Axonal sensorimotor neuropathy in patients with beta-thalassaemia.

OBJECTIVE: The purpose of this study was to investigate the prevalence of peripheral neuropathy in patients with beta-thalassaemia. METHODS: Thirty six patients with a mean age of 29.2+/-8.2 years and 17 healthy controls with a mean age of 27.6+/-9.1 were included in this study. Measurements included the neuropathy symptoms score (NSS), the neuropathy disability score (NDS) as well as nerve conduction studies of two motor (ulnar and peroneal) and two sensory (ulnar and sural) nerves of the right limbs. RESULTS: A mainly sensory axonal polyneuropathy was present in 19 out of 36 patients (52.7%). Eight out of these 19 patients also had abnormal NDS values. The neuropathy correlated significantly with the age of the patients and the hematocrit. However, it did not correlate with the presence of antibodies against HCV, the ferritin levels, or with a history of transfusions, desferrioxamine treatment, or splenectomy. CONCLUSIONS: This study showed a high prevalence of a predominantly sensory neuropathy in patients with beta-thalassaemia. The electrophysiological data suggest that the underlying pathology is an axonopathy. Chronic hypoxia of the nerves resulting from severe anaemia may contribute to the pathogenesis of this neuropathy.

Refining the primary open-angle glaucoma GLC1C region on chromosome 3 by haplotype analysis.

The GLC1C locus for primary open-angle glaucoma (POAG) is inherited as an autosomal dominant trait. This region on chromosome 3 is 11 cM long. DNA samples from members of a Greek and an American GLC1C family were obtained to determine whether additional typing of microsatellite markers in family members might narrow the region. GLC1C family members were evaluated clinically for POAG on the basis of open angles, intraocular pressures, cupping of discs, and visual fields. DNA samples from the Greek and Oregon GLC1C families were used to further refine the GLC1C region using microsatellite markers. A total of 22 affected members were identified in the two families. Common alleles for D3S3637 and D3S3612 were present in the disease haplotype from both families, suggesting that they may have a common founder. A newly diagnosed patient in the American family had a recombination in the distal portion of the GLC1C haplotype. This recombination narrows the GLC1C region from 11 to 4 cM.

Genetic linkage of autosomal dominant primary open angle glaucoma to chromosome 3q in a Greek pedigree.

A locus for juvenile onset open angle glaucoma (OAG) has been assigned to chromosome 1q in families with autosomal dominant inheritance (GLC1A), due to mutations in the TIGR/MYOC gene. For adult onset OAG, called primary open angle glaucoma or POAG, five loci have so far been mapped to different chromosomes (GLC1B-GLC1F). Except for the GLC1B locus, the other POAG loci have so far been reported only in single large pedigrees. We studied a large family identified in Epirus, Greece, segregating POAG in an autosomal dominant fashion. Clinical findings included increased cup to disc ratio (mean 0.7), characteristic glaucomatous changes in the visual field, and intraocular pressure before treatment more than 21 mmHg (mean 31 mmHg), with age at diagnosis 33 years and older. Linkage analysis was performed between the disease phenotype and microsatellite DNA polymorphisms. Linkage was established with a group of DNA markers located on chromosome 3q, where the GLC1C locus has previously been described in one large Oregon pedigree. A maximal multipoint lod score of 3.88 was obtained at marker D3S1763 (penetrance 80%). This represents the second POAG family linked to the GLC1C locus on chromosome 3q, and haplotype analysis in the two families suggests an independent origin of the genetic defect.

Prevalence of factor V Leiden, prothrombin G20210A, and MTHFR C677T mutations in a Greek population of blood donors.

The pathogenesis of venous thrombosis involves the interaction of genetic and environmental factors. In order to estimate the frequency of the factor V Leiden, the prothrombin G20210A, and the MTHFR C677T mutations in the Greek population, we analyzed 160 healthy Greek blood donors by PCR amplification and detected allele frequencies of 2.5%, 2.2%, and 35.3%, respectively. The allele frequencies were compared with reported frequencies of other populations of southern Europe. The identification of these common genetic risk factors for thrombosis should enable easy DNA diagnosis and carrier detection in a high proportion of cases and will contribute to a better understanding of the interaction of genetic and environmental risk factors.

Apolipoprotein E epsilon4 allele as a genetic risk factor for left ventricular failure in homozygous beta-thalassemia.

In homozygous beta-thalassemia, the organ damage is mainly attributed to excessive iron deposition through the formation of oxygen free radicals. Despite appropriate transfusion and chelation therapy and low ferritin levels, patients still develop organ failure, heart failure being the main cause of death. This study was designed to determine whether the decreased antioxidant activity of the apolipoprotein E (APOE) 4 allele could represent a genetic risk factor for the development of left ventricular failure (LVF) in beta-thalassemia homozygotes. A total of 251 Greek beta-thalassemia homozygotes were studied. Patients were divided in three groups: group A (n = 151) with no cardiac impairment, group C (n = 47) with LVF, and 53 patients with LV dilatation and normal LV systolic function constituted the group B. DNA was obtained from all patients, and the polymerase chain reaction was used to analyze the polymorphism at the APOE locus. The APOE allele frequencies were compared with those of a Greek control sample of 216 healthy blood donors. Patients with no cardiac impairment had an APOE 4 allele frequency (7.9%) not different from population controls (6.5%, P > .05), while patients with LVF had a significantly higher frequency of APOE 4 (12.8%) than the controls (P < .05, odds ratio = 2.11, 95% confidence interval 1.03 to 4.32). The APOE 4 allele may represent an important genetic risk factor for the development of organ damage in homozygous beta-thalassemia.

Apolipoprotein E polymorphism in the Greek population.

The APOE gene is located on chromosome 19, and the three common alleles are designated epsilon2, epsilon3, and epsilon4. The epsilon4 allele is associated with increased plasma cholesterol, atherosclerosis and cardiovascular disease, Alzheimer's disease, and decreased longevity. The objective of the present study was to estimate the distribution of APOE alleles in the Greek population by DNA analysis. The material consisted of 216 voluntary, healthy Greek blood donors (146 males/70 females). The APOE allele frequencies were epsilon2: 5.3%, epsilon3: 88.2%, epsilon4: 6.5%. The epsilon4 allele frequency of 6.5% in the Greek population is, together with the frequency in the Chinese population, among the lowest in the world.

Exclusion of one pedigree affected by adult onset primary open angle glaucoma from linkage to the juvenile glaucoma locus on chromosome 1q21-q31.

A locus for autosomal dominant juvenile onset primary open angle glaucoma (POAG) was recently assigned to chromosome region 1q21-q31. In the present study, a large Greek family with autosomal dominant adult onset POAG was investigated using microsatellite markers. Exclusion of linkage of the adult onset POAG gene to the region D1S194-D1S191 was obtained in this pedigree. Therefore, the data provide evidence that juvenile and adult onset POAG are genetically distinct disease entities.

Molecular basis for nonphenylketonuria hyperphenylalaninemia.

Nonphenylketonuria hyperphenylalaninemia (non-PKU HPA) is defined as phenylalanine hydroxylase (PAH) deficiency with blood phenylalanine levels below 600 mumol/liter (i.e., within the therapeutic range) on a normal dietary intake. Haplotype analysis at the PAH locus was performed in 17 Danish families with non-PKU HPA, revealing compound heterozygosity in all individuals. By allele-specific oligonucleotide (ASO) probing for common PKU mutations we found 12 of 17 non-PKU HPA children with a PKU allele on one chromosome. To identify molecular lesions in the second allele, individual exons were amplified by polymerase chain reaction and screened for mutations by single-strand conformation polymorphism. Two new missense mutations were identified. Three children had inherited a G-to-A transition at codon 415 in exon 12 of the PAH gene, resulting in the substitution of asparagine for aspartate, whereas one child possessed an A-to-G transition at codon 306 in exon 9, causing the replacement of an isoleucine by a valine in the enzyme. It is further demonstrated that the identified mutations have less impact on the heterozygote's ability to hydroxylate phenylalanine to tyrosine compared to the parents carrying a PKU mutation. The combined effect on PAH activity explains the non-PKU HPA phenotype of the child. The present observations that PKU mutations in combination with other mutations result in the non-PKU HPA phenotype and that particular mutation-restriction fragment length polymorphism haplotype combinations are associated with this phenotype offer the possibility of distinguishing PKU patients from non-PKU individuals by means of molecular analysis of the hyperphenylalaninemic neonate and, consequently, of determining whether a newborn child requires dietary treatment.

Molecular characterization of mild-to-moderate hemophilia A: detection of the mutation in 25 of 29 patients by denaturing gradient gel electrophoresis.

To date it has been difficult to characterize completely a genetic disorder, such as hemophilia A, in which the involved gene is large and unrelated affected individuals have different mutations, most of which are point mutations. Toward this end, we analyzed the DNA of 29 patients with mild-to-moderate hemophilia A in which the causative mutation is likely to be a missense mutation. Using computer analysis, we determined the melting properties of factor VIII gene sequences to design primer sets for PCR amplification and subsequent denaturing gradient gel electrophoresis (DGGE). A total of 45 primer sets was chosen to amplify 99% of the coding region of the gene and 41 of 50 splice junctions. To facilitate detection of point mutations, we mixed DNA from two male patients, and both homoduplexes and heteroduplexes were analyzed. With these 45 primer sets, 26 DNAs containing previously identified point mutations in the factor VIII gene were studied, and all 26 mutations were easily distinguishable from normal. After analyzing the 29 patients with unknown mutations, we identified the disease-producing mutation in 25 (86%). Two polymorphisms and two rare normal variants were also found. Therefore, DGGE after computer analysis is a powerful method for nearly complete characterization of disease-producing mutations and polymorphisms in large genes such as that for factor VIII.