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S Simsek

Publications and source records attributed to S Simsek.

At least 55 records · Page 3Linked to original sources
PubMed1995

Rh E/e genotyping by allele-specific primer amplification.

It has been shown that the Rhesus (Rh) blood group antigens are encoded by two homologous genes: the Rh D gene and the Rh CcEe gene. The Rh CcEe gene encodes different peptides: the Rh C, c, E, and e polypeptides. Only one nucleotide difference has been found between the alleles encoding the Rh E and the Rh e antigen polypeptides. It is a C-->G transition at nucleotide position 676, which leads to an amino acid substitution from proline to alanine in the Rh e-carrying polypeptide. Here we present an allele-specific primer amplification (ASPA) method to determine the Rh E and Rh e genotypes. In one polymerase chain reaction, the sense primer had a 3'-end nucleotide specific for the cytosine at position 676 of the Rh E allele. In another reaction, a sense primer was used with a 3'-end nucleotide specific for the guanine at position 676 of the Rh e allele and the Rh D gene, whereas the antisense primer had a 3'-end nucleotide specific for the adenine at position 787 of the Rh CcEe gene. We tested DNA samples from 158 normal donors (including non-Caucasian donors and donors with rare Rh phenotypes) in these assays. There was full concordance with the results of serologic Rh E/e phenotyping. Thus, we may conclude that the ASPA approach leads to a simple and reliable method to determine the Rh E/e genotype. This can be useful in Rh E/e genotyping of fetuses and/or in cases in which no red blood cells are available for serotyping. Moreover, our results confirm the proposed association between the cytosine/guanine polymorphism at position 676 and the Rh E/e phenotype.

Alleles↗
PubMed1995

Actions of 1,4-dihydropyridines in isolated mesenteric vascular beds.

Recent studies indicate that the vasorelaxation induced by nitrendipine may be mediated partially by increased release of nitric oxide (NO). To study this effect in more detail and to examine the vasodilating effect of other 1,4-dihydropyridines (1,4-DHP) with regard to a possible involvement of NO, we investigated the effects of nitrendipine, nifedipine, nisoldipine, and nimodipine on isolated mesenteric vascular bed an the influence of L-NG-nitroarginine (L-NNA) on 1,4-DHP-induced vasorelaxation. Perfusion with these 1,4-DHP resulted in a concentration-dependent increase in global flow and vascular diameter in all vessel branches. Nifedipine exhibited a more pronounced effect on G4 vessels, whereas the actions of the other 1,4-DHP on the investigated vascular tree were more homogeneous. The dilating and flow increasing effects of nitrendipine and nifedipine could be significantly antagonized by treatment with LNNA. The vasodilating effects of nisoldipine and nimodipine could also be antagonized with L-NNA. We conclude that NO release plays an additional role in the relaxation of small resistance vessels by 1,4-DHP.

Animals↗
PubMed1994

The human platelet alloantigens, HPA-5(a+, b-) and HPA-5(a-, b+), are associated with a Glu505/Lys505 polymorphism of glycoprotein Ia (the alpha 2 subunit of VLA-2).

GP Ia/IIa (also called VLA-2 or alpha 2 beta 1) is the primary receptor for collagen on platelets. The human platelet alloantigens HPA-5a(Brb) and HPA-5b(Bra) have been found to reside on the platelet GP Ia/IIa complex. In order to establish the molecular basis of the HPA-5 system, platelet RNA was isolated form HPA-5 (a+, b-) and HPA-5(a-, b+) individuals. After reverse transcription, cDNA coding for glycoprotein Ia (GP Ia) was amplified by the polymerase chain reaction (PCR). Nucleotide sequence analysis of the PCR products revealed an A-->G polymorphism at base pair 1648 of the coding region of the mature protein, resulting in a substitution of lysine (AAG) in HPA-5b(Bra) by glutamic acid (GAG) in HPA-5a(Brb) at amino acid 505. Subsequent PCR-ASRA (allele-specific restriction enzyme analysis) with Mnl I using cDNA derived from three HPA-5 (a+, b-), one HPA-5 (a+, b+) individuals demonstrated that HPA-5a and -5b alleles are distinguishable by DNA typing. In addition to the A-->G substitution at base pair 1648, three silent mutations were identified, G-->C (195 bp), C-->T (837 bp), G-->A (1041 bp).

Antigens, Human Platelet↗
PubMed1994

Human platelet antigen-5 (Br) genotyping by ASPA: allele-specific primer amplification (PCR-SSP)

We have developed a PCR assay named ASPA (allele-specific primer amplification) to determine the HPA-5a and -5b genotypes. It consists of two PCR-reactions. One primer of each primer set has a 3'-end nucleotide which is specific for A or G at position 505 of the HPA-5b or -5a allele respectively. The HPA-5 genotypes determined in this way were strictly concordant with the genotypes established by the PCR-ASRA and with the phenotypes established using MAIPA. The ASPA is a rapid and reliable technique and can be used for the determination of alleles which code for platelet antigen allotypes.

Alleles↗
PubMed1994

Cys209 Ser mutation in the platelet membrane glycoprotein Ib alpha gene is associated with Bernard-Soulier syndrome.

Molecular genetic analysis has been performed on a patient with Bernard-Soulier syndrome (BSS). The patient had characteristically giant platelets and was deficient in the glycoprotein (GP) Ib/IX/V complex, the von Willebrand factor (vWf) receptor on platelets. Previous studies with monoclonal antibodies directed against GP Ib alpha (CD 42b) and GP IX (CD 42a) demonstrated the absence of GP Ib alpha and presence of small amounts of GP IX on the surface of the patient's platelets. In this study the presence of GP V (CD 42d) is also demonstrated. This indicates a defect in the alpha-subunit of glycoprotein Ib. Therefore polymerase chain reaction (PCR)-amplification of the genomic DNA coding for GP Ib alpha was performed. Nucleotide sequence analysis of the entire coding region of GP Ib alpha revealed a homozygous single base pair mutation T-->A, leading to a single amino acid substitution cysteine-->serine at position 209 of the mature protein. We took advantage of the Mse I target site in the mutant allele, created by the T-->A mutation, to analyse all available family members. PCR-ASRA (allele-specific restriction enzyme analysis) using the restriction enzyme Mse I, revealed the heterozygosity of the mother and the two children of the patient, whereas homozygosity of the patient for the Cys209Ser mutation was confirmed. The sister of the patient was not found to be a carrier of the mutant allele. The mutation identified in the family studied, responsible for the deficiency of the GP Ib/IX/V complex, suggests that the cysteine at amino acid position 209 may be involved in disulphide bonding.

Adult↗
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

Does immediate hormone replacement therapy affect the oncologic outcome in endometrial cancer survivors?

The purpose of this study was to evaluate the effect of immediate hormone replacement therapy (HRT) on oncologic outcome of patients with endometrial cancer. The patients were recruited prospectively after extensive discussion of risks and benefits of HRT. Continuous daily regimen of 0.625 mg conjugated equine estrogen plus 2.5 mg medroxyprogesterone acetate was initiated 4-8 weeks after surgery at first postoperative visit. The patients who had the same characteristics with the HRT group were assigned as a control group. Overall, 50 patients received HRT. There was no significant difference with respect to prognosticators between the HRT users and the control group. Seven patients (14%) stopped the use of HRT. Only two patients stopped the therapy before 24 months, and all the remaining patients used HRT for at least 24 months, with a mean value of 49.1 months. Neither the patients who used HRT nor the ones who left the therapy had recurrence at the time of writing of this article. This prospective case-control study showed that immediate postoperative use of HRT did not increase the recurrence or death rates in endometrial cancer survivors.

Adult↗
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↗