Reyhan Öner

Molecular, genetic and epidemiologic studies on selective complete C1q deficiency in Turkey.

Selective complete C1q deficiencies (SCDC1q) of the complement component C1q are rare genetic disorders with high prevalence of lupus-erythematosus-like symptoms and recurrent infections. Among the 41 published cases from 23 families, 10 derive from 6 Turkish families. One particular mutation leading to a stop codon in the C1q A gene was first identified in members of a Gypsy family from the Slovac Republic. Later the same mutation has been found in all cases in four SCDC1q families from Turkey suggesting that one particular defective allele may be present in the populations of Southeastern Europe and Turkey. This study was undertaken to investigate the frequency of C-->T mutation in exon II of C1qA gene in Turkish population by using allele-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP). Among the 1544 patients from 15 pediatric departments and an additional 89 SLE patients of various ages no C1qA gene mutation was found. There were 43 heterozygous and 4 homozygous mutations in 161 family members or relatives investigated from the 4 families known with SCDC1q. Among the 223 inhabitants who were nonrelative to the 3 SCDC1q families living in the same village were screened for mutation and one heterozygous individual was observed. Although this mutant allele appears to be at a low prevalence in the population tested, individuals with recurrent infections or symptoms of lupus erythematosus-like syndrome should be tested for this mutation to rule out this type of C1q deficiency.

Mitochondrial DNA sequence variation in the Anatolian peninsula (Turkey)

Throughout human history, the region known today as the Anatolian peninsula (Turkey) has served as a junction connecting the Middle East, Europe and Central Asia, and, thus, has been subject to major population movements. The present study is undertaken to obtain information about the distribution of the existing mitochondrial D-loop sequence variations in the Turkish population of Anatolia. A few studies have previously reported mtDNA sequences in Turks. We attempted to extend these results by analysing a cohort that is not only larger, but also more representative of the Turkish population living in Anatolia. In order to obtain a descriptive picture for the phylogenetic distribution of the mitochondrial genome within Turkey, we analysed mitochondrial D-loop region sequence variations in 75 individuals from different parts of Anatolia by direct sequencing. Analysis of the two hypervariable segments within the noncoding region of the mitochondrial genome revealed the existence of 81 nucleotide mutations at 79 sites. The neighbourjoining tree of Kimura’s distance matrix has revealed the presence of six main clusters, of which H and U are the most common. The data obtained are also compared with several European and Turkic Central Asian populations.

Investigation of ascorbate-Cu (II) induced cleavage of DNA by scanning tunneling microscopy.

Scanning Tunneling Microscopy (STM) was used for the investigation of oxidative DNA damage. A PCR amplified fragment of human beta-globin gene was used as a model for time dependent cleavage reaction by ascorbate and copper. Cleavage reactions were carried out in a medium containing 0.5 microgram/20 microliters DNA, 20 nM Tris-HC1 pH, 7.8 and ascorbate-Cu (II) in the final concentrations of 1 mM and 30 microM, respectively. The mixtures were incubated at 37 degrees C for 5, 15 and 30 min. For STM studies, 3 pg/5 microliters DNA samples were deposited on the gold coated mica and dried in a water flow vacuum drier. The STM was operated in air at atmospheric pressure with a tip-to-substrate bias of 100 mV and tunneling currents of < 10 pA. Etched tips of Pt/Ir wires were used in a constant current mode. The degradated DNA structure can be distinguished from the intact DNA and the sizes of the degradation products can be identified in the STM micrographs. The size of fragments decreased from approximately 3000 A to 34 A in ascorbate-Cu (II) medium, after 30 min of incubation.

Genotype-Phenotype Analysis in HbS-Beta-Thalassemia

Genotypes and phenotypes were studied in 31 Turkish HbS-beta-thalassemia patients. In 19 patients the beta-thalassemia mutations were beta+ and in 12 the beta 0 phenotype. The IVSI-110 mutation was found in 45% of the patients. IVSI-1, beta 39, IVSII-1 and FSC8 are the genotypes associated with beta 0-thalassemia. Hematological data were evaluated at the time of diagnosis and 4 years after diagnosis. The mean HbF value was 13 +/- 7.8% at diagnosis and 9.7 +/- 7.8% 4 years later. A significant negative correlation was observed between the age of the patients and the HbF value (p < 0.05). No statistically significant differences were observed between the mean of hematological parameters in beta(+)- and beta 0-thalassemia patients except for the mean HbF value which were 10.7 +/- 6.9 and 15.9 +/- 7.7% in beta(+)- and beta 0-thalassemia, respectively (p < 0.05). The study indicated that beta-thalassemia mutations in trans to the HbS mutation do not exert any beneficial effect on the manifestation of the disease.

Molecular analysis of the Turkish form of deletion-inversion (δβ)° thalassaemia

Two unrelated (δβ)°‐thalassaemia patients from Southern Turkey are presented. DNA studies indicated that both of them are homozygous for the Turkish type of (δβ)°‐thalassaemia characterized by one large deletion of 11.5 kb including the δ and β globin genes at the 5′ end and one small deletion of 1.6 kb at the 3′ end, which are separated by an inverted 7.6 kb long DNA segment that includes L1 repetitive sequence. In the present study a PCR‐based method was performed to produce a unique deletion‐specific product and subjected to sequence analysis for the determination of the breakpoint. DNA polymorphisms in the β‐globin gene cluster of deletion‐inversion type of (δβ)°‐thalassaemia, IVS‐I‐6 and β‐39 globin genes were examined. Analysis of sequence variations in regulatory regions including the 5′ hypersensitive site‐2 of the locus control region (LCR), the δ, Gγ and Aγ 5′ flanking regions and the second intervening sequence (IVS‐II) of Aγ and Gγ genes indicated the presence of close similarities between the chromosome carrying the Turkish form of deletion‐inversion δβ)°‐thalassaemia and the chromosome associated with β‐39 nonsense mutation in haplotype II. These two chromosomes are characterized by the presence of a 4 base pair deletion in the AγT globin gene promoter. A C → T alteration at position −199 5′ to the δ gene was also found to be associated with the Turkish type of (δβ)°‐thalassaemia and β‐39 chromosome.

HOMOZYGOSITY FOR Hb E-SASKATOON [β22(B4)Glu → Lys] IN A TURKISH PATIENT

A 30-year-old female who is homozygous for a Hb E-like abnormal hemoglobin and her immediate relatives were studied. Clinical examination of the proband revealed no abnormality. Routine hematological analysis showed that her hemoglobin level was 12 g/dL, MCV 82 fL, MCH 28 pg, RDW 15%. DNA sequence analysis indicated the presence of a G → A substitution at codon 22 corresponding to an abnormal hemoglobin, namely Hb E-Saskatoon [β22(B4)Glu → Lys (GAA → AAA)]. Absence of any abnormalities in clinical and routine hematological investigations of the homozygous patient indicated that the phenotypical expression of the Hb E-Saskatoon is very mild. Using a reverse transcription-polymerase chain reaction technique, the α/β and βX/βA-mRNA (X = Hb E-Saskatoon) ratios were determined. Normal α/β and βX/βA-mRNA ratios were found in the homozygous patient and in all heterozygotes, indicating that the respective mutation did not alter the stability of the mRNA. FokI restriction enzyme analysis of the polymerase chain reaction products obtained from the genomic DNA and/or β-globin mRNA made it possible for rapid diagnosis of Hb E-Saskatoon, and for its differentiation from Hb E [β26(B8)Glu → Lys (GAG → AAG)]. Analysis of the restriction fragment length polymorphism (RFLP) in the β-globin gene complex of the index patient and of another unrelated family with a compound heterozygosity for Hb E-Saskatoon and β-thalassemia revealed that the Hb E-Saskatoon mutation shared a common allele.

Molecular Analysis of Turkish β-Thalassemia Heterozygotes with Normal Hb A2 Levels

Four parents of three unrelated families who are obligatory β-thalassemia heterozygotes and two parents with Hb Knossos are presented. In these subjects, although the red blood cell counts and red cell indices were compatible with β-thalassemia trait, the Hb A2 values were between 1.9–2.9 % of the total hemoglobin. Examination of the δ-globin gene by Southern blot, restriction endonuclease analysis, and by direct sequencing of amplified DNA revealed the presence of the (δ˚) -7.2 kb Corfu type deletion, the (δ+) codon 27 (G→T) and (δ˚) IVS-I-2 (T→C) mutations in trans or in cis with a severe β-thalassemia allele, and the (δ˚) codon 59 (-A) deletion in cis with the βKnossos allele.

Chronic hemolytic anemia associated with glucose 6-phosphate dehydrogenase (Guadalajara)1159 C → T (387 Arg → Cys) deficiency associated with Gilbert syndrome in a Turkish patient

The case of an 8-year-old male child with severe kernicterus sequelae is presented in this paper. The childs hemoglobin value varied between 6.0 and 10.8 g/dL and his reticulocyte count ranged between 3.4 and 46.0% during the steady-state condition and hyperhemolytic crisis, respectively. A chronic hemolytic typeof red cell G6PD deficiency was diagnosed. DNA studies indicate that the mutation was G6PD Guadalajara 1159 C M T (387 Arg M Cys) that is situated at the NADP binding site. Additionally, extra nucleotides of (TA) in the A(TA) n TAA motif of the promoter region of the uridine diphosphate-glucuronosyltransferase gene (UGT-1) were found to be homozygous in the patient. The coexistence of Gilbert syndrome with a chronic type of G6PD deficiency was suggested as a cause of neonatal hyperbilirubinemia leading to kernicterus.

Mutations in coagulation factor XIII A gene in three Turkish patients: two novel mutations and a known insertion.

Summary.  Molecular analysis of factor XIII A gene on three unrelated Turkish families identified two novel and one known mutations. One novel mutation is a substitution of cytidine by guanine at codon 541 in exon 12, β barrel 1 domain of the coagulation factor XIII A subunit gene resulting in the conversion of asparagine to lysine. The mutation alters the restriction site of the enzyme MboII. The second novel mutation, a 4 bp (–CAAA) deletion located in a direct repetitive sequence (CAAACAAA) between codons 466–469, results in premature termination of translation at codon 474. The third mutation is a previously reported single nucleotide (cytidine) insertion at codon 400 in exon 9 of the factor XIII gene.

Identification of an inframe deletion and a missense mutation in the factor XIIIA gene in two Turkish patients.

Abstract: We report two novel mutations in factor XIIIA (FXIIIA) gene that caused congenital factor XIII deficiency in two unrelated patients. The first alteration, a missense mutation Leu235Arg in exon 6 of FXIIIA gene, is located in the putative calcium‐binding part of the core domain of the enzyme. Replacement of non‐polar hydrophobic leucine residue with positively charged arginine residue is likely to effect protein folding thus destabilizing the molecule. The second mutation is a 3‐bp deletion in exon 14 of FXIIIA gene. This deletion is located in beta barrel 2 domain of the protein and results in translation of an aberrant FXIIIA molecule that lacks lysine residue either at positions 677 or 678. As this inframe deletion is located in a direct repetetive sequence of AAGAAG, that codes for two lysine residues, the exact location of deletion could not be detected.