Catalase Enzyme Mutations and their Association with Diseases

Mol Diagn 2004; 8 (3): 141-149 1084-8592/04/0003-0141/$31.00/0 REVIEW ARTICLE © 2004 Adis Data Information BV. All rights reserved. Catalase Enzyme Mutations and their Association with Diseases László Góth,1,2 Péter Rass3 and Anikó Páy4 1 2 3 4 Department of Clinical Analytical Chemistry, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary Sigma-Aldrich Ltd, Budapest, Hungary Biological Research Center, Hungarian Academy of Science, Szeged, Hungary Contents Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 2. Catalase Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 3. Catalase Gene and Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142 4. Benign Polymorphisms of Catalase Gene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 5. Association of Catalase Gene Mutations with Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 5.1 Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 5.2 Blood Pressure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 5.3 Vitiligo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 5.4 Alzheimers Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144 5.5 Decreased Catalase Activity in Tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6. Acatalasemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.1 Clinical Features of Acatalasemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 6.2 Catalase Gene Mutations in Acatalasemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 6.2.1 Japanese-Type Acatalasemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 6.2.2 Swiss-Type Acatalasemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 6.2.3 Hungarian-Type Acatalasemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 6.3 Other Catalase Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 7. Future Prospects for the Detection of Catalase Gene Mutations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Abstract Enzyme catalase seems to be the main regulator of hydrogen peroxide metabolism. Hydrogen peroxide at high concentrations is a toxic agent, while at low concentrations it appears to modulate some physiological processes such as signaling in cell proliferation, apoptosis, carbohydrate metabolism, and platelet activation. Benign catalase gene mutations of 5′ noncoding region (15) and intron 1 (4) have no effect on catalase activity and are not associated with disease. Catalase gene mutations have been detected in association with diabetes mellitus, hypertension, and vitiligo. Decreases in catalase activity in patients with tumors is more likely to be due to decreased enzyme synthesis rather than to catalase mutations. Acatalasemia, the inherited deficiency of catalase has been detected in 11 countries. Its clinical features might be oral gangrene, altered lipid, carbohydrate, homocysteine metabolism and the increased risk of diabetes Góth et al. 142 mellitus. The Japanese, Swiss, and Hungarian types of acatalasemia display differences in biochemical and genetic aspects. However, there are only limited reports on the syndrome causing these mutations. These data show that acatalasemia may be a syndrome with clinical, biochemical, genetic characteristics rather than just a simple enzyme deficiency. Catalase is an enzyme which converts two molecules of hydrogen peroxide into two molecules of water and one of oxygen. This heme-containing enzyme has been identified in the organs of bacteria through to humans; in humans it is distributed in virtually all aerobic tissue. During the first few decades of the last century the catalase enzyme was the subject of considerable biologic, chemical, clinical, and diagnostic research but its role remained obscure. In recent years further research has been undertaken into this ‘old’ enzyme and this paper discusses these recently identified aspects of the catalase enzyme. 2. Catalase Enzyme Enzyme catalase (C 1.11.1.6) is the main regulator of hydrogen peroxide metabolism.[1,2] For the erythrocytes and other tissues, such as the pancreas and heart, which have low catalase activity,[3-8] high concentrations of this enzyme in the erythrocytes provides a defense against high concentrations of hydrogen peroxide Recently published papers on the tissue distribution of catalase,[1,2,6] glutathione peroxidase,[9] and hemoglobin,[10,11] seem to confirm the predominant role that catalase has in the control of hydrogen peroxide concentrations. The enzymatic function of catalase has several unusual features: • its reaction with hydrogen peroxide is first order and depends entirely on the concentration of hydrogen peroxide; • at high substrate concentrations the rate of reaction is unusually rapid; • at low substrate concentrations, slow catalytic activity of the two hydrogen peroxide substrates, and probably with the peroxidatic activity with one hydrogen peroxide and one proton donor (ethanol), substrates play a role in the clearance of hydrogen peroxide.[4,11-13] These important catalase characteristics may help to explain the increasing body of evidence which indicates a new role for hydrogen peroxide as a messenger of signaling.[13-22] Hydrogen peroxide is formed by pathways such as oxidase enzymes, reactive oxygen species,[23] and human tumor cells.[24] © 2004 Adis Data Information BV. All rights reserved. Hydrogen peroxide appears to modulate the inflammatory process by regulating the expression of adhesive molecules, controlling cell proliferation, and apoptosis and modulating platelet aggregation.[25-31] A high concentration of hydrogen peroxide, due to a deficiency (acquired or inherited) of catalase, especially when reacted with redox-active metal ions such as iron or copper, yields the highly reactive hydroxyl radical in the Fenton and Haber-Weiss reaction. This radical is responsible for injury in the cell membrane, mitochondrial electron transport, homocysteine metabolism, and DNA.[32-37] Among the reactive oxygen species, hydrogen peroxide is regarded as a key substrate in oxidative stress because this small, diffusible molecule is stable under physiological conditions. Toxic effects (damage of DNA, protein, cell membrane) due to high concentrations of hydrogen peroxide can be decreased or abolished when extra catalase is added or generated.[18,32,38,39] 3. Catalase Gene and Protein The single locus coding for human catalase has been mapped to 11p13. The catalase gene is 34 kb in length and contains 12 introns, 13 exons, and encodes for a protein of 526 amino acids.[40,41] Human catalase protein is a tetramer composed of four identical subunits, each contains a heme group. The subunits have four domains: (i) an extended non-globular amino terminal arm, which stabilizes the quaternary structure; (ii) an anti-parallel eightstranded β barrel providing the residues on the distal side of the heme; (iii) a rather random ‘wrapping domain’ around the subunit exterior including the proximal heme ligand; and (iv) a final αhelix structure. The pentacoordinated iron-heme is accessible at the distal side to peroxides at the bottom of the 25Å long channel extending from the surface. This channel is critical for the molecular ruler recognition mechanism of hydrogen peroxide by the protein side-chains histidine (His)75, asparagine (Asn)148, glutamine (Glu)168, and aspartatic acid (Asp)128. The reactivity of heme iron is tuned by electron donation by the tryosine (Tyr)358 ligand and neutralization of the carboxylate charge by arginine (Arg)72, Arg112, Arg365, Mol Diagn 2004; 8 (3) Catalase Enzyme Mutations and their Association with Diseases and the hydrogen bond involves Tyr358, Arg354, His258, and Asp348.[42] 4. Benign Polymorphisms of Catalase Gene The benign polymorphisms of the catalase gene are listed in table I. They include single nucleotide substitutions in flanking, 5′ noncoding, intron 1, and exons 1, 9, and 10. For these mutations, no effect has been reported on catalase expression, decreased catalase activity, or association with disease/pathological changes.[43-46] The HinfI restriction fragment length polymorphism (RFLP; Southern blot) of the 5′ noncoding and flanking region of the catalase gene also showed the highly polymorphic characteristics of these regions.[52] In 2002 Casp et al.[53] reported on the association of a catalase gene silent mutation at position 111 in exon 9 (Asp384Asp) with vitiligo susceptibility. In case of a C to T polymorphism at –262 bp from the transcription site, the T variant showed a higher transcriptional activity in HepG2 and K562 cells.[48] A similar effect in human blood and liver cells can also be postulated. Further evidence from larger scale studies is required to prove an association between human blood catalase activity and this polymorphism. 5. Association of Catalase Gene Mutations with Disease 5.1 Diabetes Mellitus An association between the catalase mutations and type 2 diabetes mellitus has been investigated in a Moscow study.[47] 132 healthy individuals and 154 patients with type 2 diabetes were examined for the polymorphic markers C1167T and two neighboring mini-satellites (D1S5097 and D11S208). The genotype CC of C1167T polymorphism was found to be associated with a higher risk of type 2 diabetes. For the D1S5097 polymorphism, the frequency of alleles 15 and 16 and genotype 18/20 were significantly higher in diabetic patients than in controls. For the D11S208 polymorphism, the frequency of alleles 17 and 18 and genotype 18/20 were significantly higher in diabetic patients than in controls. These data could suggest an association between these three catalase loci and type 2 diabetes.[47] These polymorphisms were not associated with the development of diabetic nephropathy in patients with type 1 diabetes.[54] The first report of an association between inherited catalase deficiency and diabetes mellitus appeared in 2000.[33] An in© 2004 Adis Data Information BV. All rights reserved. 143 creased frequency of diabetes (12.7%) was found in Hungarian acatalasemic families (table II). The different catalase mutations in patients with diabetes caused a decrease in blood catalase activities which may lead to increased hydrogen peroxide concentrations in tissue and blood. These increased hydrogen peroxide levels may damage oxidationsensitive pancreatic β-cells leading to a decrease in insulin production.[33,61] The exact mechanisms by which hydrogen peroxide Table I. Benign polymorphisms of the catalase gene Mutation Position (numbering Region in the original paper) Reference Mutation in noncoding regions C to T –1167 Flanking 47 C to T –262 Flanking 48 G to A –259a (330) Flanking 49 A to T –21a Flanking 43 C to A –20 Flanking 45 C to T –18a Flanking 45 C to T 4a 5′ noncoding 46 A to G 17b (20) 5′ noncoding 53 T to C 20b 5′ noncoding 44 C to T 44b 5′ noncoding 46 T to C 49b 5′ noncoding 43 G to A –60c Intron 1 50 G to C 5 Intron 1 51 G to A 7 Intron 1 51 T to A 11 Intron 1 50 G to T 61 Intron 1 51 T to C 78 Intron 1 43 G to A 50 3′ noncoding 43 Mutations in coding regions T to C 12 Exon 1 Ser3Ser 46 A to C 27 Exon 1 Ser27Arg 46 T to C 111 Exon 9 Asp389Asp 43 T to C 60 Exon 10 Leu418Leu 44 a Numbering corresponds to the number of basepairs as denoted by Quan et al.[41] b Numbering corresponds to number of basepairs upstream from the transcription site MET (ATG-initiating codon). c Numbering corresponds to number of basepairs back from the first nucleotide of exon 1. The C mutation at position 49 of 5′ uncoding region was found to T to C by Góth[46] and this mutation was denoted to C to T earlier by Wen et al.[43] Asn = asparagine; Asp = aspartatic acid; Arg = arginine; His = histidine; Leu = leucine; Met = methionine; Ser = serine; Tyr = tyrosine. Mol Diagn 2004; 8 (3) Góth et al. 144 Table II. Reported prevalence of inherited catalase deficiency and diabetes mellitus in Hungary Activity (MU/L) Sex Diabetes Mutation References nucleotide exon/intron codon Acatalasemic 4.5 F Type 2 GA insertion e2 67 55 7.6 F Type 2 GA insertion e2 67 55 24.4 F Type 2 GA insertion e2 67 56 59.2 F Type 2 G insertion e2 48 57 30.9 F Type 2 G to A missense e9 354 58 50.8 F Type 2 G to Tt substitution i7 59 66.0 M Type 1 G to T substitution i7 59 60.3 F Type 2 T to A missense e2 53 60 58.9 F Type 2 G to C missense e2 66 60 F 13 M 20 No No Hypocatalasemic Normocatalasemic (n = 33) 102.6 ± 18.4 effects pancreatic function/insulin production are still unknown.[33-35,38,39] These data suggest that inherited catalase deficiency may be a minor risk factor for diabetes mellitus, especially for type 2 diabetes. Beyond inherited catalase deficiency, other factors such as genetics and environmental effects are responsible for the development of diabetes mellitus; 33 hypocatalasemic subjects in these families with the same mutation did not develop diabetes mellitus.[33,50,61] 5.2 Blood Pressure The first report to implicate the genetic variations of catalase in susceptibility to essential hypertension came from China in 2001.[49] Among the four single nucleotide polymorphisms (SNP; C to T at –773, G to A at –259, and T to A at –21 position of flanking region, and A to G at +17 of 5′ noncoding region) only the SNP at –773 demonstrated strong evidence (p < 0.002) of an association with essential hypertension (systolic blood pressure over 160mm Hg). This study included 37 individuals with hypertension and 22 individuals (with a systolic blood pressure below 104mm Hg) as controls. The SNP at –773 in the promoter region of the catalase gene is predicted to both create and destroy transcription factor binding sites, but it is not clear how they affect blood pressure levels.[49] Furthermore, there are no data on catalase enzyme activities. © 2004 Adis Data Information BV. All rights reserved. 55-60 5.3 Vitiligo Vitiligo susceptibility is a complex genetic trait that may be associated with the genes responsible for melanin biosynthesis, response to oxidative stress, and/or regulation of autoimmunity, as well as environmental factors. Both case-control and family-based genetic association studies showed that the T to C at position 20 of 5′ flanking region and T to C at 60 of exon 10 (Leu418Leu) were not associated with vitiligo.[53] The T to C single nucleotide polymorphism (Asp389Asp) in exon 9 suggests a possible association between this mutation and vitiligo. The C allele is transmitted more frequently in patients with vitiligo, which may contribute to the deficiency of catalase and accumulation of excess hydrogen peroxide.[53] This paper[53] revealed no data on catalase activity levels in blood while earlier studies[62] showed decreased catalase activities in the epidermis of patients with vitiligo. 5.4 Alzheimers Disease The oxidative stress hypothesis is proposed as one of a number of possible mechanisms underlying pathogenesis of Alzheimers disease. This hypothesis it suggests that the accumulation of hydrogen peroxide in the brain of affected individuals may be responsible for the development of the disease. Overproduction and/or insufficient detoxification of hydrogen peroxide may trigMol Diagn 2004; 8 (3) Catalase Enzyme Mutations and their Association with Diseases ger a cascade of neurotoxic events contributing to the neural damage characteristic of the disease.[63] In a study of 137 controls and 137 patients with Alzheimers disease, the C to T nucleotide substitution at position –262 of the catalase gene flanking region showed no difference in frequency (p > 0.5). Therefore, this mutation does not confer a protective effect in respect to Alzheimers disease.[63] 5.5 Decreased Catalase Activity in Tumors Catalase activity in the liver has been known to be reduced[64,65] in a tumor size-dependent fashion and is restored to the normal level by tumor removal.[66,67] These findings instigated extensive research into a cancer toxin (toxohormone) that might be involved in a cancer cachexia.[68,69] In 1973 Uenoyama and Ono[70] described two factors, one inhibitory and one stimulatory, which act on catalase synthesis in rat liver. In tumors, the inhibitory factor had greater effect on the catalase synthesis than the stimulatory factor. More recent studies showed that human tumor cells may produce large amounts of hydrogen peroxide[24] and that the decrease in catalase activity is due to the depression of the catalase gene transcription by an approximately 35 kDa nuclear protein bound to the silencer element present in hepatoma cells but not in liver cells.[71] There are data which suggest that catalase is also substantially modulated by signaling molecules.[72] These findings demonstrate that decreased catalase activity in tumor cells, especially in the liver, is due to a regulatory mechanism and not to catalase gene mutations, and similarly, the decreased blood catalase activity in different tumors is due to the decreased catalase synthesis.[73] 6. Acatalasemia The first human catalase deficiency was identified in Japanese patients in 1948.[74-76] Genetically, acatalasemia means the homozygous condition thus the term acatalasemia is actually a misnomer as there is usually a small amount (<10%) of residual enzyme activity in erythrocytes, consequently hypocatalasemia may be a more correct term. However, for the sake of convenience hypocatalasemia will indicate the heterozygous state with intermediate (about 50%) levels of catalase activity.[77] Acatalasemia is genetically a heterogenous condition with worldwide distribution. To date 113 cases of acatalasemia have been diagnosed in 59 families from 11 countries (Japan, Korea, Switzerland, Israel, US, Mexico, Germany, Peru, Iran, Austria, and Hungary).[75,76,78-88] The patients with acatalasemia in Japan, © 2004 Adis Data Information BV. All rights reserved. 145 Switzerland, and Hungary have been characterized[76,89-91] using clinical, biochemical and molecular genetic methods, while the identification of cases in other countries has been sporadic and relatively poorly characterized. The frequency of acatalasemia is 0.04 : 1000 in Switzerland, 0.8 : 1000 in Japan, and 0.05 : 1000 in Hungary; the frequency of hypocatalasemia is similar in Japan (2–4 : 1000) and in Hungary (2.3 : 1000). 6.1 Clinical Features of Acatalasemia In general, acatalasemia is a relatively benign syndrome.[77] Oral gangrene and ulceration (Takahara disease) are associated with Japanese patients diagnosed with acatalasemia and appear in roughly 20–50% of the cases, with a higher incidence in childhood. It might be caused by catalase deficiency at the tissue level, differences in oral flora, lack of oral hygiene, and environmental factors. Support for the latter is provided by the observation that its frequency declined in recent years.[91-93] Takahara disease has only been reported for patients with acatalasemia in Japan and Germany.[83,93] The ulceration and consequent gangrene are probably promoted by hydrogen peroxide generated by phagocytic cells (neutrophils) and bacterial (streptococci, pneumococci) actions and the associated lack of catalase in the effected tissues and/or erythrocytes. Recent findings revealed increased cholesterol, low-density lipoprotein (LDL)-cholesterol, ApoB, Lp(a), and decreased LDL oxidative resistance in the Hungarian hypocatalasemic patients when these values were compared with those of normocatalasemic family members. These changes may mean that these patients have a higher risk of coronary arterial diseases.[94] Furthermore, association of hyperhomocysteinemia and inherited catalase deficiency is associated with decreased folate and erythrocyte production. After further investigation in the Hungarian acatalasemic and hypocatalasemic patients, one of the acatalasemic patients died at the age of 60, she had had a mastectomy and hemicolectomy due to breast carcinoma and residual tumor in the colon.[87] Her hypocatalasemic brother died at the age of 77 due to prostate carcinoma (Góth, unpublished data). One hypocatalasemic female with type 2 diabetes died at the age of 73 (Góth, unpublished data). For one hypocatalasemic male with type 1 diabetes, uncontrolled living conditions and complications of his diabetes (uremia, hypertension, and subarachnoidal hemorrhage) are responsible for his early death (aged 47; Góth, unpublished data). Another hypocatalasemic male died from a cerebrovascular lesion when he was 77 years old (Góth, unpublished data). At autopsy, one acatalasemic and three Mol Diagn 2004; 8 (3) Góth et al. 146 hypocatalasemics had more atherosclerosis for their age than normal (Góth, unpublished data). Contrary to these risk factors, the mean age for the living Hungarian acatalasemic/hypocatalasemic patients (45.1 ± 19.3y; n = 62) did not differ (p = 0.522) from that of normocatalasemic family members (42.9 ± 18.5y; n = 66) [Góth, unpublished data]. New findings showed a higher incidence (12.7%) of diabetes in Hungarian acatalasemic (2/2) and hypocatalasemic (6/61) patients.[33] One hypocatalasemic patient had type 1 diabetes (onset at 7y) and two acatalasemic and 5 hypocatalasemic patients had type 2 diabetes (age of onset 43.0 ± 10.8y; range 35–56y). The other hypocatalasemic patients (n = 55) and normocatalasemic family members (n = 65) did not have diabetes. The manifestation and diagnosis of symptomatic type 2 diabetes usually occur after the age of 40;[95] two Hungarian hypocatalasemics experienced an earlier onset (35 and 36 years of age). The increased frequency of diabetes, especially the non insulindependent form, may be explained by the cumulative oxidative damage on pancreatic β-cells, especially on the mitochondria.[33-35,96,97] Recent findings[33,36,94] show that acatalasemia is not only a benign genetic polymorphism, but is also associated with changes in lipid, erythrocyte, and carbohydrate metabolism. There are reports of new techniques (from the polyethylene glycol conjugates to catalase mimics and immuno-targeting) that can treat inherited catalase deficiency.[55,56,98] It has been shown that artificial superoxide dismutase (SOD)/ catalase mimics [Mn-Salem; manganase 3-methoxy N,N′-bis(salycilidine)ethylenediamine chloride] can protect cells from oxidative stress in a large number of disease models.[55] Similar favorable effects were found when the immuno-targeted delivery of catalase to the catalase poor endothelium was performed.[56] 6.2 Catalase Gene Mutations in Acatalasemia Surprisingly, even with the early identification of the nucleotide sequence of human catalase[40,41] and the large number of acatalasemic families (n = 59), there are only a small number of published papers on the catalase mutations responsible for decreased catalase activity. 6.2.1 Japanese-Type Acatalasemia Among the 46 acatalasemic families identified with the Japanese type of acatalasemia, only two syndrome causing mutations have been reported.[43,44] A single G to A nucleotide substitution at the fifth position of intron 4 (splicing mutation) was responsible for the catalase deficiency (Japanese type A). This splicing muta© 2004 Adis Data Information BV. All rights reserved. tion was detected in unrelated acatalasemics and 1 related hypocatalasemic.[43,44] The type B of Japanese acatalasemia was detected by Hirono et [57] al. in 1 homozygote and 4 heterozygotes in the same family. This mutation showed a T deletion at 358 nucleotide position causing a frameshift mutation followed by a nonsense mutation. The truncated protein formed is unstable, with no catalase activity. 6.2.2 Swiss-Type Acatalasemia There has been only a very limited study of Swiss-type catalase mutations. This is most likely due to the early death of the program coordinator (Dr H. Aebi). Crawford et al.[59] suggested that a regulatory mutation might be responsible for a truncated catalase protein. Earlier investigations[91] showed that this truncated protein is less stable. This may explain why the catalase deficiency in the erythrocyte of long lifespan is more severe than in other cells of short lifespan. This phenomena was not detected in either the Japanese or Hungarian patients with acatalasemia. 6.2.3 Hungarian-Type Acatalasemia Four novel catalase mutations have been reported for Hungarian acatalasemic and hypocatalasemic patients. The detection of these mutations was based on a large scale catalase screening program that involved 18 200 hospital/clinic patients and 4930 healthy individuals.[58,60,87] • Hungarian Type A[99]: A GA insertion at nucleotide position 138 of exon 2 increased the GA repeat from 4 to 5. This insertion caused a frameshift in the amino acid sequence from 69 to 133 and generated a TGA termination codon at 134. This truncated protein lacks the essential amino acid (His75) in the active center.[42] This mutation was detected in one acatalasemic family (which included 2 acatalasemics and 6 hypocatalasemics) and in 3 hypocatalasmic families (totalling 23 hypocatalasemics). We used a simple PCR-heteroduplex screening method for the detection of this mutation.[100] The blood catalase activities of the acatalasemics were 4.5 MU/L (4.0%) and 7.6 MU/L (6.7%) and 49.2 ± 19.5 MU/L (45.8%; n = 23) for the hypocatalasemics and were compared with the normocatalasemic (107.6 ± 19.5; n = 26) family members. Hungarian Type B[101]: This family had three hypocatalasemic • (68.1 ± 5.91 MU/L, 52.2%) and four normocatalasemic (130.4 ± 8.7 MU/L) family members. The PCR-heteroduplex screening method yielded heteroduplex formation in exon 2. The nucleotide sequence analyzes revealed a G insertion at position 79 in exon 2 causing a frameshift of amino acid sequence from 49 to 57 with a TGA stop codon at 58. This truncated protein Mol Diagn 2004; 8 (3) Catalase Enzyme Mutations and their Association with Diseases • • with its 58 amino acids instead of the 526 for the regular catalase protein is not able to maintain the enzymatic function. Hungarian Type C[102]: The PCR single-strand conformational polymorphism (SSCP) screening method demonstrated a mutation in intron 7. The nucleotide sequence analyzes showed a G to T substitution at position 5 of intron 7. The effect of this splice site mutation on the decreased catalase protein was confirmed by Western blot analyzes. This mutation was detected in 7 hypocatalasemics (58.5 ± 11.5 MU/L, 60.6%) from two hypocatalasemic families. The catalase activity of normocatalasemic family members was 96.9 ± 4.1 MU/L (n = 7). Hungarian Type D[50]: PCR-SSCP screening method showed a mutation in exon 9. The G to A substitution at position 5 of exon 9 is a missense mutation changing Arg354 to histidine. The Arg354, His218, Asp348 effect the promotion sites which stabilize the electrostatic field generated by different iron oxidation states in the active center of catalase protein.[42] This mutation was found in 4 hypocatalasemics (55.6 ± 16.9 MU/L, 53.7%) of one hypocatalasemic family with 6 normocatalasemic (103.6 ± 23.5 MU/L) family members. 6.3 Other Catalase Mutations Three hundred and eight patients with type 2 diabetes were examined for catalase gene mutations, two novel mutations were detected.[51] The first was a T to A missense mutation at position 96 of exon 2 causing the change of Asp53Glu. The second one is a G to C missense mutation at 135 nucleotide position of exon 2 which causes the substitution of Glut66Cys (cysteine). These amino acids are localized in the neighboring region of valine (Val)44, Arg72, Val73, Val74, and His75 which are important in the heme-protein interaction[42] and could cause decreased (58.7% and 48.2%) catalase enzyme activities. These mutations have been detected in one person without family pedigree. These results show the heterogenous feature of the acatalasemic syndrome which requires further examination for the other five hypocatalasemic families in Hungary. The Human Genome Mutation Database (Cardiff)[103] contains the mutations of Japanese type A, B, and Hungarian type A, B, C of acatalasemia and catalase association with hypertension.[49] 7. Future Prospects for the Detection of Catalase Gene Mutations Enzyme catalase were first detected in 1819,[104] first used in clinical laboratory practice in 1910,[64] and first reported as an inherited deficiency in 1948.[74,75] It has recently gained attention © 2004 Adis Data Information BV. All rights reserved. 147 again because of its role as the main regulator of hydrogen peroxide metabolism.[1,2,5,6,8,10-12] Recent findings show that catalase has a role in the regulation of hydrogen peroxide concentrations in signaling.[13-22,25-31] However, there are only limited papers on catalase mutations and their association with diseases such as diabetes mellitus,[33,54,61] hypertension,[49] and vitiligo[53] and only six syndrome causing mutations (two for Japanese and four for Hungarian patients) have been identified.[43,44,50,57,99-102] Acatalasemia was thought to be a relatively benign enzyme deficiency,[77] but recent findings in Hungarian acatalasemic/hypocatalasemic patients revealed its association with diabetes[33,61] and biochemical changes in lipid,[94] homocysteine, and erythrocyte metabolism.[36] Therefore, further studies are required to examine catalase mutations in acatalasemia and diseases which are associated with decreased catalase activity, such as diabetes, atherosclerosis, or tumors.[73] The regulatory role of catalase via hydrogen peroxide in signaling might be a new field of examination for catalase mutations. 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Ann Chim Phys 1819; 11: 85 Correspondence and offprints: Dr László Góth, Department of Clinical Biochemistry and Molecular Pathology, Medical and Health Science Center, University of Debrecen, Debrecen PO Box 40, H-4012, Hungary. E-mail: [email protected] Mol Diagn 2004; 8 (3)