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M Tzetis

Publications and source records attributed to M Tzetis.

At least 19 recordsLinked to original sources
PubMed2005

Asporin and knee osteoarthritis in patients of Greek origin.

Ostearthritis (OA) is characterized by focal areas of loss of the articular cartilage in synovial joints, associated with varying degrees of osteophyte formation, subchondral bone change and synovitis. The Asporin (ASPN) gene which encodes a protein of the extracellular cartilage matrix contains a triplet repeat encoding for aspartic acid (D) within exon 2 The D14 allele was found associated with knee and hip osteoarthritis in case-control study in the Japanese population. Genotyping Greek knee OA patients for the D repeats we determined that the D15 allele could be considered a risk allele for our population.

Aged↗
PubMed2003

Cystic fibrosis in Greece: molecular diagnosis, haplotypes, prenatal diagnosis and carrier identification amongst high-risk individuals.

Cystic fibrosis (CF) mutation analysis on 437 CF patients, characterized 80 different mutations (20 so far specific to our population) accounting for 91% of CF genes and generating 103 different genotypes. Eight mutations were common [F508del (53.4%), 621+1G>T (5.7%), G542X (3.9%), N1303K (2.6%), 2789+5G>A (1.7%), 2183AA>G (1.4%), E822X (1.4%), R1158X (1%)], 12 showed frequencies between 0.5% and 1%, while the remaining (60) were very rare (1 to 3 alleles). Denaturing gradient gel electrophoresis (DGGE) screening of 12 exons (3, 4, 7, 10, 11, 13, 14b, 16, 17b 20 and 21) detected 85.5% of CF alleles. Haplotypes for eight diallelic and three microsatellite markers have been characterized for the common, a few rare and novel Greek mutations. Results of 165 prenatal diagnoses (including 49 due to bowel hyperechogenicity), testing a total of 41 different parental genotypes, are reported. One hundred and sixteen prenatal tests resulted in 22 affected, 59 heterozygous, 34 normal fetuses and one incomplete diagnosis. Of the 49 echogenic bowel fetuses, 3 were heterozygotes. Carrier screening was initiated, with emphasis on individuals and couples in high-risk groups - with a family history of CF, one partner with CF, and couples with male infertility seeking in vitro fertilization (IVF). Mutation analysis on 672 individuals (120 couples, 91 unaffected CF siblings, 283 CF family relatives and 58 general population subjects), identified a total of 176 heterozygotes and 7 couples where both partners were CF heterozygotes. Prenatal diagnosis was performed in 4 cases and 3 were counseled on the availability of a prenatal test.

Alleles↗
PubMed2002

Abnormal mRNA splicing resulting from consensus sequence splicing mutations of ATP7B.

More than 200 Wilson disease (WD) disease-causing mutations have been defined to date. Missense mutations are largely prevalent while splice-site mutations are limited in number. Most reside in the splice donor or acceptor sites and only a minority are detected in splicing consensus sequences. Furthermore, only a few splicing mutations have been studied at the RNA level to date. In this study, using the RT-PCR method we performed the molecular characterization of four consensus splice-site mutations identified by DNA analysis in patients with WD. One of them, previously described 1707+3insT, occurred at position 3 in the donor splice site of intron 4, while the other three, 2122-8T>G, 2866-6T>G, and 3061-12T>A, are novel and occurred in the acceptor splice sites of introns 7, 12, and 13, respectively. Analysis revealed a prevalently abnormal splicing in the samples carrying the mutations compared to the normal controls. Comparison of RNA splicing with normal controls in liver and lymphocytes further suggests that abnormal splicing of the WD gene is also present and differentially regulated in normal tissues. The results produced in this study strongly suggest that DNA mutations residing in the consensus sequence of WD gene splice sites result in the WD phenotype by interfering with the production of the normal WD protein. Further studies are necessary to better quantify the amount of different transcripts produced by these mutations, and establish their correlation with the disease phenotype.

Adenosine Triphosphatases↗
PubMed2002

Pregnancies following blastocyst stage transfer in PGD cycles at risk for beta-thalassaemic haemoglobinopathies.

BACKGROUND: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i. METHODS: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis. RESULTS: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%). CONCLUSIONS: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation.

Biopsy↗
PubMed2001

Qualitative and quantitative analysis of mRNA associated with four putative splicing mutations (621+3A-->G, 2751+2T-->A, 296+1G-->C, 1717-9T-->C-D565G) and one nonsense mutation (E822X) in the CFTR gene.

The effects of four splicing mutations and one nonsense mutation on cystic fibrosis transmembrane conductance regulator ( CFTR) gene expression were investigated by reverse transcription-polymerase chain reaction analysis of mRNA extracted from nasal epithelial cells harvested from patients harbouring the mutations. We studied four subjects with 621+3A-->G, two with 2751+2T-->A, one with 296+1G-->C, two with 1717-9T-->C-D565G and seven with E822X and compared the results with CFTR mRNA from normal subjects. Our results showed that mutations 621+3A-->G, 2751+2T-->A, and 296+1G-->C, which disrupt the 5' splice donor sites of introns 4, 14a, and 2, respectively, and 1717-9T-->C-D565G, which possibly disrupts the exonic splicing enhancer sequences of exon 12 (owing to the missense mutation in cis), lead to the production of aberrantly spliced mRNA in nasal epithelial cells. Three of the splicing mutations (621+3A-->G, 2751+2T-->A, and 296+1G-->C) result in severe deficiency of normal CFTR mRNA and severe phenotype in the patients. This information is especially useful for mutation 621+3A-->G, which is found in other populations as well, and was initially reported as a polymorphism. The complex allele 1717-9T-->C-D565G results in aberrant splicing of CFTR mRNA with production of transcripts lacking exon 12 (major product), with minor amounts of transcripts revealing joint exon 11 and 12 skipping. Nonsense mutation E822X results in a severe reduction in mRNA levels to about 6% of wild type. Patients with the mutation have a severe clinical phenotype, with both the pancreatic and the pulmonary function affected.

Adolescent↗
PubMed2001

CFTR gene mutations--including three novel nucleotide substitutions--and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease.

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.

Adolescent↗
PubMed2001

Gilbert syndrome associated with beta-thalassemia.

The authors investigated whether the considerable variability in serum bilirubin levels (STB) found in transfusion-dependent beta-thalassemia, beta-thal intermedia, and heterozygous beta-thalassemia individuals could be related to the coexistence of Gilbert syndrome (GS). The promoter region [A(TA)nTAA] of the bilirubin UDP-glucuronosyltransferase gene (UGT1A1) was analyzed in a total of 128 beta-thalassemia individuals (108 transfusion-dependent beta-thal patients, 20 very mild beta-thal intermedia) and in 33 beta-thal heterozygotes. The control group consisted of 70 healthy children with no history of anemia. The frequency of GS genotype (TA)7/(TA)7 did not differ significantly between the groups studied. A significant difference was observed between serum bilirubin levels (STB) and GS genotypes (TA)7/(TA)7 and (TA)6/(TA)7 and also between (TA)7/(TA)7 and (TA)6/(TA)6 for all groups examined. These results confirm that the (TA)7/(TA)7 GS genotype is one of the factors accounting for the hyperbilirubinemia observed in beta-thalassemia major, intermedia, and heterozygous individuals.

Bilirubin↗
PubMed2000

Delineation of the spectrum of Wilson disease mutations in the Greek population and the identification of six novel mutations.

In this study, we report the further results of an ongoing project on the delineation of the spectrum of mutations on the ATP7B gene in Wilson disease (WD) patients of Greek origin. We have analyzed 24 additional families and detected 16 mutations (five frameshifts, two splice site, two nonsense, and seven missense), of which six are novel. On adding these results to the ones already published by us, we conclude that WD shows a marked allelic heterogeneity in the Greek population. Of the total number of mutations so far detected, the most common eight account for the molecular defect in 72.8% of the WD chromosomes. The most frequent mutation is the His0169Gln, which has a frequency of 28.5%, similar to those reported in North European populations. Using these data, an efficient strategy of mutation screening for WD is possible in this population, thus improving the possibility of preclinical diagnosis.

Adenosine Triphosphatases↗
PubMed1999

Preimplantation genetic diagnosis in 10 couples at risk for transmitting beta-thalassaemia major: clinical experience including the initiation of six singleton pregnancies.

Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies, since it allows the selection of unaffected IVF embryos for transfer. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage stage embryos, followed by polymerase chain reaction (PCR)-based DNA analysis. However, PCR-based DNA analysis of single cells is subject to several problems including sample contamination, total PCR failure, or failure of one allele to amplify--a phenomenon known as allelic drop-out (ADO). Furthermore, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a spectrum of mutations and compound genotypes. With the above considerations we developed an accurate and reliable strategy for analysis of beta-globin gene mutations, applicable for PGD for the wide spectrum of beta-thalassaemia major genotypes in the Greek population. The strategy involves nested PCR followed by denaturing gradient gel electrophoresis (DGGE) analysis. DGGE is an advantageous method for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it identifies the presence of normal alleles and in addition can monitor the occurrence of ADO. This report describes the application of the DGGE-based diagnostic strategy in 11 clinical IVF/PGD cycles, in 10 couples at risk for transmitting beta-thalassaemia major. The transfer of at least one embryo diagnosed as unaffected for beta-thalassaemia major in nine couples has resulted in the initiation of six pregnancies. Four pregnancies have so far been confirmed as unaffected for beta-thalassaemia major by first or second-trimester prenatal diagnosis, two of which have resulted in the birth of two healthy babies. Three singleton pregnancies are still on-going and one ectopic pregnancy was terminated.

Biopsy↗
PubMed1999

Molecular, haematological and clinical studies of the -101 C --> T substitution of the beta-globin gene promoter in 25 beta-thalassaemia intermedia patients and 45 heterozygotes.

We report the clinical, haematological, biosynthetic and molecular data of 25 double heterozygote beta-thalassaemia intermedia patients and 45 beta-thalassaemia heterozygotes with the C --> T substitution at nucleotide position -101 from the Cap site, in the distal CACCC box of the beta-globin gene promoter. This mutation is considered the most common amongst the silent beta-thalassaemia mutations in Mediterranean populations. Of the 25 compound heterozygotes for the beta -101 C --> T and common severe beta-thalassaemia mutations, all but one had mild thalassaemia intermedia preserving haemoglobin levels around 9.5 g/dl and haemoglobin F levels < 25%. The only transfused patient was characterized to have an additional alpha-globin gene. Strict assessment of haematological and biosynthetic findings in the heterozygotes for the beta -101 C --> T mutation (excluding six cases with an alpha-globin gene defect) demonstrated that less than half of them had completely normal (silent) haematology; the remainder had either high haemoglobin A2 values (in the range of 3.7-5.1%) and/or low red cells indices and/or raised haemoglobin F values. The alpha/non-alpha-globin chain synthesis ratios were generally raised, with mean 1.44 (1.07-2.10). Amongst the parents of the compound heterozygotes, who were not selected for molecular analysis following haematological screening, half of the cases were completely silent. Interaction with severe beta-thalassaemia mutations always resulted in the clinical phenotype of mild non-transfusion-dependent thalassaemia intermedia.

Adolescent↗
PubMed1999

A widely applicable strategy for single cell genotyping of beta-thalassaemia mutations using DGGE analysis: application to preimplantation genetic diagnosis.

Preimplantation genetic diagnosis (PGD) allows the selection of unaffected IVF embryos for transfer in couples that are at risk for transmitting genetic diseases. For monogenic diseases, polymerase chain reaction (PCR)-based diagnosis is usually performed on single blastomeres. In Greece, up to 10 per cent of the population are carriers for beta-thalassaemia and related haemoglobinopathies, and more than 20 pathological mutations in the beta-globin gene have been described. In this study we report a strategy which includes a first round of PCR, allowing subsequent nested PCR and DGGE analysis for at least 95 per cent of beta-thalassaemia major genotypes in the Greek population. The use of DGGE for beta-globin genotype analysis is advantageous: it facilitates simultaneous analysis of more than one mutation in a single PCR fragment, it detects the presence of normal alleles and monitors the occurrence of allelic drop-out (ADO) through the expectation that heterozygous samples have more than one electrophoretic band on DGGE analysis. The optimization, accuracy and reliability of the method was evaluated by genotyping 325 single blastomeres, 110 amniocytes and 55 lymphocytes. Results confirmed that PCR efficiency and occurrence of ADO are improved by higher denaturation temperatures in the first cycles of first-round PCR, influenced by the size of the fragment amplified in the first round of PCR and additionally by the quality and type of cells being genotyped. The proposed strategy was accurate and reliable, and thus for application to PGD should ensure the transfer of unaffected embryos. Furthermore it is widely applicable in most of the populations worldwide where beta-thalassaemia is common.

Blastomeres↗
PubMed1998

Dating the origin of the CCR5-Delta32 AIDS-resistance allele by the coalescence of haplotypes.

The CCR5-Delta32 deletion obliterates the CCR5 chemokine and the human immunodeficiency virus (HIV)-1 coreceptor on lymphoid cells, leading to strong resistance against HIV-1 infection and AIDS. A genotype survey of 4,166 individuals revealed a cline of CCR5-Delta32 allele frequencies of 0%-14% across Eurasia, whereas the variant is absent among native African, American Indian, and East Asian ethnic groups. Haplotype analysis of 192 Caucasian chromosomes revealed strong linkage disequilibrium between CCR5 and two microsatellite loci. By use of coalescence theory to interpret modern haplotype genealogy, we estimate the origin of the CCR5-Delta32-containing ancestral haplotype to be approximately 700 years ago, with an estimated range of 275-1,875 years. The geographic cline of CCR5-Delta32 frequencies and its recent emergence are consistent with a historic strong selective event (e.g. , an epidemic of a pathogen that, like HIV-1, utilizes CCR5), driving its frequency upward in ancestral Caucasian populations.

Acquired Immunodeficiency Syndrome↗
PubMed1998

Cystic fibrosis mutation screening in CBAVD patients and men with obstructive azoospermia or severe oligozoospermia.

Congenital bilateral absence of the vas deferens (CBAVD) found in otherwise healthy infertile males, is associated with a high incidence of mutated cystic fibrosis transmembrane conductance regulator (CFTR) alleles, and is considered a genital form of cystic fibrosis (CF). The CF gene may also be involved in the aetiology of male infertility in cases other than CBAVD. The present study was undertaken to test the involvement of CFTR gene mutations in 14 CBAVD males and additionally in cases of male infertility caused by obstructive azoospermia (n = 10) and severe oligozoospermia (n = 3). The entire coding region of the CFTR gene was analysed using denaturing gradient gel electrophoresis (DGGE). The three allele (5T, 7T, 9T) polymorphic tract of thymidines in intron 8 (IVS8-polyT) of which the 5T allele acts as a mild mutation, causing reduced levels of normal CFTR mRNA due to deletion of exon 9, was also analysed. Of the 14 CBAVD cases, four (28.6%) were found to have mutations in both copies of the CFTR gene, six (42.8%) had one CFTR mutation, and in the remaining four (28.6%) no CFTR mutations were found. Of the 10 cases with obstructive azoospermia, three (30%) had one CFTR mutation and in the remaining seven (70%) no mutations were found. None of the three severe oligozoospermia cases carried a CFTR mutation. The frequency of the IVS8(5T) allele was 14.3% (4/28) for the CBAVD cases and 5% (1/20) for the obstructive azoospermia cases, none of the severe oligozoospermia males carried the IVS8-5(5T) allele. The data indicate that while there is a strong association between male infertility caused by CBAVD and mutations in the CFTR gene, cases of obstructive azoospermia without CBAVD also seem to be associated with CFTR gene mutations.

Cystic Fibrosis↗
PubMed1997

Characterization of more than 85% of cystic fibrosis alleles in the Greek population, including five novel mutations.

To completely characterize the spectrum of mutations in the cystic fibrosis transmembrane conductance regulator gene in Greek cystic fibrosis (CF) patients, we screened 500 CF chromosomes by denaturing gradient gel electrophoresis followed by direct sequencing. We identified 48 mutations, accounting for 85.6% of CF chromosomes. They included eight novel mutations, three of which we have described before and five (E822X, Y247X, 2752-26A-->G, 3152delT, and 2751 + T-->A), which are described in this report. The detection of such a high proportion of Greek CF mutations is important for improving prenatal and genetic diagnosis of CF in Greece.

Adult↗