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P Ioannou

Publications and source records attributed to P Ioannou.

At least 19 recordsLinked to original sources
PubMed2005

Alphoid DNA from different chromosomes forms de novo minichromosomes with high efficiency.

Clones from one BAC and one PAC library carrying centromeric alphoid DNA were characterized and found to be stable but to differ according to the enzyme used to make the library. Five different clones with homogeneous alphoid DNA, derived from chromosomes 13/21, 14/22, 17 and 18, were all shown to form minichromosomes de novo after transfection into the human cell line HT1080 in greater than 29% of the cell lines analysed. Similarly sized alphoid arrays (110-160 kb) from chromosomes 17, 13/21 and 14/22 all formed minichromosomes in about 50% of the cell lines analysed while a smaller array (50 kb) of 14/22 alphoid was less efficient (29% of cell lines) and a larger array (200 kb) from chromosome 18 was more efficient (2/2 cell lines). Thus the larger arrays of alphoid DNA gave higher percentages of cell lines with minichromosomes. However, smaller arrays may be preferable for gene expression as there appeared to be more EGFP expression from these minichromosomes.

Centromere↗
PubMed2001

Alpha-thalassaemia prenatal diagnosis by two PCR-based methods.

In Cyprus all couples carrying alpha0-thalassaemia mutations are detected in the course of the thalassaemia carrier screening program and prenatal diagnosis is offered to all of them. Prenatal diagnosis for alpha-thalassaemia is routinely done by two independent molecular methods. With the first method, the mutations of the parents are directly determined by gap-PCR and then the chorionic villus sample (CVS) is examined for the presence of these mutations. With the other method, a (CA)n repeat polymorphic site located between the psialpha1- and alpha2-globin genes is used for determining the presence or absence of the normal and mutant alleles. In the period from 1995 to 1999, molecular analysis of 46 couples in which haematological data were consistent with deletion of two alpha-globin genes in both partners indicated that only 13 of them were actually at risk for haemoglobin (Hb) Bart's hydrops fetalis and prenatal diagnosis was provided in 16 pregnancies. The molecular diagnosis was possible in all cases with the use of both gap-PCR and (CA)n repeat polymorphisms analysis. No misdiagnosed cases for alpha-thalassaemia have been reported to date.

Adult↗
PubMed2001

Mapping of the second Friedreich's ataxia (FRDA2) locus to chromosome 9p23-p11: evidence for further locus heterogeneity.

Friedreich's ataxia (FRDA), the most-common form of autosomal recessive ataxia, is inherited in most cases by a large expansion of a GAA triplet repeat in the first intron of the frataxin (X25) gene. Genetic heterogeneity in FRDA has been previously reported in typical FRDA families that do not link to the FRDA locus on chromosome 9q13. We report localization of a second FRDA locus (FRDA2) to chromosome 9p23-9p11, and we provide evidence for further genetic heterogeneity of the disease, in a family with the classic FRDA phenotype.

Adult↗
PubMed2001

A multi-center study in order to further define the molecular basis of beta-thalassemia in Thailand, Pakistan, Sri Lanka, Mauritius, Syria, and India, and to develop a simple molecular diagnostic strategy by amplification refractory mutation system-polymerase chain reaction.

The spectrum of the beta-thalassemia mutations of Thailand, Pakistan, India, Sri Lanka, Mauritius and Syria has been further characterized by a multi-center study of 1,235 transfusion-dependent patients, and the mutations discovered used to assess the fidelity of a simple diagnostic strategy. A total of 44 beta-thalassemia mutations were identified either by allele-specific oligonucleotide hybridization, amplification with allele-specific primers, or DNA sequencing of amplified product. The results confirm and extend earlier findings for Thailand, Pakistan, India, Mauritius and Syria. This is the first detailed report of the spectrum of mutations for Sri Lanka. Two novel mutations were identified, codon 55 (-A) and IVS-I-129 (A-->C), both found in Sri Lankan patients. Two beta-thalassemia mutations were found to coexist in one beta-globin gene: Sri Lankan patients homozygous for the beta0 codon 16 (-C) frameshift were also homozygous for the beta+ codon 10 (C-->A) mutation. Studies of Sri Lankan, Pakistani, and Indian carriers suggest the codon 10 (C-->A) mutation is just a rare polymorphism on an ancestral allele, on which the beta0 codon 16 (-C) mutation has arisen. Each country was found to have only a few common mutations accounting for 70% or more of the beta-thalassemia alleles. A panel of primers to diagnose the majority of the mutations by the amplification refractory mutation system was developed, enabling a simple molecular diagnostic strategy to be introduced for each country participating in the multi-center study.

Asia↗
PubMed2001

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non-invasive prenatal diagnosis.

Fetal nucleated red blood cells (n-rbc) occur in the maternal circulation from 7 weeks of pregnancy. The enrichment of these cells from maternal blood will depend upon their stage of differentiation, which changes during development. We characterised the fetal n-rbc from chorionic villus sample (CVS) washings and used them to model first trimester non-invasive prenatal diagnosis. The ratio of epsilon- to gamma-globin-producing cells declined rapidly from 10 to 13 weeks, as did the ratio of nucleated to non-nucleated rbc. By 13 weeks the great majority of cells containing gamma- or epsilon-globin are anucleate. The fetal n-rbc were highly variable in size and density and sedimented over a wide density range with a high proportion (>80%) at a density overlapping with that of maternal rbc. We have devised an enrichment procedure using Orskoff lysis to differentially lyse the maternal cells followed by density centrifugation and separation using magnetic beads. This simple protocol allowed recovery of 70% (69+/-22%) of fetal cells when added at approximately 10 fetal cells/ml maternal blood. When 1 fetal cell/ml millilitre maternal blood was added (total volume 10 ml) the recovery was more variable but remained at approximately 70% (72+/-47%), with at least one fetal cell recovered in all cases.

Cell Nucleus↗
PubMed2000

Molecular characterization of beta-thalassemia in Syria.

This study concerns the determination of beta-thalassemia alleles and other hemoglobin variants in 82 patients from Syria. We have characterized 146 chromosomes and found 17 different beta-thalassemia mutations, and one beta-globin chain variant that gives rise to the abnormal Hb S. The eight most common beta-thalassemia mutations were the IVS-I-110 (G-->A), IVS-I-1 (G-->A), codon 5 (-CT), -30 (T-->A), codon 39 (C-->T), IVS-I-6 (T-->C), IVS-II-1 (G-->A), and codon 15 (TGG-->TAG). These mutations accounted for almost 75% of the total beta-thalassemia chromosomes. We identified 34 different genotypes with a high level of homozygosity. The various beta-thalassemia mutations were characterized using gene amplification with specific oligonucleotide primers, restriction enzyme analysis, denaturing gradient gel electrophoresis and direct sequencing. By combining these three approaches we were able to detect mutations in almost 90% of the chromosomes studied. Our findings provide a sound foundation on which to base a preventive program for thalassemia and we believe that the data that we present will facilitate the improvement of medical services such as carrier screening, genetic counseling, and prenatal diagnosis. Furthermore a detailed knowledge of the molecular pathology of beta-thalassemia will strongly improve the prenatal diagnosis services in Syria.

Adolescent↗
PubMed1999

Genetic variation and intergenerational FMR1 CGG-repeat stability in 100 unrelated three-generation families from the normal population.

In order to identify genetic factors governing expansion of the CGG repeat in the FMR1 gene and to determine what predisposes or causes a normal stable allele to change to an unstable premutation allele, it is essential to study and understand the basis of normal variation. The aim of this study was to investigate genetic variation and intergenerational stability of the FMR1 CGG-repeat region in 100 unrelated three-generation families from the general population (651 meioses). The number of CGG-repeats in the FMR1 gene was determined in all 750 individuals from the 100 families (a total of 1,132 X-chromosomes), and the allele frequencies and variability were analyzed. Thirty-six different alleles (12-60 repeats) were seen with 30 (45.8%) as the most common allele; overall female heterozygosity was 73%. Most (>96%) of the normal array lengths were less than 40 repeats. Fifteen families with at least one allele equal to or greater than 40 repeats (40-60) were identified; in one of these families there was an increase of one triplet repeat during transmission from a mother to son. These findings, together with future molecular analyses, may provide data to test proposed models that attempt to explain the mutational process and the population dynamics of the triplet repeat region of the FMR1 gene, including the transition from normal to unstable alleles, or to test other putative cis-acting sequences that may be involved with instability in the FMR1 gene.

Alleles↗
PubMed1999

Genetic and physical analyses of the centromeric and pericentromeric regions of human chromosome 5: recombination across 5cen.

Human centromeres are poorly understood at both the genetic and the physical level. In this paper, we have been able to distinguish the alphoid centromeric sequences of chromosome 5 from those of chromosome 19. This result was obtained by pulsed-field gel electrophoresis after cutting genomic DNA with restriction endonucleases NcoI (chromosome 5) and BamHI (chromosome 19). We could thus define a highly polymorphic marker, representing length variations of the D5Z1 domain located at the q arm boundary of the chromosome 5 centromere. The centromeric region of chromosome 5 was then analyzed in full detail. We established an approximately 4.6-Mb physical map of the whole region with five rare-cutting enzymes by using nonchimeric YACs, two of which were shown to contain the very ends of 5cen on both sides. The p-arm side of 5cen was shown to contain an alphoid subset (D5Z12) different from those described thus far. Two genes and several putative cDNAs could be precisely located close to the centromere. Several L1 elements were shown to be present within alpha satellites at the boundary between alphoid and nonalphoid sequences on both sides of 5cen. They were used to define STSs that could serve as physical anchor points at the junction of 5cen with the p and q arms. Some STSs were placed on a radiation hybrid map. One was polymorphic and could therefore be used as a second centromeric genetic marker at the p arm boundary of 5cen. We could thus estimate recombination rates within and around the centromeric region of chromosome 5. Recombination is highly reduced within 5cen, with zero recombinants in 58 meioses being detected between the two markers located at the two extremities of the centromere. In its immediate vicinity, 5cen indeed exerts a direct negative effect on meiotic recombination within the proximal chromosomal DNA. This effect is, however, less important than expected and is polarized, as different rates are observed on both arms if one compares the 0 cM/Mb of the p proximal first 5.5 Mb and the 0.64 cM/Mb of the q proximal first 5 Mb to the sex-average 1.02 cM/Mb found throughout the entire chromosome 5. Rates then become close to the average when one goes further within the arms. Finally, most recombinants (21/22), irrespective of the arm, are of female origin, thus showing that recombination around 5cen is essentially occurring in the female lineage.

Blotting, Southern↗
PubMed1999

Novel splice site mutation at IVS8 nt 5 of HEXB responsible for a Greek-Cypriot case of Sandhoff disease.

Sandhoff disease is caused by abnormalities in HEXB gene encoding the beta-subunit of beta-hexosaminidase. In this study, we analyzed the HEXB gene of a Sandhoff carrier in the Greek-Cypriot community. A G to C transversion was identified in one allele of her HEXB gene at position 5 of the 5'-splice site of intron 8 (IVS8 nt5). One of 13 cDNA clones derived from her lymphocyte HEXB mRNA lacked the last four nucleotides "GTTG" of exon 8, which created a premature termination codon at 11 codons downstream. In vivo transcription of the mutant HEXB gene fragment in CHO cells resulted in deletion of the "GTTG." The mutation has not been found in 40 DNA samples from anonymous donors, indicating that this is not a polymorphism in the Cypriot population. These results clearly indicate that the splice site mutation at IVS8 nt5 is responsible for this case of Sandhoff disease.

Bacterial Proteins↗
PubMed1999

Comparative in vivo expression of beta(+)-thalassemia alleles.

Double heterozygotes who inherit one abnormal though stable beta-globin variant in association with a molecularly identified beta(+)-thalassaemia allele provide unique opportunities to quantify the in vivo expression of particular beta(+)-thalassemia alleles. The globin products of the two alleles can be separated, quantified and the output of the beta(+)-thalassaemia allele expressed as the MCH-beta(A) in pg beta(A)-globin/beta(+)-thalassemia allele/RBC = 0.5 MCH x Hb A%. In this communication we provide new quantitative data on the expression of five mutations as follows: the beta(+)-87 (C-->G) = 3.8 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-1 (G-->A) = 0.2 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 1); the beta(+) IVS-I-6 (T-->C) = 2.9 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 7); the beta(+) IVS-I-110 (G-->A) = 1.1 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 13), and the beta(+) IVS-II-745 (C-->G) = 1.74 pg beta(A)-globin/beta(+)-thalassemia allele/RBC (n = 2). The values obtained are compared with those of other beta(+)-thalassemia alleles from the literature. It can be seen that the MCH-beta(A) value may be a correct index of thalassemia severity useful for the correlation of genotype with phenotype, and for understanding the effects of mutations in beta-globin genes on pathophysiologically meaningful beta-globin gene expression.

Adolescent↗
PubMed1998

A humane alternative to the measurement of the lethal effects (LD50) of non-neurotoxic venoms using hens' eggs.

The accurate measurement of venom lethality is the basis of clinical treatment of snakebite and of much venom-related research. Lethality tests are necessarily carried out in animal models and the results extrapolated to man. While we may be confounded by the obvious limitations of this approach, we can improve the situation by using a non-sentient living system, such as the very early developmental stage of the chick embryo, as an alternative to lethality testing in mammals. The continuing need for lethality testing of venoms and their isolated components, which underpins the development and assessment of antivenoms, currently accounts for thousands of mice annually; this is becoming increasingly unacceptable, first because of the amount of suffering caused and second, because of the high cost incurred. We describe here the use of 4 6 d old chick embryos as a system for estimating venom lethality. The shell-less yolk sac membrane offers a vascular system which develops before intact nervous reflex arcs are functional and therefore the embryo is incapable of experiencing pain. Venom is applied to the membrane on a filter paper disc and its effects on vascular and cardiac function are easily observed throughout the 6 h experiment. Eight venoms tested on eggs and by conventional LD50 assays in mice were compared. A highly significant correlation was obtained suggesting that this simple and inexpensive test would be a far more acceptable alternative for non-neurotoxic venoms.

Animal Testing Alternatives↗
PubMed1998

Neonatal screening for Duchenne muscular dystrophy: a novel semiquantitative application of the bioluminescence test for creatine kinase in a pilot national program in Cyprus.

The objectives of this study were to evaluate a novel semiquantitative application of the bioluminescence test for screening newborns for Duchenne muscular dystrophy (DMD) and to use this technique in a pilot national program. The study was performed on the island of Cyprus, which provides ideal conditions for maximizing the prevention rate due to the small size of the country, the well-defined population, and the high degree of awareness of the public concerning genetic diseases. Guthrie spots were obtained through the national screening center for phenylketonuria and congenital hypothyroidism. The bioluminescence method for measuring creatine kinase (CK) in dried blood spots was adapted for use in a semiquantitative way. During the first 6 years of the program (1992-1997), we screened 30,014 samples and found 43 with initially high CK values. We were able to obtain repeat specimens in 35 cases. Of the repeat samples, 30 were found to have normal activity, giving a false-positive rate of 0.10%. Five boys had persistent CK elevations and were confirmed to be DMD or Becker (BMD) cases by DNA analysis and/or dystrophin analysis. The semiquantitative application of the bioluminescence assay of CK that we have introduced has proved to be a fast and reliable method for screening large numbers of samples for DMD. It has a low rate of false positives, which compares favorably with that of other DMD screening programs. Although it is early to evaluate its impact fully, the program seems to be bringing about the anticipated benefits to affected families.

Creatine Kinase↗
PubMed1998

Chromosome 1 localization of a gene for autosomal dominant medullary cystic kidney disease.

There is a group of inherited cystic nephropathies that are characterized by juvenile onset recessive inheritance (familial juvenile nephronophthisis, FJN) or by adult onset dominant inheritance (medullary cystic disease, MCD) and share similar clinico-pathological presentation to the extent that they are usually grouped together under the term FJN/MCD complex. The main symptoms consist of renal cyst formation in the medulla or the corticomedullary junction and salt wasting. Although earlier reports had suggested that one single gene may be responsible for this pathology, recent reports have shown that the FJN complex itself comprises a genetically heterogeneous group. Here we are presenting two large Cypriot families that segregate autosomal dominant medullary cystic kidney disease (ADMCKD) with hyperuricemia and gout and with very late age of onset (mean 62.2 and 51.5 years). We performed DNA linkage mapping using highly polymorphic microsatellite markers and found linkage to marker locus D1S1595 at 1q21 with a two-point lod score of 6.45 at Theta = 0.00. Analysis of haplotypes and of critical recombinants enabled confinement of the disease locus within an approximately 8 cM region between marker loci D1S498 and D1S2125. FISH mapping with a large P1 clone confirmed the physical localization within 1q21. The two families share the same disease haplotype, thus suggesting their relationship through a common ancestor and the possible existence of a single ADMCKD-causing mutation within these families. To our knowledge this is the first genetic locus identified to cause FJN/MCD pathology of the dominant adult type.

Chromosome Mapping↗
PubMed1997

New coagulation factor V gene polymorphisms define a single and infrequent haplotype underlying the factor V Leiden mutation in Mediterranean populations and Indians.

Two novel polymorphisms were identified in the factor V gene by direct sequencing of intronic areas. One of them, located in intron 9, is the marker closest to the Leiden mutation ever described, whereas the other, in intron 16, displays a rare allele invariantly associated to the mutation. Allele-specific amplification protocols were designed to perform extensive screenings for both polymorphic sites. The new markers were used in combination with six previously described polymorphisms to define specific factor V gene haplotypes. Haplotype investigations in 506Q homozygous thrombotic patients and normal controls showed the presence of a single haplotype underlying the factor V Leiden mutation in Mediterranean populations (among which Greek Cypriots, where the R506Q mutation is particularly frequent) and Indians. When traced in the absence of the Leiden mutation, the background haplotype was found to be present and roughly as frequent as the mutation itself in these populations. These findings indicate a single mutational event, that probably occurred outside Europe, as the cause of the Leiden mutation and provide a powerful tool to investigate its evolutionary history.

Cyprus↗
PubMed1995

alpha-Thalassaemia in the population of Cyprus.

We have determined the alpha-thalassaemia (alpha-thal) determinants in 78 patients with Hb H disease from Cyprus; 25 were Turkish Cypriots and 53 were Greek Cypriots. Four deletional and three non-deletional alpha-thal alleles were present; the -alpha(3.7 kb) alpha-thal-2 and the --MED-I alpha-thal-1 were most frequently seen; --MED-II and -(alpha)20.5 deletions occurred at considerably lower frequencies. About 15% of all chromosomes carried a non-deletional alpha-thal-2 allele; of these the 5 nucleotide (nt) deletion at the first intervening sequence (IVS-I) donor splice site was present in approximately 8% of all chromosomes. Two types of polyadenylation signal (poly A) mutations were observed. No striking frequency differences were seen between Greek and Turkish Cypriot patients. Combinations of the various types of alpha-thal resulted in eight different forms of Hb H disease. The phenotypes were comparable except for great variations in the level of Hb H which was highest (average approximately 22%) in the 12 patients with the alpha 5nt alpha/--MED-I combination. One patient with the same form of Hb H disease but with an additional beta-thal (IVS-I-110,G-->A) heterozygosity had a most severe microcytosis and hypochromia with < 1% Hb H. Variations in the level of Hb H might correlate with the severity of the disease, although this was not evident from the haematological data.

Alleles↗