, between the platelets of the parents. However, crossmatching with the serum of the mother and the platelets of the child and the father was strongly positive, suggesting a new platelet antibody specificity. To investigate the inheritance of the 'Groa' antigen involved, the available family members were tested in the platelet immunofluorescence test (PIFT) and the monoclonal antibody-specific immobilization of platelet antigens (MAIPA) assay. The Groa antigen was found to be inherited in an autosomal-codominant fashion. In the MAIPA, we localized the Groa antigen on the glycoprotein IIb/IIIa complex (alpha IIb beta 3). The GP IIb/IIIa localization was confirmed in immunoprecipitation studies. In Western blotting experiments, we further localized the Groa antigen on the GP IIIa (beta 3) subunit of the GP IIb/IIIa complex. Until now we have tested approximately 400 unrelated donors. None of these appeared to be positive for the Groa antigen, suggesting a phenotype frequency in the Dutch population of less than 0.01.

Adult↗

PubMed1994

Sequence analysis of cDNA derived from reticulocyte mRNAs coding for Rh polypeptides and demonstration of E/e and C/c polymorphisms.

RNA derived from enriched reticulocytes of Rh-phenotyped donors was isolated, reversely transcribed into cDNA and amplified with Rh-specific primers by polymerase chain reaction. Nucleotide sequence analysis of the entire coding region of the Rh cDNAs was carried out. Four types of cDNAs were identified, tentatively designated as RhSCI, RhSCII, RhSCIII and RhSCIV. Comparison of RhSCII with RhSCI (identical to the previously reported RhIXb/30A cDNA), showed single base pair difference. Since RhSCI and RhSCII were found to be related to the presence of E or e antigen, respectively, the P226A amino acid polymorphism appears to be the genetic basis of the E/e polymorphism. RhSCIII was demonstrated to be a transcript derived from the RhD gene, with 35 amino acid substitutions as compared to RhSCI. RhSCIV was found to be present only in RhC-positive individuals, indicating that RhSCIV encodes a polypeptide carrying the C antigen. Six nucleotide changes, resulting in four amino acid substitutions W16C, L60I, N68S and P103S, were observed between RhSCII and RhSCIV, probably representing the C/c polymorphism.

Amino Acid Sequence↗

PubMed1994

Molecular genetics of human platelet antigens.

The purpose of this article is to review the molecular genetics of human platelet antigens, the application of molecular biological techniques to detect mutations underlying polymorphisms and the importance of these techniques for clinical medicine of immune-mediated platelet destruction. Review articles, original papers and preliminary (unpublished) observations from our own laboratory are the main source for this article. The nomenclature and phenotype frequency of the platelet alloantigens in different ethnic groups are described. Recent molecular biological advances are also reviewed. It appears that the human platelet antigen systems are due to single base pair substitutions. These mutations create or are responsible for the loss of a target site for a restriction enzyme in one of the alleles. Thus, DNA typing by polymerase chain reaction and subsequently allele-specific restriction enzyme analysis (PCR-ASRA) can be performed.

Antigens, Human Platelet↗

PubMed1994

Identification of a homozygous single base pair deletion in the gene coding for the human platelet glycoprotein Ib alpha causing Bernard-Soulier syndrome.

Bernard-Soulier Syndrome (BSS) is a hereditary bleeding disorder which is caused by the absence or the dysfunction of the platelet glycoprotein Ib/IX/V (GP Ib/IX/V) complex, the major receptor for von Willebrand factor (vWf). BSS is characterized by the presence of giant platelets that show a reduced binding of vWf. Although BSS is a well-characterized disease, and many cases have been described in the literature, the molecular genetic basis of this disorder has been studied in only a few patients. We have studied the genetic basis of the defect in a BSS patient. Flow cytometric analysis of the platelet membrane glycoproteins revealed a significant decrease or absence of GP Ib alpha on the platelet surface, and low levels of GP V and GP IX. In subsequent immunoprecipitation experiments, we confirmed the presence of GP V (although in significantly decreased amounts) on the platelet surface. These results indicated a defect in the GP Ib alpha chain. Genomic DNA coding for GP Ib alpha was amplified, using the polymerase chain reaction (PCR). Subsequent direct sequence analysis demonstrated a homozygous deletion of T317 resulting in a frameshift deletion and predicting a substitution of Arg for Leu76. This deletion causes a shift in the reading frame, predicting a premature stop codon after 19 altered amino-acids, leading to a severily truncated molecule. The molecular genetic defect found in this patient differed from the mutations observed in three other BSS patients described in the literature. This points to a marked hetereogeneity of this disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Association of a variable number of tandem repeats (VNTR) in glycoprotein Ib alpha and HPA-2 alloantigens.

The human platelet alloantigen HPA-2(Koa/Kob) system is involved in two clinical syndromes, neonatal alloimmune thrombocytopenia and platelet transfusion refractoriness. We have previously described that the human platelet alloantigens HPA-2a(Kob) and HPA-2b(Koa), are caused by a Thr145Met amino acid polymorphism in the N-terminal globular domain of the human platelet glycoprotein (GP) Ib alpha. In the present study the question was addressed as to whether a genetic association exists between this Thr145Met polymorphism and the recently described variable number of tandem repeat (VNTR) polymorphism in GP Ib alpha. Such an association has already been suggested by serological analysis (Ishida et al., 1991). This VNTR polymorphism results from a 13-amino-acid sequence repeat in the macroglycopeptide region of GP Ib alpha. Therefore, we developed a PCR method to analyze the VNTR region of 106 normal individuals who were also analyzed for the HPA-2 polymorphism. In this method genomic DNA derived from mononuclear cells was purified, the polymorphic region was amplified by PCR and was electrophoresed on agarose gels. Differences in the size of the PCR products made VNTR typing possible. Genotyping for the HPA-2 system was done by allele-specific restriction site analysis of PCR products with the restriction enzyme Sfa NI. The DNA derived from 12 HPA-2(a-b+) subjects, contained only the B variant (with 3 repeats) of the VNTR polymorphism. The D variant (with 1 repeat) was only found in HPA-2a positive individuals. The C variant (with 2 repeats) was found to be strongly associated with HPA-2a. However, two members of a family with a HPA-2(a+b+) genotype were found to be homozygous for the C variant of the VNTR polymorphism. This shows that the C variant can also be associated with HPA-2b. The A variant (with 4 repeats) was not encountered in the population studied. The strong association of HPA-2 and VNTR polymorphism, lying 761 bp apart on the GP Ib alpha gene, indicates linkage disequilibrium.

Alleles↗

PubMed1993

Glanzmann's thrombasthenia caused by homozygosity for a splice defect that leads to deletion of the first coding exon of the glycoprotein IIIa mRNA.

Glanzmann's thrombasthenia (GT) is the result of the absence or of an altered and dysfunctional expression on the platelet membrane of the fibrinogen receptor (glycoprotein [GP] IIb/IIIa complex). Various molecular genetic mechanisms have been found to be responsible for this inherited disease. In a patient with a severe type of GT, we have found a splice variant in the GP IIIa gene that leads to premature chain termination. Immunoprecipitation experiments, using monoclonal antibodies specific for GP IIb/IIIa, showed that GP IIb/IIIa was not detectable on the platelet membrane. Amplification of reversely transcribed platelet GP IIIa mRNA by the polymerase chain reaction and subsequent sequence analysis showed a 86-bp deletion, which corresponds to exon i of the GP IIIa gene. This deletion results in a shift of the reading frame leading to eight altered amino acids followed by a premature termination codon. Analysis of the corresponding genomic DNA fragments showed three mutations in the exon i-intron i boundary region of the GP IIIa gene. One of these mutations is a G-->T transition that eliminates the GT splice donor site in the wild type. This base pair change creates a restriction site for the enzyme Mse I. Allele-specific restriction enzyme analysis (ASRA) with Mse I of amplified genomic DNA of the parents and the proposita showed that both parents (who are first cousins) are heterozygous, whereas the proposita is homozygous for the G-->T substitution.

Adult↗

PubMed1993

Determination of human platelet antigen frequencies in the Dutch population by immunophenotyping and DNA (allele-specific restriction enzyme) analysis.

Platelets from 200 random Dutch blood donors were typed for the human platelet alloantigens HPA-1 to -5 recognized at present and for Naka. Naka is an epitope on glycoprotein IV, not expressed on the platelet of individuals with hereditary GP IV deficiency. Platelet immunofluorescence and monoclonal antibody-specific immobilization of platelet antigens (MAIPA) were applied for this purpose. The observed phenotype frequencies were 97.86% and 28.64% for HPA-1a and -1b, 100% and 13.15% for HPA-2a and -2b, 80.95% and 69.84% for HPA-3a and -3b, 100% and 0% for HPA-4a and -4b, 100% and 19.7% for HPA-5a and HPA-5b, respectively. Platelets from all donors reacted with the anti-Naka antibodies. To determine the gene frequencies for the HPA-1, HPA-2 and HPA-3 systems directly, DNA from 98 of these donors was isolated from peripheral blood mononuclear leucocytes and specific fragments were amplified by polymerase chain reaction (PCR). The fragments were analyzed using allele-specific restriction enzymes (ASRA). In all amplified PCR products an "internal control" for each assay, ie, a restriction site for the applied enzyme independent from the phenotype of the donor was present. In all donors tested, phenotypes, as determined by serological methods and genotypes, directly determined by the ASRA, were identical. Thus, the PCR-ASRA described in this report is a practical and reliable technique for the determination of alleles that code for platelet antigen allotypes, at least in the Dutch population.

Alleles↗

PubMed1993

Single point mutation in human glycoprotein IIIa is associated with a new platelet-specific alloantigen (Mo) involved in neonatal alloimmune thrombocytopenia.

Here we describe a new platelet-specific alloantigen that was identified in a case of neonatal alloimmune thrombocytopenia. This antigen has provisionally been called "Mo." By studying the Mo family, it was shown to be inherited in an autosomal dominant manner. Immunoprecipitation and Western blot analysis showed that the antigen resides on platelet glycoprotein IIIa (GP IIIa). Genomic analysis, performed by applying polymerase chain reaction and sequencing, showed a C-->G substitution of base pair 1267 of the coding region of the DNA for GP IIIa, resulting in a substitution of Proline407 by Alanine407. That this substitution is associated with the antigen could be demonstrated by restriction fragment length polymorphism analysis of cDNA, prepared from platelet RNA, and of genomic DNA. It was confirmed by dot-blot hybridization with allele-specific oligonucleotides. All family members, also those being Mo antigen-positive, were healthy. None of them appeared to suffer from increased tendency of bleeding or thrombosis. Thus, the Mo mutation does not lead to significant platelet dysfunction in vivo with heterozygous carriers. One of 450 random healthy blood donors who were tested was positive for the Mo antigen. Typing was performed by the classical serologic methods as well as by DNA analysis.

Base Composition↗

PubMedDate not supplied

Effect of sex in branch retinal vein occlusion.

PURPOSE: To evaluate the effect of sex in relation to crossing numbers, arterial overcrossings and occlusion sites in patients with branch retinal vein occlusion (BRVO). METHODS: Fifty-one eyes with BRVO (study group) and 48 eyes with retinal problems other than BRVO (control group) were investigated. All venous occlusions were examined in detail with regard to their anatomical positions, sites and arteriovenous crossings on the retinal surrounding area two disc diameters nasally, four disc diameters temporally, three disc diameters superiorly and three inferiorly from the optic disc, in the arteriovenous phase of fundus fluorescein angiography and fundus color photography. RESULTS: The study group comprised 29 women (56.86%) and 22 men (43.14%). All occlusions were unilateral and located at the crossing areas. BRVO had arterial overcrossing in 28 eyes (96.6%) in females and 22 eyes (95.5%) in males (p>0.005). The average numbers of crossings were 7.79 +/- 1.57 in females and 7.82 +/- 1.05 in males in the study group (p>0.05), and respectively 7.30 +/- 1.19 and 6.95 +/- 1.13 (p>0.05) in the control group. The average arterial overcrossing ratios were 75% in females and 71% in males in the study group (p>0.05), and 74% and 65% in the control group (p<0.05). CONCLUSIONS: Our data suggest that females have a higher risk than males because of their arterial overcrossing ratio and BRVO prefer arterial overcrossing. However the insignificant difference between the female and male patients leads us to assume that the effect of sex on BRVO cannot be explained only by local anatomical factors since their effect is only slight.

Adult↗

PubMedDate not supplied

Intraoperative application of mitomycin C in the surgical treatment of pterygium.

PURPOSE: To investigate the effectiveness of intraoperative mitomycin C in pterygium surgery. METHODS: The effectiveness of intraoperatively administered mitomycin C and the occurrence of postoperative complications were evaluated in 17 patients with two recurrences of pterygium. The authors employed the "bare-sclera technique" and placed a sterile sponge soaked in a 0.02% mitomycin C solution intraoperatively in the episcleral space for 3 minutes. The control group (15 patients) underwent only surgical excision. Patients were followed for 21 to 30 months. RESULTS: The pterygium recurred in one (5.9%) of the 17 patients in group 1 and in six (40%) of the 15 controls. Statistical analysis using Fisher's exact test showed a significant (p = 0.027) reduction of recurrences of pterygium in the group treated intraoperatively with mitomycin C. No serious complications or side effects arose during the follow-up period. CONCLUSIONS: Mitomycin C administered intraoperatively can be considered an effective treatment to improve the success rate after surgical excision.

Adult↗

PubMedDate not supplied

The effect of 0.25% apraclonidine in preventing intraocular pressure elevation after Nd:YAG laser posterior capsulotomy.

PURPOSE: The efficacy and adverse effects of 0.25% apraclonidine on intraocular pressure (IOP) after Nd:YAG laser posterior capsulotomy were investigated, and the results were compared with placebo, 0.50% timolol maleate and 1% apraclonidine. METHODS: Eighty eyes were randomly assigned to four groups of 20 eyes. In a double-masked design, the groups were treated with placebo (group 1), 0.50% timolol maleate (group 2), 1% apraclonidine (group 3), 0.25% apraclonidine (group 4) one hour before and five minutes after Nd:YAG laser posterior capsulotomy. IOP was measured by applanation tonometry 1 hour before (baseline IOP) and 1, 3, 24 hours after capsulotomy. RESULTS: The average baseline IOP increased respectively 3.90 +/- 5.35, 5.95 +/- 5.32, 1.15 +/- 3.20 mmHg in the first group 1, 3 and 24 hours post-treatment. There were significant differences between baseline IOP and 1 and 3 hours but not at 24 hours (p = 0.004, p = 0.001, p = 0.13). IOP increased 0.40 +/- 4.08, 0.75 +/- 5.33, 0.80 +/- 6.03 mmHg in the second group at the same times. The differences between the average baseline IOP and the 1, 3 and 24 h measurement were not significant (p = 0.83, p = 0.65, p = 0.93). In the third group, IOP decreased 3.70 +/- 2.40, 3.30 +/- 2.47, 2.65 +/- 1.56 mmHg at the measurement times, with significant differences between the average baseline IOP and the 1, 3 and 24 hour measurements (p = 0.001, p = 0.0001, p = 0.01). In the fourth group IOP increased 0.35 +/- 3.32 mmHg at 1 hour, but decreased 1.25 +/- 3.41, 0.90 +/- 2.07 mmHg at 3 and 24 hours. The differences were not significant (p = 0.94, p = 0.16, p = 0.08). When the 0.25% and 1% apraclonidine groups were compared, there were significant differences between the average IOP at 1 hour in both groups but not at 3 and 24 hours (p = 0.01, p = 0.17, p = 0.21). Similarly, there were no significant differences between the average IOP at the same times when the 0.25% apraclonidine group was compared with the timolol group (p = 0.30, p = 0.08, p = 0.16). Some systemic and local side effects were seen in the timolol and 1% apraclonidine groups, but none with 0.25% apraclonidine. CONCLUSIONS: It was concluded that 0.25% apraclonidine is effective in preventing the early elevation of IOP after Nd:YAG laser posterior capsulotomy and may offer an alternative to 0.50% timolol maleate and 1% apraclonidine.

Adrenergic alpha-Agonists↗