Identification of point mutations in Turkish DMD/BMD families using multiplex-single stranded conformation analysis (SSCA).

Small mutations are the cause of the disease in one third of cases of Duchenne and Becker muscular dystrophy (DMD/BMD). The identification of point mutations in the dystrophin gene is considered to be very important, because it may provide new insights into the function of dystrophin and direct information for genetic counselling. In this study, we have screened 18 deletion-prone exons (25.5% of the coding region) of the dystrophin gene by using a modified non-isotopic multiplex single-stranded conformation analysis (SSCA). Mutations responsible for the disease phenotype could be identified in five out of 56 unrelated DMD/BMD patients without detectable deletions. Two of these mutations, 980-981delCC and 719G > C, are novel mutations which have not been described previously. Four of the five mutations, including 980-981delCC detected in this study are found to be nonsense or frameshift mutations leading to the synthesis of a truncated dystrophin protein. The missense mutation, 719G > C, causing the substitution of highly conserved alanine residue at 171 with proline in the actin binding domain of the dystrophin, is associated with a BMD phenotype. This study also revealed the presence of six polymorphisms in Turkish DMD/BMD patients.

Identification of Deletion Carriers in Duchenne/Becker Muscular Dystrophy Families Using a Digoxigenin-Labeled Quantitative Polymerase Chain Reaction Technique.

Background: A quantitative polymerase chain reaction (PCR) technique based on the incorporation of digoxigenin (DIG), and visualization of the labeled fragments for the detection of deletion carriers in Duchenne/Becker muscular dystrophy families has been developed. Methods and Results: Sixty-five DNA samples taken from mothers and/or sisters of familial and sporadic deletion patients were investigated in the exponential phase of amplification. All obligate carriers were correctly identified using this technique. In more than 95% of deletion families, possible carriers could be screened by using four different multiplex systems specifically designed to increase the efficiency of the detection. Deletions were found to be present in 42% of possible carriers. All these results were confirmed by computer-assisted laser densitometry. Conclusions: Dosage analysis by DIG-labeled quantitative PCR is a reliable and accurate technique for detecting Duchenne/Becker muscular dystrophy deletion carriers.

Binding of protein uH2A and histone H2A to DNA.

The binding properties of protein uH2A and histone H2A to DNA were investigated by filter binding assays. Both proteins revealed similar affinity for native and denatured DNA. Competition with increasing amounts of repetitive and nonrepetitive DNA has shown that protein uH2A binds selectively to nonrepetitive sequences. When poly d(A-T) was used as a competitor, uH2A bound to this polynucleotide with much greater affinity than histone H2A. These findings suggest a selective binding to regulatory A-T rich intergenic sequences in native DNA.

The effect of cycloheximide on nuclear uH2A content.

The effect of intraperitonal cycloheximide administration on acid-soluble rat liver chromatin proteins has been investigated by electrophoresis in acetic acid-urea polyacrylamide gels. A nonhistone protein, which migrates between oxidized histone H3 and histone H1 has been found to be increased in amount following cycloheximide treatment. This protein seems to be identical with semihistone protein H24 (uH2A). A possible relationship of uH2A to the inhibition of rRNA synthesis is discussed.

A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G.

A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying alpha-amylase and glucoamylase activities. Cell growth is modulated mainly by intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular substrate and biomass concentrations. Extracellular alpha-amylase and glucoamylase activities are taken to depend on biomass concentration. The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, alpha-amylase and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement.

Polymorphisms associated with the FVIII and FIX genes in the Turkish population.

In order to determine the gene frequencies of nine polymorphic sites associated with FVIII and FIX genes in the Turkish population a sample of 50-235 unrelated X chromosomes from healthy individuals were analysed by using PCR-based assays. The Turkish population was found to be as polymorphic as Europeans in the FVII and FIX genes. Analysis of FIX haplotypes revealed that the most frequent haplotype observed in European populations and Anglo-Americans was also very common among Turks. The present population-based study indicates that two marker loci, namely HindIII and St14 in the factor VIII gene and DdeI and HhaI in the factor IX gene, are highly informative and useful markers that can be used in DNA linkage analysis for the assessment of haemophilia carriers and affected fetuses in the Turkish population.

Modeling of recombinant yeast cells: reduction of phase space.

The mechanism of starch fermentation by recombinant Saccharomyces cerevisiae in batch reactor is studied. Experiments were carried in the presence and absence of oxygen, with different initial starch concentrations. A variety of data concerning biotic and abiotic phases are collected. Nonlinear data analysis techniques are used to determine the block diagram of the system under study. Data analysis and processing reported here, are believed to form a basis in further work in structured modeling of biological systems, recombinant yeast cultures in particular.

High salt effect on RNA synthesis in Krebs ascites tumor cells.

The incubation of Krebs ascites tumor cells in medium with a high salt concentration resulted in a partial inhibition of nuclear RNA synthesis. The residual RNA polymerase activity in such nuclei was only slightly inhibited by low concentrations (50 nM) of alpha-amanitin. This finding suggested an inhibition of RNA polymerase II activity under conditions of medium hypertonicity. Indeed, RNA polymerase II, isolated from the nuclei of cells exposed to hypertonicity, revealed only half of the control activity. On the other hand, RNA polymerase I was not affected by hypertonicity. Moreover, chromatin fractions isolated from cells incubated in hypertonic or isotonic medium were equally template-active in RNA synthesis.

High-level production of TaqI restriction endonuclease by three different expression systems in Escherichia coli cells using the T7 phage promoter.

Three different expression systems were constructed for the high-level production of TaqI restriction endonuclease in recombinant Escherichia colicells. In system [R], the TaqI endonuclease gene was cloned and expressed under the control of the strong T7 RNA polymerase promoter. To protect cellular DNA, methylase protection was provided by constitutive co-expression of TaqI methylase activity either by cloning the TaqI methylase gene on a second plasmid (system [R,M]) or by constructing a recombinant plasmid harboring both the endonuclease and methylase genes (system [R+M]). In batch shake flasks containing complex media, co-expression of the methylase gene in systems [R,M] and [R+M] resulted in a 2- and 3-fold increase in volumetric productivity over system [R], yielding activities of 250x10(6) U l(-1) and 350x10(6) U l(-1), which were 28 and 39 times higher than the data in the literature, respectively. Under controlled bioreactor conditions in chemically defined medium, co-expression of methylase activity greatly improved the yield and specific TaqI endonuclease productivity of the recombinant cells, and reduced acetic acid excretion levels. System [R,M] is preferable for high expression levels at longer operation periods, while system [R+M] is well-suited for high expression levels in short-term bioreactor operation.

Isolation of protein uH2A using a one step preparative gel electrophoresis.

A one step electrophoretic procedure for the isolation of protein uH2A has been devised which may improve the overall yield. The improvement involves elimination of intermediate steps which might result in the decrease of the yield. The method may serve as an alternate to the conventional methods and can also be used successfully for the isolation of several different proteins.

Study of 15 protein polymorphisms in a sample of the Turkish population.

Anatolia, because of its geographic position and its use as an area of settlement, was also a land of transit that accommodated a succession of populations. The last important invasion occurred in the Middle Ages with the arrival of the Turks, an Altaic-speaking nomadic population descended from the Oguz tribes and originating in Mongolia. Although the Turks imposed their culture, their genetic contribution seems to have been modest. To validate this hypothesis, we studied the genetic structure of the Turkish population by examining 15 genetic markers in a sample of 93 subjects. The allele frequencies observed were HP*1 = 0.240; GLO1*1 = 0.344, ESD*2 = 0.134, GC*1S = 0.613, GC*1F = 0.129, PGM1*2S = 0.322, PGM1*2F = 0.041, PGM1*1F = 0.027, F13B*1 = 0.762, F13B*2 = 0.101, ORM1*S = 0.327, AHSG*2 = 0.181, C6*B = 0.239, C7*1 = 0.983, APOC2*1 = 1.0, APOE*3 = 0.868, APOE*2 = 0.063, BF*F = 0.258, BF*S07 = 0.017, BF*SQ0 = 0.011, C4A*Q0 = 0.145, C4A*2 = 0.070, C4A*5 = 0.012, C4A*6 = 0.023, C4B*Q0 = 0.101, C4B*2 = 0.048, C4B*3 = 0.005, and C4B*11 = 0.005. The present Turkish population was compared to other European, Middle Eastern, and North African populations by means of correspondence analysis. Turks cluster with Turkomans, who share the ancient Turks' derivation from the Oguz tribe. Moreover, Turks clearly belong to European groups and resemble the populations of neighboring countries. Therefore the present data support the hypothesis that the ancient Turkish tribes, who started to enter Anatolia 1000 years ago, contributed little to the gene pool of the preexisting Anatolian populations. Alternatively, if the genetic structure of the invading Turks resembled that of the ancient Anatolians, it will be impossible to find traces of their admixture with the autochthonous inhabitants of Anatolia. However, further analysis of other samples from Turkey and from populations living in the homelands of the Turkish tribes, namely, the eastern area of the Caspian Sea and Mongolia, is needed.

Screening of deletions and RFLP analysis in Turkish DMD/BMD families by PCR.

We have screened 76 DMD and 5 BMD patients for deletions, using two separate Multiplex gene amplification systems. The use of both systems together revealed deletions in 52% of the cases in the Turkish population. The majority of these deletions (33/37) were found to be localized within the central region of the dystrophin gene. The remaining deletions were mapped to the proximal hotspot. Deletion end-points were identified by PCR and/or by Southern blot analysis with cDNA probes, and exceptions to the Open Reading Frame (ORF) hypothesis are discussed. PCR-based techniques to screen the pERT87.15/XmnI, pERT87.15/BamHI, and pERT87.8/TaqI polymorphisms were used for linkage analysis in the Turkish DMD/BMD families, and approximately 70% of the mothers at risk were found to be informative for at least one of these polymorphisms studied.

Screening of deletions in SMN, NAIP and BTF2p44 genes in Turkish spinal muscular atrophy patients.

Deletions of the spinal muscular atrophy (SMA)-determining gene, SMN1, NAIP, and a third multicopy gene, BTF2p44tel were investigated in 60 unrelated Turkish SMA patients. SMN1 was deleted for at least exons 7 and 8 in 85% of the Turkish SMA patients. The NAIP gene was deleted in 75 and 33% of type I and type II SMA patients, respectively. Analysis of the 5'end of the BTF2p44tel gene indicated the extension of deletion in 13.3% of the cases, mainly in type I patients. Deletions of the NAIP and BTF2p44tel genes were detected in 1.3 and 3.9% of carrriers, respectively, in Turkish SMA families. Two patients were detected to harbor the hybrid SMN gene, one type II with deletion of the NAIP gene, and one type III without deletion of the NAIP gene.

Prenatal prediction of childhood-onset spinal muscular atrophy (SMA) in Turkish families.

Childhood-onset spinal muscular atrophy (SMA) is one of the most common neurodegenerative genetic disorders. SMN1 is the SMA-determining gene deleted or mutated in the majority of SMA cases. There is no effective cure or treatment for this disease yet. Thus, the availability of prenatal testing is important. Here we report prenatal prediction for 68 fetuses in 63 Turkish SMA families using direct deletion analysis of the SMN1 gene by restriction digestion. The genotype of the index case was known in 40 families (Group A) but unknown in the remaining 23 families (Group B). A total of ten fetuses were predicted to be affected. Eight of these fetuses were derived from Group A and two of these fetuses were from Group B families. Two fetuses from the same family in Group A had the SMNhyb1 gene in addition to homozygous deletion of the NAIP gene. One fetus from Group A was homozygously deleted for only exon 8 of the SMN2 gene, and further analysis showed the presence of both the SMN1 and SMNhyb1 genes but not the SMN2 gene. In addition, one carrier with a homozygous deletion of only exon 8 of the SMN1 gene was detected to have a SMNhyb2 gene, which was also found in the fetus. To our knowledge, these are the first prenatal cases with SMNhyb genes. Follow-up studies demonstrated that the prenatal predictions and the phenotype of the fetuses correlated well in 33 type I pregnancies demonstrating that a careful molecular analysis of the SMN genes is very useful in predicting the phenotype of the fetus in families at risk for SMA.

Identification of the parental origin of polysomy in two 49,XXXXY cases.

The parental origin and mechanism of formation of polysomy X were studied in two polysomic cases, using four X-linked restriction fragment length polymorphisms, three (CA)n dinucleotide repeat sequences and one variable number tandem repeat (VNTR) locus as genetic markers. A nonradioactive technique based on the hybridization of the polymerase chain reaction (PCR) product was developed for the analysis of dinucleotide repeats. Segregation analysis using different nonradioactive approaches based on the PCR, revealed that all four X chromosomes were of maternal origin. These data provide additional evidence of an identical mechanism of successive nondisjunctions in maternal meiosis I and II.

Cystic fibrosis mutations and associated haplotypes in Turkish cystic fibrosis patients.

Identification of mutations causing cystic fibrosis (CF) in the Turkish population is essential for assessment of the molecular basis of CF in Turkey and the development of strategies for prenatal diagnosis and genetic counseling. Here, we present an updated report of mutations found in the Turkish CF population from an extensive screening study of the entire coding region, including exon-intron boundaries and the promoter region. Cases for which mutations could not be identified were also screened for previously defined large alterations and (TG)mTn-M470V loci. This study revealed a total of 27 different mutations accounting for almost 60% of disease genes in the Turkish population. In this study, we also identified the haplotypes associated with 17 mutations and those associated with unknown mutations. The mutation spectrum of CF in Turkey and its associated haplotypes indicated the presence of a major Mediterranean component in the contemporary Turkish population.

Analysis of the CFTR gene in Turkish cystic fibrosis patients: identification of three novel mutations (3172delAC, P1013L and M1028I).

In order to determine the spectrum of cystic fibrosis (CF) mutations in the Turkish population, a complete coding region of the cystic fibrosis transmembrane conductance regulator (CFTR) gene including exon-intron boundaries, on 122 unrelated CF chromosomes from 73 Turkish CF families was analysed by denaturing gradient gel electrophoresis and multiplex heteroduplex analysis on MDE gel matrix. In addition to 15 previously reported mutations and 12 polymorphisms, three novel mutations, namely 3172delAC, P1013L and M1028I, were detected. DeltaF508 was found to be present on 18.8% of CF chromosomes. The second most common mutation was 1677delTA, with a frequency of 7.3%, followed by G542X and 2183AA-->G mutations, with frequencies of 4.9%. These four most common mutations in Turkish CF population account for approximately 36% of mutations. This study could only detect 52.5% of disease-causing mutations in this population; 47.5% of CF alleles remain to be identified, reflecting the high molecular heterogeneity of the Turkish population.