Tiivistelmä Lasten liikehäiriöt ovat etiologialtaan ja taudinkuvaltaan heterogeeninen ryhmä. Vaik... more Tiivistelmä Lasten liikehäiriöt ovat etiologialtaan ja taudinkuvaltaan heterogeeninen ryhmä. Vaikeissa häiriöissä vaste lääkehoitoon on usein vähäinen. Syväaivostimulaatio voi merkittävästi auttaa osaa potilaista. Hoitopäätös vaatii moniammatillisen työryhmän arvion liikehäiriön laadusta, neurofysiologiset tutkimukset ja korkealaatuisen kuvantamisen. Lapsia ja nuoria hoitavien yksiköiden on tärkeää verkostoitua kansallisesti ja kansainvälisesti.Abstract Movement disorders are disabling conditions that can cause abnormal posture, torsion, repetitive movement or tremor via the disruption of regulatory areas in the brain. Among paediatric patients, generalised dystonia is the most common primary movement disorder, and dyskinetic CP the most common secondary phenotype, both with limited or unsatisfactory traditional treatment options. The prevalence of dystonia is suggested to be higher than reported since it is not always easy to recognise. Deep brain stimulation (DBS) is an establishe...
Background The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and in... more Background The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and information regarding cytokine production outside of FS episodes is scarce. Methods In our controlled follow-up study of patients with FSs, we compared the levels of 12 serum cytokines after the patients’ first FSs, during febrile episodes without FSs, after recurrent FSs, during healthy periods after FSs, and between patients and controls. Results Two-hundred fifty-one patients with first FS participated in the study, of whom 17 (mean age 1.6 years, SD 0.7) with recurrent FSs completed the protocol as required by the sample size calculations. The mean IL-1RA level was higher after the first FSs (2580 pg/mL, SD 1516) than during febrile episodes without FSs (1336 pg/mL, SD 1364, P = 0.006) and healthy periods after FSs (474 pg/mL, SD 901, P = 0.001). IL-1RA levels were also higher during first (2580 pg/mL) and recurrent FSs (2666 pg/mL, SD 1747) in comparison with febrile controls (746 pg/...
The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spo... more The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.
Lasten ja nuorten migreeni • Lasten ja nuorten migreenin esiintyvyys ja ilmaantuvuus ovat lisäänt... more Lasten ja nuorten migreeni • Lasten ja nuorten migreenin esiintyvyys ja ilmaantuvuus ovat lisääntyneet. • Perusterveydenhuollossa on tärkeää selvittää migreenin taustasyitä ja esiintymistä. • Migreenin diagnostiikassa tarvitaan lääkärin tutkimuksen lisäksi harvoin muita tutkimuksia. • Lapsen ja nuoren on tärkeä saada tietoa päänsäryn hoito-ja itsehoitokeinoista. • Estolääkitys voidaan aloittaa perusterveydenhuollossa, mikäli migreenikohtaukset ovat viikoittaisia. Tuuli immonen LT, lastenneurologian erikoislääkäri ja kliininen opettaja HUS, lasten ja nuorten sairaudet ja Helsingin yliopisto Hannu Heiskala lastenneurologian dosentti Lastentautien tutkimuskeskus ja Helsingin yliopisto Heli Helander LT, lastenneurologian ja lastentautien erikoislääkäri OYS, lasten ja nuorten vastuualue ja Oulun yliopisto, PEDEGOtutkimusyksikkö Veera HölTTä LT, lastenneurologian erikoislääkäri HUS, lasten ja nuorten sairaudet ja Helsingin yliopisto milla Ylijoki LT, lastenneurologian erikoislääkäri TYKS, lasten ja nuorten klinikka, lastenneurologinen yksikkö ja Turun yliopisto Positiivinen sukuanamneesi tukee diagnoosia. kirjallisuuTTa 1 Gormley P, Anttila V, Winsvold BS ym. Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine.
␣-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid ... more ␣-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver. Immunoelectron microscopy and subcellular fractionation located racemase to mitochondria and peroxisomes. The enzymes were purified from both organelles with immunoaffinity chromatography. The isolated proteins were of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with ␣-methylacyl-CoA racemase activity was excluded. A racemase gene of about 15 kilobases was isolated. Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15B1. The putative initial ATG in the racemase gene was preceded by a functional promotor as shown with the luciferase reporter gene assay. The corresponding cDNAs were isolated from rat and mouse liver. The recombinant rat protein was overexpressed in active form in Pichia pastoris. The presented data suggest that the polypeptide encoded by the racemase gene can alternatively be targeted to peroxisomes or mitochondria without modifications. It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxisomal targeting signal.
WereporttheisolationandcharacterizationofcDNAclones for a novel isoform of lysyl hydroxylase (lys... more WereporttheisolationandcharacterizationofcDNAclones for a novel isoform of lysyl hydroxylase (lysylhydroxylase2),aposttranslationalenzymeofcollagenbiosynthesis.Theopenreadingframepredictedapro-tein of 737 amino acids, including an amino-terminalsignal peptide. The amino acid sequence has overallsimilarity of over 75% to the lysyl hydroxylase (lysylhydroxylase1)characterizedearlier.Thissimilarityisevenhigherinthecarboxyl-terminalendofthemole-cules.Lysylhydroxylase2containsninecysteineresi-dues,whichareconservedinlysylhydroxylase1.Fur-thermore, the conserved histidines and aspartateresidues required for lysyl hydroxylase activity arepresentinthesequence.Northernanalysisidentifiedatranscriptof4.2kilobases,whichwashighlyexpressedinpancreasandmuscletissues.ExpressionofcDNAininsectcellsusingabaculovirusvectoryieldedproteinswith lysyl hydroxylase activity and an antiserumagainstasyntheticpeptideofthededucedaminoacidsequencerecognizedproteinswithmolecularweightsof88and97kDainhomogenatesofth...
The contribution of de novo and ultra-rare genetic variants in severe and moderate intellectual d... more The contribution of de novo and ultra-rare genetic variants in severe and moderate intellectual disability (ID) has been extensively studied whereas the genetic architecture of mild ID has been less well characterized. To elucidate the genetic background of milder ID we studied a regional cohort of 442 ID patients enriched for mild ID (>50%) from a population isolate of Finland. We analyzed rare variants using exome sequencing and CNV genotyping and common variants using common variant polygenic risk scores. As controls we used a Finnish collection of exome sequenced (n=11311) and GWAS chip genotyped (n=11699) individuals.We show that rare damaging variants in genes known to be associated with cognitive defects are observed more often in severe (27%) than in mild ID (13%) patients (p-value: 7.0e-4). We further observed a significant enrichment of protein truncating variants in loss-of-function intolerant genes, as well as damaging missense variants in genes not yet associated wit...
The contribution of de novo variants in severe intellectual disability (ID) has been extensively ... more The contribution of de novo variants in severe intellectual disability (ID) has been extensively studied whereas the genetics of mild ID has been less characterized. To elucidate the genetics of milder ID we studied 442 ID patients enriched for mild ID (>50%) from a population isolate of Finland. Using exome sequencing, we show that rare damaging variants in known ID genes are observed significantly more often in severe (27%) than in mild ID (13%) patients. We further observe a significant enrichment of functional variants in genes not yet associated with ID (OR: 2.1). We show that a common variant polygenic risk significantly contributes to ID. The heritability explained by polygenic risk score is the highest for educational attainment (EDU) in mild ID (2.2%) but lower for more severe ID (0.6%). Finally, we identify a Finland enriched homozygote variant in the CRADD ID associated gene.
Background Methionine synthase deficiency is a rare inborn error of intracellular cobalamin metab... more Background Methionine synthase deficiency is a rare inborn error of intracellular cobalamin metabolism caused by mutations in the MTR (5-methyltetrahydrofolate-homocysteine S-methyltransferase) gene, resulting in megaloblastic anemia and neurologic symptoms. Methods and Results We describe for the first time a homozygous MTR gene c.3518C > T (p.P1173L) mutation in a patient with severe megaloblastic anemia, developmental delay, and drug-resistant seizures associated with hyperhomocysteinemia and hypomethioninemia. Methionine synthase activity was only 9% of the reference value, and MTR protein expression was decreased in the fibroblasts of the patient. The clinical features of our patient are similar to previously published patients with the complementation type G disorder of methionine synthase deficiency with the exception of drug-resistant seizures. However, intramuscular injections of hydroxocobalamin (OHCbl) in conjunction with betaine and folic acid provided verified clinical and electrophysiological treatment response. Conclusion This study emphasizes the importance of early diagnosis of patients having neurologic symptoms due to methionine synthase deficiency where early treatment has significant effects on the clinical outcome of the patients. Elevated level of plasma homocysteine together with low methionine in plasma amino acid analysis should raise a suspicion of remethylation disorder.
Objective We conducted a randomized controlled trial to evaluate whether a combination of repeate... more Objective We conducted a randomized controlled trial to evaluate whether a combination of repeated botulinum toxin A (BTX-A) and conservative treatment is more effective in decreasing toe-walking than conservative treatment alone at 24 months follow-up. Patients and Methods Children between 2 and 9 years of age were randomized either into the conservative (CO) or botulinum treatment (BTX) group. The treatment in the CO group consisted of firm shoes, night splints, a home stretching program and physiotherapy. The BTX arm had all the same conservative treatments added with calf muscle BTX-A injections repeated in 6 months intervals if needed. Change in toe-walking pattern, ankle range of movement (ROM), and overall function were assessed at baseline and 6, 12, 18, and 24 months posttreatment. Results A total of 30 toe-walkers participated: 14 in CO and 16 in BTX group. At 24 months, all children in the BTX group and 85% in the CO group evaluated by the blinded physiotherapist (p ¼ 0.065), 75% in the BTX group and 70% in the CO group graded by the research physiotherapist (p ¼ 0.730), and 50% in the BTX group and 54% in the CO group reported by the parents ceased toe-walking (p ¼ 0.837). The most prominent change was noted during the 1st year. The BTX group seemed to reach the goal earlier. No significant differences between the treatment groups in function or in ankle ROM ensued. Conclusion Adding BTX injections did not significantly enhance the goal to walk either flat foot or with heel strike at 24 months posttreatment.
cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA l... more cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver lambdagt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur. J. Biochem.231, 815-822]. Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39679 Da. The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence. The cDNA coding for mouse racemase was cloned from a mouse liver lambdaZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) that shares 89.7% similarity with the rat protein. Expression of the rat racemase as are combinant protein in Escherichia coli with the pTrcHisB-expression ve...
In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enz... more In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hyd...
Recently it has been shown that peroxisomes interact with microtubules which affect the structure... more Recently it has been shown that peroxisomes interact with microtubules which affect the structure and intracellular distribution of the organelle (Schrader, M., J. K. Burkhardt, E. Baumgart, G. Lüers, H. Spring, A. Völkl, H. D. Fahimi: Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs. Eur. J. Cell Biol. 69, 24-35 (1996)). In the present work, we have applied immunological techniques to study the organization of peroxisomes within the rat liver cell. Antibodies to a pentadecapeptide corresponding to amino acid residues 403-417 of the 70 kDa integral peroxisomal membrane protein (Kamijo, K., S. Taketani, S. Yokota, T. Osumi, T. Hashimoto: The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein super family. J. Biol. Chem. 265, 4534-4540 (1990)) were raised in rabbits and affinity purified. This antibody was found to be highly specific for peroxisomes as determined by ELISA and Western blot analysis. Immunoelectron microscopy of tissue sections from rat liver revealed that peroxisomal membranes were labeled with this antibody and, in addition, labeling was found on tubular extensions often connecting peroxisomes. Antibodies to alpha-tubulin were used to locate the microtubular system. Microtubules were often found in close connection to peroxisomes, suggesting interaction between peroxisomes and the cytoskeleton. Double-labeling experiments for the 70 kDa integral peroxisomal membrane protein and alpha-tubulin demonstrated that the tubular structures connecting peroxisomes did not colocalize with microtubules. These results suggest that peroxisomes are organized in reticular structures within rat liver cells and that the structure and localization of these reticuli may be determined by their association to the microtubular network.
In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enz... more In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.
Tiivistelmä Lasten liikehäiriöt ovat etiologialtaan ja taudinkuvaltaan heterogeeninen ryhmä. Vaik... more Tiivistelmä Lasten liikehäiriöt ovat etiologialtaan ja taudinkuvaltaan heterogeeninen ryhmä. Vaikeissa häiriöissä vaste lääkehoitoon on usein vähäinen. Syväaivostimulaatio voi merkittävästi auttaa osaa potilaista. Hoitopäätös vaatii moniammatillisen työryhmän arvion liikehäiriön laadusta, neurofysiologiset tutkimukset ja korkealaatuisen kuvantamisen. Lapsia ja nuoria hoitavien yksiköiden on tärkeää verkostoitua kansallisesti ja kansainvälisesti.Abstract Movement disorders are disabling conditions that can cause abnormal posture, torsion, repetitive movement or tremor via the disruption of regulatory areas in the brain. Among paediatric patients, generalised dystonia is the most common primary movement disorder, and dyskinetic CP the most common secondary phenotype, both with limited or unsatisfactory traditional treatment options. The prevalence of dystonia is suggested to be higher than reported since it is not always easy to recognise. Deep brain stimulation (DBS) is an establishe...
Background The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and in... more Background The role of cytokines in the pathogenesis of febrile seizures (FSs) is unclear, and information regarding cytokine production outside of FS episodes is scarce. Methods In our controlled follow-up study of patients with FSs, we compared the levels of 12 serum cytokines after the patients’ first FSs, during febrile episodes without FSs, after recurrent FSs, during healthy periods after FSs, and between patients and controls. Results Two-hundred fifty-one patients with first FS participated in the study, of whom 17 (mean age 1.6 years, SD 0.7) with recurrent FSs completed the protocol as required by the sample size calculations. The mean IL-1RA level was higher after the first FSs (2580 pg/mL, SD 1516) than during febrile episodes without FSs (1336 pg/mL, SD 1364, P = 0.006) and healthy periods after FSs (474 pg/mL, SD 901, P = 0.001). IL-1RA levels were also higher during first (2580 pg/mL) and recurrent FSs (2666 pg/mL, SD 1747) in comparison with febrile controls (746 pg/...
The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spo... more The ND1 subunit gene of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a hot spot for mutations causing Leber hereditary optic neuropathy and several mutations causing the mitochondrial encephalopathy, lactic acidosis and stroke-like episodes syndrome (MELAS). We have used Escherichia coli and Paracoccus denitrificans as model systems to study the effect of mutations 3946 and 3949, which change conserved residues in ND1 and cause MELAS. The vicinity of these mutations was also explored with a series of mutations in charged residues. The 3946 mutation results in E214K substitution in human ND1. Replacement of the equivalent residue in E. coli with lysine or glutamine detracted from enzyme assembly and the assembled enzyme was inactive. However, the equivalent E234Q mutant enzyme in P. denitrificans failed to assemble completely (or was rapidly degraded). Also the corresponding substitution with aspartate decreased the enzyme activity in P. denitrificans and E. coli. The 3949-equivalent substitution, Y229H in E. coli, lowered the catalytic activity by 30%. In addition, an activation of the enzyme during catalytic turnover was seen in this bacterial NDH-1, something that was even more pronounced in another mutant in the same loop, D213E. Several other mutations in this region decreased the enzyme activity. The studied MELAS mutations are situated in a matrix-side loop, which appears to be highly sensitive to structural perturbations. The results provide new information on the function of the region affected by the MELAS mutations 3946 and 3949 that is not obtainable from patient samples or current eukaryote models.
Lasten ja nuorten migreeni • Lasten ja nuorten migreenin esiintyvyys ja ilmaantuvuus ovat lisäänt... more Lasten ja nuorten migreeni • Lasten ja nuorten migreenin esiintyvyys ja ilmaantuvuus ovat lisääntyneet. • Perusterveydenhuollossa on tärkeää selvittää migreenin taustasyitä ja esiintymistä. • Migreenin diagnostiikassa tarvitaan lääkärin tutkimuksen lisäksi harvoin muita tutkimuksia. • Lapsen ja nuoren on tärkeä saada tietoa päänsäryn hoito-ja itsehoitokeinoista. • Estolääkitys voidaan aloittaa perusterveydenhuollossa, mikäli migreenikohtaukset ovat viikoittaisia. Tuuli immonen LT, lastenneurologian erikoislääkäri ja kliininen opettaja HUS, lasten ja nuorten sairaudet ja Helsingin yliopisto Hannu Heiskala lastenneurologian dosentti Lastentautien tutkimuskeskus ja Helsingin yliopisto Heli Helander LT, lastenneurologian ja lastentautien erikoislääkäri OYS, lasten ja nuorten vastuualue ja Oulun yliopisto, PEDEGOtutkimusyksikkö Veera HölTTä LT, lastenneurologian erikoislääkäri HUS, lasten ja nuorten sairaudet ja Helsingin yliopisto milla Ylijoki LT, lastenneurologian erikoislääkäri TYKS, lasten ja nuorten klinikka, lastenneurologinen yksikkö ja Turun yliopisto Positiivinen sukuanamneesi tukee diagnoosia. kirjallisuuTTa 1 Gormley P, Anttila V, Winsvold BS ym. Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine.
␣-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid ... more ␣-Methylacyl-CoA racemase, an enzyme of the bile acid biosynthesis and branched chain fatty acid degradation pathway, was studied at the protein, cDNA, and genomic levels in mouse liver. Immunoelectron microscopy and subcellular fractionation located racemase to mitochondria and peroxisomes. The enzymes were purified from both organelles with immunoaffinity chromatography. The isolated proteins were of the same size, with identical N-terminal amino acid sequences, and the existence of additional proteins with ␣-methylacyl-CoA racemase activity was excluded. A racemase gene of about 15 kilobases was isolated. Southern blot analysis and chromosomal localization showed that only one racemase gene is present, on chromosome 15, region 15B1. The putative initial ATG in the racemase gene was preceded by a functional promotor as shown with the luciferase reporter gene assay. The corresponding cDNAs were isolated from rat and mouse liver. The recombinant rat protein was overexpressed in active form in Pichia pastoris. The presented data suggest that the polypeptide encoded by the racemase gene can alternatively be targeted to peroxisomes or mitochondria without modifications. It is concluded that the noncleavable N-terminal sequence of the polypeptide acts as a weak mitochondrial and that the C-terminal sequence acts as a peroxisomal targeting signal.
WereporttheisolationandcharacterizationofcDNAclones for a novel isoform of lysyl hydroxylase (lys... more WereporttheisolationandcharacterizationofcDNAclones for a novel isoform of lysyl hydroxylase (lysylhydroxylase2),aposttranslationalenzymeofcollagenbiosynthesis.Theopenreadingframepredictedapro-tein of 737 amino acids, including an amino-terminalsignal peptide. The amino acid sequence has overallsimilarity of over 75% to the lysyl hydroxylase (lysylhydroxylase1)characterizedearlier.Thissimilarityisevenhigherinthecarboxyl-terminalendofthemole-cules.Lysylhydroxylase2containsninecysteineresi-dues,whichareconservedinlysylhydroxylase1.Fur-thermore, the conserved histidines and aspartateresidues required for lysyl hydroxylase activity arepresentinthesequence.Northernanalysisidentifiedatranscriptof4.2kilobases,whichwashighlyexpressedinpancreasandmuscletissues.ExpressionofcDNAininsectcellsusingabaculovirusvectoryieldedproteinswith lysyl hydroxylase activity and an antiserumagainstasyntheticpeptideofthededucedaminoacidsequencerecognizedproteinswithmolecularweightsof88and97kDainhomogenatesofth...
The contribution of de novo and ultra-rare genetic variants in severe and moderate intellectual d... more The contribution of de novo and ultra-rare genetic variants in severe and moderate intellectual disability (ID) has been extensively studied whereas the genetic architecture of mild ID has been less well characterized. To elucidate the genetic background of milder ID we studied a regional cohort of 442 ID patients enriched for mild ID (>50%) from a population isolate of Finland. We analyzed rare variants using exome sequencing and CNV genotyping and common variants using common variant polygenic risk scores. As controls we used a Finnish collection of exome sequenced (n=11311) and GWAS chip genotyped (n=11699) individuals.We show that rare damaging variants in genes known to be associated with cognitive defects are observed more often in severe (27%) than in mild ID (13%) patients (p-value: 7.0e-4). We further observed a significant enrichment of protein truncating variants in loss-of-function intolerant genes, as well as damaging missense variants in genes not yet associated wit...
The contribution of de novo variants in severe intellectual disability (ID) has been extensively ... more The contribution of de novo variants in severe intellectual disability (ID) has been extensively studied whereas the genetics of mild ID has been less characterized. To elucidate the genetics of milder ID we studied 442 ID patients enriched for mild ID (>50%) from a population isolate of Finland. Using exome sequencing, we show that rare damaging variants in known ID genes are observed significantly more often in severe (27%) than in mild ID (13%) patients. We further observe a significant enrichment of functional variants in genes not yet associated with ID (OR: 2.1). We show that a common variant polygenic risk significantly contributes to ID. The heritability explained by polygenic risk score is the highest for educational attainment (EDU) in mild ID (2.2%) but lower for more severe ID (0.6%). Finally, we identify a Finland enriched homozygote variant in the CRADD ID associated gene.
Background Methionine synthase deficiency is a rare inborn error of intracellular cobalamin metab... more Background Methionine synthase deficiency is a rare inborn error of intracellular cobalamin metabolism caused by mutations in the MTR (5-methyltetrahydrofolate-homocysteine S-methyltransferase) gene, resulting in megaloblastic anemia and neurologic symptoms. Methods and Results We describe for the first time a homozygous MTR gene c.3518C > T (p.P1173L) mutation in a patient with severe megaloblastic anemia, developmental delay, and drug-resistant seizures associated with hyperhomocysteinemia and hypomethioninemia. Methionine synthase activity was only 9% of the reference value, and MTR protein expression was decreased in the fibroblasts of the patient. The clinical features of our patient are similar to previously published patients with the complementation type G disorder of methionine synthase deficiency with the exception of drug-resistant seizures. However, intramuscular injections of hydroxocobalamin (OHCbl) in conjunction with betaine and folic acid provided verified clinical and electrophysiological treatment response. Conclusion This study emphasizes the importance of early diagnosis of patients having neurologic symptoms due to methionine synthase deficiency where early treatment has significant effects on the clinical outcome of the patients. Elevated level of plasma homocysteine together with low methionine in plasma amino acid analysis should raise a suspicion of remethylation disorder.
Objective We conducted a randomized controlled trial to evaluate whether a combination of repeate... more Objective We conducted a randomized controlled trial to evaluate whether a combination of repeated botulinum toxin A (BTX-A) and conservative treatment is more effective in decreasing toe-walking than conservative treatment alone at 24 months follow-up. Patients and Methods Children between 2 and 9 years of age were randomized either into the conservative (CO) or botulinum treatment (BTX) group. The treatment in the CO group consisted of firm shoes, night splints, a home stretching program and physiotherapy. The BTX arm had all the same conservative treatments added with calf muscle BTX-A injections repeated in 6 months intervals if needed. Change in toe-walking pattern, ankle range of movement (ROM), and overall function were assessed at baseline and 6, 12, 18, and 24 months posttreatment. Results A total of 30 toe-walkers participated: 14 in CO and 16 in BTX group. At 24 months, all children in the BTX group and 85% in the CO group evaluated by the blinded physiotherapist (p ¼ 0.065), 75% in the BTX group and 70% in the CO group graded by the research physiotherapist (p ¼ 0.730), and 50% in the BTX group and 54% in the CO group reported by the parents ceased toe-walking (p ¼ 0.837). The most prominent change was noted during the 1st year. The BTX group seemed to reach the goal earlier. No significant differences between the treatment groups in function or in ankle ROM ensued. Conclusion Adding BTX injections did not significantly enhance the goal to walk either flat foot or with heel strike at 24 months posttreatment.
cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA l... more cDNA species coding for alpha-methylacyl-CoA racemase were cloned from rat and mouse liver cDNA libraries and characterized. The rat liver lambdagt11 cDNA expression library was screened with anti-racemase IgG [Schmitz, Albers, Fingerhut and Conzelmann (1995) Eur. J. Biochem.231, 815-822]. Several full-length clones were obtained that contained an open reading frame of 1083 bp, coding for a protein of 361 amino acid residues with a predicted molecular mass of 39679 Da. The sequences of three peptides that were isolated by HPLC from a tryptic digest of purified rat liver racemase fully matched the cDNA-derived amino acid sequence. The cDNA coding for mouse racemase was cloned from a mouse liver lambdaZAP cDNA expression library and sequenced. The coding region of 1080 bp codes for a 360-residue protein (molecular mass 39558 Da) that shares 89.7% similarity with the rat protein. Expression of the rat racemase as are combinant protein in Escherichia coli with the pTrcHisB-expression ve...
In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enz... more In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hyd...
Recently it has been shown that peroxisomes interact with microtubules which affect the structure... more Recently it has been shown that peroxisomes interact with microtubules which affect the structure and intracellular distribution of the organelle (Schrader, M., J. K. Burkhardt, E. Baumgart, G. Lüers, H. Spring, A. Völkl, H. D. Fahimi: Interaction of microtubules with peroxisomes. Tubular and spherical peroxisomes in HepG2 cells and their alterations induced by microtubule-active drugs. Eur. J. Cell Biol. 69, 24-35 (1996)). In the present work, we have applied immunological techniques to study the organization of peroxisomes within the rat liver cell. Antibodies to a pentadecapeptide corresponding to amino acid residues 403-417 of the 70 kDa integral peroxisomal membrane protein (Kamijo, K., S. Taketani, S. Yokota, T. Osumi, T. Hashimoto: The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein super family. J. Biol. Chem. 265, 4534-4540 (1990)) were raised in rabbits and affinity purified. This antibody was found to be highly specific for peroxisomes as determined by ELISA and Western blot analysis. Immunoelectron microscopy of tissue sections from rat liver revealed that peroxisomal membranes were labeled with this antibody and, in addition, labeling was found on tubular extensions often connecting peroxisomes. Antibodies to alpha-tubulin were used to locate the microtubular system. Microtubules were often found in close connection to peroxisomes, suggesting interaction between peroxisomes and the cytoskeleton. Double-labeling experiments for the 70 kDa integral peroxisomal membrane protein and alpha-tubulin demonstrated that the tubular structures connecting peroxisomes did not colocalize with microtubules. These results suggest that peroxisomes are organized in reticular structures within rat liver cells and that the structure and localization of these reticuli may be determined by their association to the microtubular network.
In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enz... more In the present study we have cloned and characterized a novel rat peroxisomal multifunctional enzyme (MFE) named perMFE-II. The purified 2-enoyl-CoA hydratase 2 with an M(r) of 31500 from rat liver [Malila, Siivari, Mäkelä, Jalonen, Latipää, Kunau and Hiltunen (1993) J. Biol. Chem. 268, 21578-21585] was subjected to tryptic fragmentation and the resulting peptides were isolated and sequenced. Surprisingly, the full-length cDNA, amplified by PCR, had an open reading frame of 2205 bp encoding a polypeptide with a predicted M(r) of 79,331 and contained a potential peroxisomal targeting signal in the C-terminus (Ala-Lys-Leu). The sequenced peptide fragments of hydratase 2 gave a full match in the middle portion of the cDNA-derived amino acid sequence. The predicted amino acid sequence showed a high degree of similarity with pig 17 beta-hydroxysteroid dehydrogenase type IV and MFE of yeast peroxisomal beta-oxidation. Recombinant perMFE-II (produced in Pichia pastoris) had 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacyl-CoA dehydrogenase activities and was catalytically active with several straight-chain trans-2-enoyl-CoA, 2-methyltetradecenoyl-CoA and pristenoyl-CoA esters. The results showed that in addition to an earlier described multifunctional isomerase-hydratase-dehydrogenase enzyme from rat liver peroxisomes (perMFE-I), another MFE exists in rat liver peroxisomes. They both catalyse sequential hydratase and dehydrogenase reactions of beta-oxidation but through reciprocal stereochemical courses.
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Papers by Heli Helander