"IZ3204"
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Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : il. ; 32 cm
Projekt má za cíl objasnit patogenetické důsledky mutace A625 na genu pro short chain acyl CoA dehydrogenásu. Znalosti o mutaci, přítomné v homozygotním stavu u 3% populace napomohou k prevenci a účinné terapii symptomů s ní spojených.
We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele, and a single point mutation, 511C-->T, in the other. The 1147C-->T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C-->T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant ( P < 0.02), suggesting that the allele 511T-625G-like 511C-625A-confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37 degrees C in comparison with the wild-type protein, comparable levels of activity at 26 degrees C, and 13% activity when incubated at 41 degrees C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37 degrees C, and 58% activity was present at 41 degrees C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C-->T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.
- MeSH
- acyl-CoA-dehydrogenasa MeSH
- acyl-CoA-dehydrogenasy genetika nedostatek MeSH
- alely MeSH
- COS buňky MeSH
- fibroblasty metabolismus MeSH
- frekvence genu MeSH
- kojenec MeSH
- komplementární DNA analýza MeSH
- kultivované buňky MeSH
- lidé MeSH
- malonáty * moč MeSH
- mutace MeSH
- novorozenec MeSH
- teplota MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- kazuistiky MeSH
- práce podpořená grantem MeSH
Medium chain acyl-CoA dehydrogenase (MCAD) deficiency is an inborn error of fatty acid metabolism. It is one of the most frequent genetic metabolic disorders among Caucasian children. The G985 allele represented 90% of all the variant alleles of the MCAD gene in an extensive series of retrospective studies. To study the distribution of the G985 allele, newborn blood samples from the following countries were tested; 3000 from Germany (1/116). 1000 each from Belgium (1/77). Poland (1/98), Czech Republic (1/240). Hungary (1/168), Bulgaria (1/91), Spain (1/141). Turkey (1/216), and 500 from Japan (none). The frequency is shown in parentheses. The haplotype of G985 alleles in 1 homozygote and 57 heterozygote samples were then analyzed using two intragenic MCAD gene polymorphisms (Iaq1 and GT-repeat). The result indicated that only 1 of the 10 known haplotypes was associated with the G985 mutation, suggesting that G985 was derived originally from a single ancestral source. We made a compilation of the G985 frequencies in these countries and those in nine other European countries studied previously. The G985 distribution was high in the area stretching from Russia to Bulgaria in the east and in all northern countries in western and middle Europe, but low in the southern part of western and middle Europe. The incidence among ethnic Basques appeared to be low. This distribution pattern and the fact that all G985 alleles belong to a single haplotype suggest that G985 mutation occurred later than the delta F508 mutation of the CFTR, possibly in the neolithic or in a later period, and was brought into Europe by IndoEuropean-speaking people. The panEuropean distribution of the G985 allele, including Slavic countries from which patients with MCAD deficiency have rarely been detected, indicates the importance of raising the level of awareness of this disease.
- MeSH
- acyl-CoA-dehydrogenasy * genetika MeSH
- alely MeSH
- biologická evoluce * MeSH
- frekvence genu MeSH
- genetická variace * MeSH
- genetické testování * metody MeSH
- haplotypy * MeSH
- lidé MeSH
- mapování chromozomů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, P.H.S. MeSH
- Geografické názvy
- Evropa MeSH
- Japonsko MeSH
- Turecko MeSH
- východní Evropa MeSH
Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease-causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.
- MeSH
- acyl-CoA-dehydrogenasa MeSH
- acyl-CoA-dehydrogenasy * genetika metabolismus nedostatek MeSH
- aktivace enzymů MeSH
- alely MeSH
- chaperon hsp60 genetika metabolismus MeSH
- chaperonin 10 genetika metabolismus MeSH
- dítě MeSH
- Escherichia coli genetika MeSH
- exony MeSH
- fenotyp MeSH
- heterozygot * MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mutace * MeSH
- novorozenec MeSH
- polymerázová řetězová reakce MeSH
- předškolní dítě MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- restrikční mapování MeSH
- rodokmen MeSH
- sekvenční analýza DNA MeSH
- sekvenční delece MeSH
- teplota MeSH
- western blotting MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
Ethylmalonic aciduria is a common biochemical finding in patients with inborn errors of short chain fatty acid beta-oxidation. The urinary excretion of ethylmalonic acid (EMA) may stem from decreased oxidation by short chain acyl-CoA dehydrogenase (SCAD) of butyryl-CoA, which is alternatively metabolized by propionyl-CoA carboxylase to EMA. We have recently detected a guanine to adenine polymorphism in the SCAD gene at position 625 in the SCAD cDNA, which changes glycine 209 to serine (G209S). The variant allele (A625) is present in homozygous and in heterozygous form in 7 and 34.8% of the general population, respectively. One hundred and thirty-five patients from Germany, Denmark, the Czech Republic, Spain, and the United States were selected for this study on the basis of abnormal EMA excretion ranging from 18 to 1185 mmol/mol of creatinine (controls < 18 mmol/mol of creatinine). Among them, we found a significant overrepresentation of the variant allele. Eighty-one patients (60%) were homozygous for the A625 allele, 40 (30%) were heterozygous, and only 14 (10%) harbored the wild-type allele (G625) in homozygous form. By overexpressing the wild-type and variant protein (G209S) in Escherichia coli and COS cells, we showed that the folding of the variant protein was slightly compromised in comparison to the wild-type and that the temperature stability of the tetrameric variant enzyme was lower than that of the wild type. Taken together, the over-representation and the biochemical studies indicate that the A625 allele confers susceptibility to the development of ethylmalonic aciduria.
- MeSH
- acyl-CoA-dehydrogenasa MeSH
- acyl-CoA-dehydrogenasy * genetika MeSH
- bodová mutace MeSH
- Cercopithecus aethiops MeSH
- DNA analýza MeSH
- exprese genu MeSH
- genetická variace MeSH
- malonáty * metabolismus MeSH
- messenger RNA MeSH
- polymorfismus konformace jednovláknové DNA MeSH
- transformované buněčné linie MeSH
- vazebná místa MeSH
- vrozené poruchy metabolismu tuků * enzymologie genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- MeSH
- acyl-CoA-dehydrogenasa genetika MeSH
- alely MeSH
- antikonvulziva * terapeutické užití MeSH
- epilepsie * farmakoterapie genetika MeSH
- fibroblasty metabolismus MeSH
- genetická variace * MeSH
- kultivované buňky MeSH
- kyseliny mastné těkavé metabolismus MeSH
- lidé MeSH
- následné studie MeSH
- oxidace-redukce MeSH
- předškolní dítě MeSH
- riboflavin * terapeutické užití MeSH
- Check Tag
- lidé MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky MeSH
- MeSH
- acyl-CoA-dehydrogenasa MeSH
- alely MeSH
- desaturasy mastných kyselin genetika nedostatek MeSH
- DNA primery genetika MeSH
- frekvence genu MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- malonáty moč MeSH
- molekulární sekvence - údaje MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus genetický * MeSH
- sekvence aminokyselin MeSH
- sekvence nukleotidů MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
