Decreased blood catalase activity is not related to specific beta-thalassemia mutations in Hungary.

INTRODUCTION: Thalassemia erythrocytes are exposed to oxidative stress especially to hydrogen peroxide, which is regulated with the enzyme catalase. The aim of this study was to examine blood catalase activity and the relationship of blood catalase and beta-thalassemia gene mutations. METHODS: Blood catalase activity, hemoglobin, HbA(2) , HbF, and beta-globin gene mutations were determined in 43 Hungarian patients with beta-thalassemia trait. RESULTS: Compared to controls, the beta-thalassemia trait patients showed a low mean (P < 0.001) of blood catalase (men: 84 ± 29 MU/L vs. sex-matched controls: 118 ± 18 MU/L and women: 74 ± 18 MU/L vs. 108 ± 114 MU/L) and a low mean of blood catalase-to-blood hemoglobin ratio (men: 0.72 ± 0.22 MU/g vs. 0.85 ± 0.12 MU/g, women: 0.77 ± 0.26 MU/g vs. 0.84 ± 0.11 MU/g). The HbA(2) determination showed high sensitivity and specificity for the detection of beta-thalassemia trait patients. Mutation analyses revealed 13 beta-thalassemia trait mutations, of which six have not been reported before in Hungarian beta-thalassemia trait patients. Each group of mutations revealed decreased (P < 0.01) mean of blood catalase and catalase-to-hemoglobin ratio. Acatalasemia mutations were not found in beta-thalassemia trait patients. CONCLUSION: The decrease in blood catalase activity might be due to the damaging effects of free radicals on the catalase protein. Consequently, these beta-thalassemia trait patients may be relatively susceptible to damage caused by oxidative stress.

Effect of C111T polymorphism in exon 9 of the catalase gene on blood catalase activity in different types of diabetes mellitus.

Hydrogen peroxide plays a major role in the pathomechanism of diabetes mellitus and its main regulator is enzyme catalase. The blood catalase and the C111T polymorphism in exon 9 was examined in type 1, type 2 and gestational diabetes mellitus. Compared to the control group (104.7 +/- 18.5 MU/l) significantly decreased (p < 0.001) blood catalase activities were detected in type 2 (71.2 +/- 14.6 MU/l), gestational (68.5 +/- 12.2 MU/l) diabetes mellitus and without change in type 1 (102.5 +/- 26.9 MU/l). The blood catalase decreased (p = 0.043) with age for type 2 diabetics and did not change (p>0.063) for type 1, gestational diabetic patients and controls. Blood catalase showed a weak association with hemoglobin A1c for type 1 diabetic patients (r = 0.181, increasing). The mutant T allele was increased in type 1 and gestational diabetes mellitus, and CT+TT genotypes showed decreased blood catalase activity for type 1 and increased activities for type 2 diabetic patients. The C111T polymorphism may implicate a very weak effect on blood catalase activity in different types of diabetes mellitus.

A new type of inherited catalase deficiencies: its characterization and comparison to the Japanese and Swiss type of acatalasemia.

Thirteen Hungarian families that exhibited inherited catalase deficiencies have been detected. Differences between the deficiencies reported from Hungary and the previously reported Swiss acatalasemia were characterized using biochemical analysis of the catalase proteins. Molecular biological methods were used to compare the previously reported types of catalase deficiencies in Japan and the Hungarian deficiencies. Three mutations (a GA insertion in exon 2, a G insertion in exon 2, and a T to G substitution in intron 7) are responsible for decreased catalase activity in 7 of the 13 Hungarian kindreds; the other 6 families have not yet been characterized. These are not the mutations observed in Japan. Changes in lipid and carbohydrate metabolism and the high incidence (12.7%) of diabetes mellitus in the Hungarian kindreds suggest that individuals with inherited catalase deficiency are at risk of atherosclerosis and diabetes mellitus. The Hungarian subjects were detected during screening of a large population for catalase activity; no overt disease state was associated with the deficiencies. We hypothesize that the increased risk of disease may be due to prolonged exposure to elevated levels of blood hydrogen peroxide due to the lack of normal removal of hydrogen peroxide by blood catalase.

A novel catalase mutation detected by polymerase chain reaction-single strand conformation polymorphism, nucleotide sequencing, and western blot analyses is responsible for the type C of Hungarian acatalasemia.

Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) screening was used for searching mutations of the catalase gene in two Hungarian hypocatalasemic families. A syndrome-causing mutation was found in a PCR product containing exon 7 and its boundaries. Nucleotide sequence analyses detected a G to T substitution at position 5 of intron 7. The effect of this splice site mutation was confirmed by Western blot analyses demonstrating a decreased catalase protein level in these patients. These findings represent a novel type (C) of catalase mutations in the Hungarian acatalasemic/hypocatalasemic patients.

Hereditary catalase deficiencies and increased risk of diabetes.

Partial or near-total lack of erythrocyte catalase activity is a rare condition, generally thought to be benign. However, little is known of the frequency of common diseases of adult onset in human beings with catalase deficiency. We report that, in a series of Hungarian patients with catalase deficiency, there is a higher frequency of diabetes than in unaffected first-degree relatives and the general Hungarian population. We speculate that quantitative deficiency of catalase might predispose to cumulative oxidant damage of pancreatic beta-cells and diabetes.

Anovel catalase mutation (a GA insertion) causes the Hungarian type of acatalasemia.

Acatalasemia, a deficiency of enzyme catalase, is an autosomal recessive syndrome with an incidence of 5:106 in Hungary. We have examined the first Hungarian acatalasemic family for the disease-causing mutation. All exons of the catalase gene were screened by PCR-SSCP, PCR-heteroduplex, and nucleotide sequence analysis. The heteroduplex formation detected in exon 2 was verified by nucleotide sequence analysis. We found a GA insertion at nucleotide position 138, increasing the GA repeat number from 4 to 5. This GA insertion caused a frameshift in the amino acid sequence from position 68 to 133 and generated a TGA terminating codon at amino acid position 134. This truncated protein lacks the essential amino acid (histidine 74) in the active center. This finding can explain the decreased blood catalase activity in the Hungarian acatalasemic family.

Genetic heterogeneity of the 5' uncoding region of the catalase gene in Hungarian acatalasemic and hypocatalasemic subjects.

The 5' uncoding region (165 bp), exon 1 (63 bp) and part of intron 1 (20 bp) of the catalase gene was amplified by PCR in acatalasemic (2), hypocatalasemic (19) patients and healthy individuals (10). The single strand conformational polymorphism of PCR products showed a highly polymorphic pattern. This polymorphism was supported by nucleotide sequence analyses yielding eight mutations. They are A to T, C to A and C to T at positions -21, -20, -18 of the 5' flanking region, T to C at positions 4, 44, 49 of the non-coding region and C to T and C to A at positions 12, 27 of exon 1. Of these nucleotide substitutions, the fourth, fifth, seventh and eighth are novel mutations. The mutations 1, 3, 6, 8 were present at least at heterozygous level in all acatalasemics and hypocatalasemics. None of these mutations may be the causal mutation(s) of acatalasemia as each of these nucleotide substitutions were detected in healthy subjects with normal blood catalase activity.

Polymorphism of 5' of the catalase gene in Hungarian acatalasemia and hypocatalasemia.

The amplified fragment length polymorphism of Hinf1 on the promoter region of the catalase gene in Hungarian acatalasemic and hypocatalasemic patients yielded three different patterns with five bands in total. The sequence analyses revealed A-to-T, C-to-A, and C-to-T mutations at positions -21, -20, and -18 upstream of the translational initiation site. The -21 A-to-T mutations were more frequent in acatalasemic and hypocatalasemic patients (36/2) than in controls (18/14). This mutation had been detected in Japanese acatalasemic patients while the other two are novel mutations. Two extra bands in the Hinf1 pattern are due to star-like activity that cleaved a G/ATTT sequence at position -4 to 0 upstream of the initiation site.

Reference ranges of normal blood catalase activity and levels in familial hypocatalasemia in Hungary.

In 1756 healthy individuals the mean and S.D. values of blood catalase activity were 111.3 +/- 16.5 MU/l with lower blood catalase for females (107.7 +/- 14.4 MU/l, n = 880) than for males (117.9 +/- 16.8 MU/l, n = 876) while the ratios of blood catalase activity to blood hemoglobin concentration were not different (0.841 +/- 0.107 MU/g versus 0.849 +/- 0.119 MU/g). The decrease of blood catalase with age was greater in males (b = -0.084 MU/l year) than in females (b = -0.016 MU/l year). The screening of 3300 healthy citizens for hypocatalasemia yielded six families (0.18%), and three families were identified out of 1630 clinic patients. These nine families revealed 37 hypocatalasemic patients with 57.5 +/- 11.7 MU/l mean and S.D. of blood catalase activity. Similarly to the Japanese and the Hungarian actalasemic patients, the electrophoretic mobilities of catalase in erythrocytes of hypocatalasemic patients were indistinguishable from that of healthy controls.

Further genetic heterogeneity in acatalasemia.

A T-deletion at position 10 of exon 4 for catalase gene was reported as a novel mutation, causing a new genetic type of acatalasemia in Japan. This mutation, destroying a Hinf1 recognition site, was searched for in Hungarian acatalasemic (2) and hypocatalasemic (22) patients and in controls (27) by Hinf1 digestion and sequence analyses of a 203 bp polymerase chain reaction (PCR) product containing the entire exon 4. The Hinf1 polymorphism did not reveal any difference between controls and hypocatalasemic as well as acatalasemic patients. These results were confirmed by sequence analyses showing the T nucleotide for the two acatalasemic and for one unrelated hypocatalasemic patient, as well as for two controls. These findings represent further evidence that acatalasemia is heterogeneous at the DNA level.

Genetic heterogeneity in acatalasemia.

203 bp long products containing exon 4 and its junctions from the catalase gene were generated by polymerase chain reaction (PCR). These products were analyzed by single strand conformational polymorphism (SSCP), hetero-duplex formation and nucleotide sequencing. No polymorphism was detected when the Hungarian acatalasemic sisters, their family members and normocatalasemic controls were analyzed. Sequence analyses did not show the G to A point mutation at position 5 of intron 4. This splicing mutation characterizes the Japanese-type of acatalasemia.

Hungarian hereditary acatalasemia and hypocatalasemia are not associated with chronic hemolysis.

Two Hungarian acatalasemic and eight hypocatalasemic patients revealed normal erythropoesis. Contrary to their decreased defence system against the toxic hydrogen peroxide, the biochemical tests (serum catalase, serum hemoglobin, serum lactate dehydrogenase (LDH) ratio of LDH1 and LDH2 isoenzymes and serum haptoglobin) excluded hemolysis. The normal activity of glutathione peroxidase and the decreased catalase activity could prevent the lysis of the erythrocytes. In the presence of extremely high levels of hydrogen peroxide acute hemolysis may not be excluded; therefore, follow-up of these patients is required.

Further characterization of Hungarian acatalasemia by Hinf1 polymorphism of catalase gene.

An Hinf1 associated restriction length polymorphism pattern is reported for the catalase gene of Hungarian normocatalasemic individuals and acatalasemic patients. The 2.4-kb pCAT 10 probe revealed 9 bands (2.1, 1.5, 1.2, 1.1, 0.9, 0.8, 0.6, 0.5 and 0.4 kb) with 9 distinct patterns for the controls. The same patterns were detected for the Hungarian acatalasemic patients. The examination of the A to T mutation of the Hungarian acatalasemic patients and their relatives at position -21 in the flanking region with Hinf1 polymorphism could not reveal any difference between the acatalasemic and the normocatalasemic catalase gene.