Prevalence of genetic mutations that predispose to thrombophilia in a Greek Cypriot population.

Several hereditary disorders, particularly those affecting the physiological anticoagulation systems, have been well established as risk factors for venous thromboembolism. In the present study, we investigated the prevalence of the following thrombogenic mutations in a Greek-Cypriot population: the G1691 factor V Leiden mutation, the G20210A mutation in the prothrombin gene, and the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR). All three variants have been documented to be significant risk factors for various cardiovascular conditions. Ninety unrelated subjects were screened. For the Leiden mutation, 11 subjects (12.2%) were heterozygous and one (1.1%) was homozygous. Seven subjects (7.8%) were heterozygous for the G20210A variant in prothrombin; no homozygotes were identified. The C677T mutation in MTHFR was found in 40 individuals in the heterozygous state (44.4%), and in 16 individuals in the homozygous state (17.8%). These data demonstrate that Greek-Cypriots have an increased frequency of thrombogenic mutations, and suggest that screening for these mutations should be seriously considered, especially when surgery or pregnancy is planned. This is the first study for the frequency of mutations in risk factors that predispose to thrombophilia on the island of Cyprus.

Germinal and somatic mutations in the PKD2 gene of renal cysts in autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in one of three genes: PKD1 on chromosome 16 accounts for approximately 85% of cases whereas PKD2 on chromosome 4 accounts for approximately 15%. Mutations in the PKD3 gene are rare. All patients present with similar clinical phenotypes, and the cardinal symptom is the formation of fluid-filled cysts in the kidneys. Previous work has provided data supporting the notion that cysts in ADPKD1 are focal in nature and form after loss of function of polycystin 1. This became evident by demonstrating that the normal PKD1 allele was inactivated somatically by loss of heterozygosity or by mutagenesis in a subset of renal or liver cysts examined. We show in this report, for the first time, multiple novel somatic mutations within the PKD2 gene of epithelial cells, in both kidneys of an ADPKD2 patient. From a total of 21 cysts examined, seven (33%) had the same C insertion within the inherited wild-type allele. In two other cysts, a nonsense mutation and a splice site AG deletion had occurred in a PKD2 allele that could not be identified as the inherited wild-type or mutant. We suggest that the autosomal dominant form of ADPKD2 occurs by a cellular recessive mechanism, supporting a two-hit model for cyst formation.

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.

Evidence for high-risk haplotypes and (CGG)n expansion in fragile X syndrome in the Hellenic population of Greece and Cyprus.

The expansion of the trinucleotide repeat (CGG)n in successive generations through maternal meiosis is the cause of fragile X syndrome. Analysis of CA repeat polymorphisms flanking the FMR-1 gene provides evidence of a limited number of "founder" chromosomes and predisposing high-risk haplotypes related to the mutation. To investigate the origin of mutations in the fragile X syndrome in the Hellenic populations of Greece and Cyprus, we studied the alleles and haplotypes at DXS548 and FRAXAC2 loci of 16 independent fragile X and 70 normal control chromosomes. In addition, we studied 191 unrelated normal X chromosomes for the distribution and frequencies of CGG alleles. At DXS548, 6 alleles were found, 2 (194 and 196) of which were represented on fragile X chromosomes. At FRAXAC2, 6 alleles were found, 4 of which were present on fragile X chromosomes. Sixteen haplotypes were identified, but only 5 were present on fragile X chromosomes. The highest number of CGG repeats (> or = 33) were associated with haplotypes 194-147, 194-151, 194-153, and 204-155. The data provide evidence for founder chromosomes and high-risk haplotypes in the Hellenic population.

Complete coding sequence, exon/intron arrangement and chromosome location of ZNF45, a KRAB-domain-containing gene.

Zinc finger genes represent a large multigene family present in mammalian and other genomes. A subgroup of these genes contain a conserved motif, the KRAB domain, at the NH2-terminal region, which was recently shown to posses a potent transcriptional repression activity. For one such gene, ZNF45, we determined the complete coding sequence, along with the exon/intron arrangement and the splice junctions. Exon 2 codes exclusively for the KRAB-A element of 42 amino acids, and exon 4 contains the 19 zinc finger repeats. In addition, we generated an EST (expressed sequence tag) from the 3'-end of ZNF45 that we used for polymerase chain reaction screening of a P1-derived genomic library. We isolated a 65-kb clone that was used for localizing this gene on chromosome 19q13.2 by fluorescence in situ hybridization.

Genetic heterogeneity in adult dominant polycystic kidney disease in Cypriot families.

Polycystic kidney disease is an inherited heterogeneous disorder that affects approximately 1:1000 Europeans. It is characterized mainly by the formation of cysts in the kidney that lead to end-stage renal failure with late age of onset. Three loci have been identified, PKD1 on the short arm of chromosome 16, which has recently been isolated and characterized, PKD2 on the long arm of chromosome 4, and a third locus of unknown location, that is apparently much rarer. In families that transmit the PKD2 gene there is a significantly later age of onset of symptoms, compared with families that transmit the PKD1 gene, and in general they present with milder progression of symptomatology. For the first time we attempted molecular genetic analysis in seven Cypriot families using highly polymorphic markers around the PKD1 and PKD2 genes. Our data showed that there is genetic and phenotypic heterogeneity among these families. For four of the families we obtained strong evidence for linkage to the PKD1 locus. In two of these families linkage to PKD1 was strengthened by excluding linkage to PKD2 with the use of marker D4S423. In three other families we showed linkage to the PKD2 locus. In the largest of these families one recombinant placed marker D4S1534 distal to D4S231, thereby rendering it the closest proximal marker known to us to date. The application of molecular methods allowed us to make presymptomatic diagnosis for a number of at-risk individuals.

A base substitution at IVS-19 3'-end splice junction causes exon 20 skipping in pro alpha 2(I) collagen mRNA and produces mild osteogenesis imperfecta.

Molecular investigations on a young patient and her family were undertaken to identify the molecular defect responsible for a mild form of osteogenesis imperfecta (OI) with blue sclerae, dentinogenesis imperfecta and joint laxity. Analysis of collagenous proteins from the proband's fibroblasts showed the presence of two populations of alpha 2(I) chains, one normal and one migrating faster on SDS gels, thereby suggesting deletion of amino acid sequences. The faster migrating chains were retained mainly in the cell layer and not found in the extracellular matrix deposited by cultured fibroblasts. Chemical cleavage of mismatch (CCM) analysis on the patient's pro alpha 2(I) mRNA: normal cDNA heteroduplexes localized the molecular defect. cDNA sequencing revealed a deletion of exon 20 (54 bp) in about half of the molecules. Genomic DNA sequencing revealed heterozygosity for a G-to-C transversion of the last nucleotide of intron 19, which changed the 3' consensus splicing site. As a consequence pro alpha 2(I)mRNA was abnormally spliced from the last codon of exon 19 to the first codon of exon 21. To our knowledge, this is the first acceptor site mutation so far described in an OI patient. Restriction analysis indicated that the mutation was present also in three other affected family members. The full sequence of COL1A2 introns 19 and 20 are reported.

Mutation analysis of coding sequences for type I procollagen in individuals with low bone density.

Mutations in one of the two genes encoding type I procollagen (COL1A1 and COL1A2) are frequently the cause of osteogenesis imperfecta (OI), a disorder characterized by brittle bones. Here we tested whether patients with low bone density also have mutations in these genes. The 26 patients studied had no apparent metabolic bone disease, but most had a positive family history of osteopenia or osteoporosis. Although a diagnosis of OI was considered by the clinician in some cases, the clinical criteria for OI were not satisfied. Our strategy for mutation analysis consisted of PCR amplification of cDNA made to fibroblast mRNA using primers specific for the coding regions of COL1A1 and COL1A2. The PCR products were then sequenced directly with primers located within each PCR product. We found that 3 of 26 patients had mutations that altered the encoded amino acid. One mutation, at position alpha 2(I)-661 has been reported (Spotila et al. 1991 Proc Natl Acad Sci USA PNAS 88:5423). The other 2 patients, who were not related to each other, had a mutation that altered the proline codon at alpha 1(I)-27 to alanine. This mutation was not found in 81 normal individuals or in 37 additional osteopenic individuals. However, its effect on the biologic function of type I collagen, as well as its role in osteopenia, is uncertain. In addition to the two mutations, we found a polymorphism in codon alpha 2(I)-459. Although this polymorphism involved an amino acid substitution, it was present with equal frequency in the patient and the normal population. By analyzing this and previously reported neutral sequence variants in the COL1A2 gene, we determined that all patients expressed both alleles of the COL1A2 gene. The 12 patients who were heterozygous for a COL1A1 neutral sequence variant also expressed both alleles. Here we present all PCR primer and sequencing primer information. The results suggest that surveying a larger group of similarly selected individuals may reveal additional mutations in the COL1A1 or COL1A2 genes.

Novel cystic fibrosis mutation associated with mild disease in Cypriot patients.

Cyprus is an island in the eastern Mediterranean basin inhabited by people of Caucasian extraction, mostly Greek-Cypriots. The most common inherited disease among Caucasians is cystic fibrosis (CF). Although no careful scientific study had ever been done the impression was that CF was extremely rare among the Greek-Cypriots, with an incidence estimated at around 1:30,000. About 2 years ago, we introduced molecular diagnostic methodology in an effort to assist clinicians in safer diagnosis of patients presenting with atypical CF symptomatology, and also for testing the hypothesis that mutations that cause milder phenotypes might be responsible for misdiagnosis or for missing entirely some cases of CF. Initial screening for delta F508 revealed that it is indeed rare in the general population. Further screening of suspected CF patients revealed a novel mutation that converted leucine at position 346 to proline (L346P) in two unrelated families. The second CF mutation was delta F508 and 1677delTA in the two families respectively, both reportedly associated with severe phenotypes. Yet our patients did not present with typical CF pictures possibly because of the dominant nature of this novel mild mutation in exon 7. Symptoms included failure to thrive, chest infections and electrolyte disturbances. These findings raise the possibility that Cyprus might have been spared very severe CF phenotypes but not cystic fibrosis transmembrane conductance regulator (CFTR) mutations.

Chromosome 4 localization of a second gene for autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disorder. A gene defect located on the short arm of chromosome 16 is responsible for the disease in roughly 86% of affected European families. Using highly polymorphic microsatellite DNA markers, we have assigned a second gene for ADPKD to chromosome 4. In eight families with clear evidence against linkage to chromosome 16 markers, linkage analysis with the markers D4S231 and D4S423, demonstrated a multipoint lod score of 22.42.

Somatic cell mosaicism: another source of phenotypic heterogeneity in nuclear families with osteogenesis imperfecta.

Mutations in the genes coding for the pro alpha 1 and pro alpha 2 chains of type I procollagen have been found in many patients with osteogenesis imperfecta (OI), a heritable disorder of connective tissue. The severity of the disease varies between families and even among members of the same family. This phenotypic variability covers a spectrum extending from very mild forms that cannot be easily detected to perinatally lethal forms. One explanation for this phenotypic variability is the nature of the mutation in the type I procollagen genes. Another explanation is mosaicism. Here we report on 2 families with propositi who have OI, whereas their mothers had a milder form of the disease. In one family, the molecular defect was previously shown to be a substitution of alpha 1(904) by cysteine [Constantinou et al., 1990]. The biochemical phenotype was characterized by significant post-translational overmodification of the mutated type 1 collagen molecules which also had a 3-4 degrees C decrease in their thermal unfolding. Also, secretion of the procollagen into the culture media was delayed. In the second family, the proposita's muscle fibroblasts synthesized and secreted type I procollagen molecules that were highly over-modified along the entire length of their triple-helical domain. Cells from the mother also synthesized normal and over-modified protein, although the amount of over-modified protein was less than that synthesized by her daughter's cells. The exact molecular defect has not yet been defined.(ABSTRACT TRUNCATED AT 250 WORDS)

The identification and characterization of KRAB-domain-containing zinc finger proteins.

The zinc finger motif is a highly conserved tandemly repeated sequence of 28-30 amino acids that was first identified in transcription factor TFIIIA from Xenopus laevis. Subsequently, similar motifs were found and characterized in many genes from mammalian genomes and the genomes of lower eukaryotes such as Drosophila and yeast, thereby defining a large superfamily of genes. Non-finger-coding modules conserved among members of subfamilies of zinc finger genes have been described in the murine genome (finger-associated boxes, or FAX domain) and the human genome (Krüppel-associated boxes, or KRAB domain). Here we report the identification and partial characterization of more members of the human KRAB-containing subfamily of genes. Based on Southern blot hybridization experiments, they also are zinc-finger-coding genes. All members share a highly homologous 42-amino-acid-long A element of the described KRAB domain. The conservation extends to the murine developmentally expressed zinc finger gene, mKr2. The homologous sequences, however, are part of the 5'-untranslated region. In all cases for which there is adequate information, the KRAB domain is found at the NH2-terminus of the respective protein. In one zinc-finger-encoding cDNA clone that we characterized further in this work, BRc1744 (ZNF45), the KRAB domain most probably constitutes the entire second exon of the gene. Based on the data, it is tempting to speculate that the FAX- and KRAB-containing zinc finger genes define subfamilies of genes with overlapping functions that participate in the regulation of common or similar developmental programs.