The cblD Defect Causes Either Isolated or Combined Deficiency of Methylcobalamin and Adenosylcobalamin Synthesis
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m407733200
ISSN1083-351X
AutoresTerttu Suormala, Matthias R. Baumgartner, David Coelho, Petra Zavadáková, Viktor Kožich, Hans Georg Koch, Martin Berghaüser, J. E. Wraith, Alberto Burlina, A Sewell, Jürgen Herwig, Brian Fowler,
Tópico(s)Metabolism and Genetic Disorders
ResumoIntracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism. Intracellular cobalamin is converted to adenosylcobalamin, coenzyme for methylmalonyl-CoA mutase and to methylcobalamin, coenzyme for methionine synthase, in an incompletely understood sequence of reactions. Genetic defects of these steps are defined as cbl complementation groups of which cblC, cblD (described in only two siblings), and cblF are associated with combined homocystinuria and methylmalonic aciduria. Here we describe three unrelated patients belonging to the cblD complementation group but with distinct biochemical phenotypes different from that described in the original cblD siblings. Two patients presented with isolated homocystinuria and reduced formation of methionine and methylcobalamin in cultured fibroblasts, defined as cblD-variant 1, and one patient with isolated methylmalonic aciduria and deficient adenosylcobalamin synthesis in fibroblasts, defined as cblD-variant 2. Cell lines from the cblD-variant 1 patients clearly complemented reference lines with the same biochemical phenotype, i.e. cblE and cblG, and the cblD-variant 2 cell line complemented cells from the mutant classes with isolated deficiency of adenosylcobalamin synthesis, i.e. cblA and cblB. Also, no pathogenic sequence changes in the coding regions of genes associated with the respective biochemical phenotypes were found. These findings indicate heterogeneity within the previously defined cblD mutant class and point to further complexity of intracellular cobalamin metabolism. Cobalamin (cbl, 1The abbreviations used are: cbl, cobalamin; AdoCbl, 5-deoxyadenosylcobalamin or adenosylcobalamin; CN-Cbl, cyanocobalamin; MeCbl, methylcobalamin; OH-Cbl, hydroxocobalamin; MMA, methylmalonyl or methylmalonic acid; MTHFR, methylenetetrahydrofolate reductase; MTR, gene coding for methionine synthase; MTRR, gene coding for methionine synthase reductase; NR1, novel reductase 1; MRI, magnetic resonance imaging; SNP, single nucleotide polymorphism. 1The abbreviations used are: cbl, cobalamin; AdoCbl, 5-deoxyadenosylcobalamin or adenosylcobalamin; CN-Cbl, cyanocobalamin; MeCbl, methylcobalamin; OH-Cbl, hydroxocobalamin; MMA, methylmalonyl or methylmalonic acid; MTHFR, methylenetetrahydrofolate reductase; MTR, gene coding for methionine synthase; MTRR, gene coding for methionine synthase reductase; NR1, novel reductase 1; MRI, magnetic resonance imaging; SNP, single nucleotide polymorphism. vitamin B12) serves as the cofactor for two enzymes in humans. Mitochondrial methylmalonyl-CoA mutase (MMA-CoA mutase; EC 5.4.99.2) requires 5-deoxyadenosylcobalamin (AdoCbl), and N5-methyltetrahydrofolate:homocysteine methyltransferase (methionine synthase, EC 2.1.1.13) is dependent on methylcobalamin (MeCbl). To fulfill its cofactor function dietary cbl must be absorbed, transported in the bloodstream, and taken up into cells in a complex series of processes involving specific receptors and carrier proteins (1Rosenblatt D.S. Fenton W.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 3897-3933Google Scholar). Intracellular processing is also complex and not fully understood. After release of lysosomal cbl into the cytosol, reduction of cob(III)alamin to cob(I)alamin is followed by reductive methylation on the methionine synthase enzyme in the cytosol, or by attachment of an adenosyl group in the mitochondrion. A number of rare defects in these intracellular processing steps are known, causing disease of varying severity. So far eight cbl complementation groups have been linked to these disorders (see "Discussion" for further details). The cblE (MIM 236270) and cblG (MIM 250940) complementation groups represent defects of methionine synthase reductase (EC 2.1.1.135; MTRR gene) and methionine synthase (MTR gene), respectively, both causing isolated homocystinuria (2Gulati S. Baker P. Li Y.N. Fowler B. Kruger W. Brody L.C. Banerjee R. Hum. Mol. Genet. 1996; 5: 1859-1865Crossref PubMed Scopus (93) Google Scholar, 3Leclerc D. Wilson A. Dumas R. Gafuik C. Song D. Watkins D. Heng H.H.Q. Rommens J.M. Scherer S.W. Rosenblatt D.S. Rozen R. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3059-3064Crossref PubMed Scopus (351) Google Scholar, 4Watkins D. Rosenblatt D.S. J. Clin. Invest. 1988; 81: 1690-1694Crossref PubMed Scopus (70) Google Scholar). Banerjee and coworkers have shown that another system, comprising soluble cytochrome b5 and novel reductase 1 (NR1), is able to reductively activate methionine synthase, in addition to methionine synthase reductase. However, the functional significance of this system remains unclear (5Olteanu H. Banerjee R. J. Biol. Chem. 2003; 278: 38310-38314Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The cblA (MIM 251100), cblB (MIM 251110), and cblH (MIM 606169) complementation groups are linked to processes unique to AdoCbl synthesis and cause isolated methylmalonic aciduria (MMA-uria) (6Fenton W.A. Gravel R.A. Rosenblatt D.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 2165-2193Google Scholar, 7Watkins D. Matiaszuk N. Rosenblatt D.S. J. Med. Genet. 2000; 37: 510-513Crossref PubMed Scopus (47) Google Scholar). Genes for the cblA (MMAA) and cblB (MMAB) groups have recently been described, although the corresponding proteins have not yet been characterized. The deduced amino acid sequence of the MMAA gene points to a transporter or accessory protein involved in translocation of cbl into the mitochondrion (8Dobson C.M. Wai T. Leclerc D. Wilson A. Wu X. Dorè C. Hudson T. Rosenblatt D.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15554-15559Crossref PubMed Scopus (118) Google Scholar). The MMAB gene appears to code for cbl adenosyltransferase (EC 2.5.1.7) (9Dobson C.M. Wai T. Leclerc D. Kadir H. Narang M. Lerner-Ellis J.P. Hudson T.J. Rosenblatt D.S. Gravel R.A. Hum. Mol. Genet. 2002; 26: 3361-3369Crossref Scopus (135) Google Scholar). Only one patient has been designated to the cblH complementation group, and nothing is known about the corresponding gene or protein (7Watkins D. Matiaszuk N. Rosenblatt D.S. J. Med. Genet. 2000; 37: 510-513Crossref PubMed Scopus (47) Google Scholar). Isolated MMA-uria is also caused by defects of the MMA-CoA mutase apoenzyme (EC 5.4.99.2), designated the mut complementation group. The majority of patients have complete enzyme deficiency (mut0), whereas some (mut–) show residual enzyme activity with reduced affinity for AdoCbl, and hydroxocobalamin (OH-Cbl) responsiveness in fibroblasts (6Fenton W.A. Gravel R.A. Rosenblatt D.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 2165-2193Google Scholar). The cblF (MIM 277380), cblC (MIM 277400), and cblD (MIM 277410) complementation groups affect early steps of intracellular cbl processing and cause combined homocystinuria with MMA-uria (1Rosenblatt D.S. Fenton W.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 3897-3933Google Scholar). The cblF defect is due to disturbed lysosomal release of OH-Cbl into cytoplasm (10Watkins D. Rosenblatt D.S. Am. J. Hum. Genet. 1986; 39: 404-408PubMed Google Scholar). The defect in the cblC and cblD groups is likely to involve cytosolic reduction of cbl as suggested by two reports (11Pezacka E.H. Biochim. Biophys. Acta. 1993; 1157: 167-177Crossref PubMed Scopus (53) Google Scholar, 12Watanabe F. Saido H. Yamaji R. Miyatake K. Isegawa Y. Ito A. Yubisui T. Rosenblatt D.S. Nakano Y. J. Nutr. 1996; 126: 2947-2951Crossref PubMed Scopus (25) Google Scholar), but the exact metabolic steps and mechanisms remain unclear. Cbl-β-ligand transferase activity was shown to be reduced to 2–34% of the mean control value in four cblC cell lines but was also low (30%) in a cblD cell line (11Pezacka E.H. Biochim. Biophys. Acta. 1993; 1157: 167-177Crossref PubMed Scopus (53) Google Scholar). Thus the role of this enzyme in the cblC disorder remains to be confirmed. The cblD complementation group has so far only been assigned to two siblings (13Willard H.F. Mellman I.S. Rosenberg L.E. Am. J. Hum. Genet. 1978; 30: 1-13PubMed Google Scholar) described in 1970 (14Goodman S.I. Moe P.G. Hammond K.B. Mudd S.H. Uhlendorf B.W. Biochem. Med. 1970; 4: 500-515Crossref PubMed Scopus (83) Google Scholar), whereas more than a hundred patients belonging to the cblC group have been reported (6Fenton W.A. Gravel R.A. Rosenblatt D.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 2165-2193Google Scholar). Studies of enzyme activities and cbl metabolism in fibroblasts of the cblD siblings revealed similar but less severe abnormalities compared with those found in cblC cells (15Fenton W.A. Rosenberg L.E. Annu. Rev. Genet. 1978; 12: 223-248Crossref PubMed Scopus (28) Google Scholar, 16Mellman I.S. Willard H.F. Youngdahl-Turner P. Rosenberg L.E. J. Biol. Chem. 1979; 254: 11847-11853Abstract Full Text PDF PubMed Google Scholar). Clinical presentation varies greatly both between and within the complementation groups. Patients with homocystinuria, isolated or in combination with MMA-emia, present with megaloblastic anemia and various neurological abnormalities (1Rosenblatt D.S. Fenton W.A. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 3897-3933Google Scholar, 17Watkins D. Rosenblatt D.S. Am. J. Med. Genet. 1989; 34: 427-434Crossref PubMed Scopus (138) Google Scholar) and have, at least to some degree, responded biochemically and clinically to vitamin B12 therapy. Patients with isolated MMA-emia usually present with metabolic decompensation and show lethargy, failure to thrive, feeding problems, and muscular hypotonia but no signs of megaloblastic anemia (6Fenton W.A. Gravel R.A. Rosenblatt D.S. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. 8th Ed. McGraw-Hill, Inc., New York2001: 2165-2193Google Scholar). We present evidence for heterogeneity within the cblD complementation group based on biochemical findings and detailed enzyme studies in cultured skin fibroblasts in three new unrelated patients. In contrast to the previously reported cblD siblings, one patient revealed isolated MMA-uria, the other two isolated homocystinuria, although each belongs to the cblD complementation group when investigated by somatic complementation in cultured fibroblasts. Patient 1, born after uneventful pregnancy, was of Irish origin. Family relationships were complicated and suggested high grade consanguinity. At 6 years of age he presented with global developmental delay, severe learning difficulties and spastic ataxia, absence of ankle jerks, and rapid deterioration of gait. His weight and head circumference were at the 50th percentile. He had no vocal skills, made no eye contact, and on attempting to walk he held his arms abducted and flexed at the elbow. Reflexes were present in the upper limbs but absent in the lower limbs. A cranial MRI scan revealed cerebral and cerebellar atrophy. Visually evoked responses were poorly formed and delayed, and an electroretinogram analysis was normal. The mean corpuscular volume was elevated (94 fl; normal range, 70–87), but hemoglobin (11.9 g/dl) and plasma B12 (787 ng/liter; normal range, 170–900) were normal. The folate content was elevated in the serum (20 μg/liter; normal range, 1.6–13.2) and in red cells (1114 μg/liter; normal range, 125–600). Plasma non-protein-bound homocystine was elevated (9 μm; normal, not detectable), and methionine was low (14 μm; normal range, 15–40). MMA was not elevated in urine. Treatment consisted of betaine (9–15 g/day), folinic acid, which was later changed to folic acid (15 mg/day), and OH-Cbl (1 mg intramuscularly daily, later 1 mg weekly). After 1 week on therapy, plasma non-protein-bound homocystine was not detectable and the methionine level was normal (37 μm). The patient appeared more alert and had increased muscle tone in all limbs with brisk tendon reflexes. Six months later he was able to walk and became very vocal. At 16 years of age, compliance with medication was good, weight and height were between the 10th and 25th, and head circumference was at the 10th percentile. He had nearly normal gait, could speak a number of single words, but required help with dressing and washing; he could finger-feed and drink from a cup but is incontinent. He showed erratic behavior, hyperactivity, and aggression, and his sleep pattern was poor. Although the intelligence quotient could not be formally assessed, the patient was clearly severely mentally retarded. Patient 2 was the first child of healthy unrelated Italian parents. Pregnancy and delivery were unremarkable. At the age of 3 months he presented with severe hypotonia, nystagmus, dystonic movements, and seizures that were difficult to control with anticonvulsants. Brain MRI analysis showed reduced myelination and a small cerebellar vermis. Megaloblastic anemia (mean corpuscular volume, 105 fl; hemoglobin, 8.5 g/dl; and hematocrit, 25.4%) with normal plasma folate and slightly reduced B12 (156 ng/liter; normal range, 200–1000) results were found. Plasma total homocysteine was elevated (128 μm; normal range, 5–16), and methionine decreased (4 μm; normal range, 15–54). MMA was not elevated in urine. Betaine (Cystadane®, Orphan Europe, 200 mg/kg/day), folic acid (15 mg/day), and OH-Cbl (1 mg/day) treatment resulted in clinical and biochemical improvement. Seizures disappeared within 10 days, and hematological status normalized. Plasma total homocysteine decreased to 23 μm, and methionine became normal (38 μm). At the age of 3 years, betaine was stopped and OH-Cbl injections were reduced to 1 mg twice a week resulting in slightly higher values of plasma total homocysteine (42 μm). Reintroduction of betaine (100 mg/kg/day) lowered plasma total homocysteine to 25 μm. At the age of 4 years his gross and fine motor skills were normal. An MRI showed normal development of the brain, however, speech delay was present and the intelligence quotient assessed by the Stanford-Binet scale was 84. Patient 3 was the second child of unrelated parents of Indian origin, born at 32 weeks of gestation with a grade II respiratory distress syndrome requiring artificial ventilation. Further complications were grade I cranial hemorrhage, necrotizing enterocolitis, and neonatal convulsions. MMA (978 mmol/mol creatinine; normal, <4) and methylcitrate (197 mmol/mol creatinine; normal, not detectable) were markedly elevated in urine. Hemoglobin and differential blood count were normal. Therapy with intramuscular OH-Cbl (1 mg/week, later 2 × 0.5 mg/week), carnitine, protein restriction, and phenobarbital resulted in normalization of metabolite excretion and the electroencephalogram. Non-protein-bound homocystine was not detected in the plasma, and later normal plasma total homocysteine (8.9 μm) was found even when treatment compliance was poor. Withdrawal of B12 therapy resulted in an increase of metabolite excretion (MMA: 18,425 mmol/mol creatinine; methylcitrate: 67 mmol/mol creatinine), which was readily reversed upon reintroduction of protein restriction and B12 therapy. At the age of 12 year the patient attended a special school because of mild learning problems. Treatment continued as done previously but compliance was poor and clinic attendance was sporadic. Accordingly, MMA excretion was usually above 1000 mmol/mol creatinine, and his electroencephalogram was abnormal. The intelligence quotient was 69 (Hawik-III test). Cell Lines—Fibroblasts were grown from skin biopsies obtained with informed consent of the patients or their parents. Cells were cultured in Earle's minimal essential medium supplemented with 10% fetal calf serum, 2 mm l-glutamine, and antibiotics as described earlier (18Suormala T. Gamse G. Fowler B. Clin. Chem. 2002; 48: 835-843Crossref PubMed Scopus (22) Google Scholar). Reference cell lines belonging to different cbl mutant classes were characterized with methods described below and included five cblA, six cblB, five cblC, nine cblE, and six cblG cell lines. The complementation group of each of these cell lines was established by complementation analysis using defined cell lines obtained from the Montreal Cell Repository (cblE and cblG mutants) or cell lines in which complementation analysis was earlier performed in the laboratory of Prof. D. Rosenblatt (cblA, cblB, and cblC mutants). Cell lines WG1220 (one of the original siblings with cblD defect), designated here as the original cblD cell line, WG1575 (cblE), and WG1308 (cblG) were obtained from the Repository for Mutant Human Cell Strains (The McGill University-Montreal Children's Hospital Research Institute, Montreal, Canada). Assays in Intact Fibroblast Monolayers—Incorporation of [14C]propionate was assayed by a slight modification of the method described by Willard et al. (19Willard H.F. Ambani L.M. Hart A.C. Mahoney M.J. Rosenberg L.E. Hum. Genet. 1976; 34: 277-283Crossref PubMed Scopus (90) Google Scholar), and formation of [14C]methionine from [14C]formate was determined as described earlier (20Fowler B. Whitehouse C. Wenzel F. Wraith J.E. Pediatr. Res. 1997; 41: 145-151Crossref PubMed Scopus (32) Google Scholar). Both assays were performed in intact fibroblasts grown in normal and OH-Cbl-supplemented media. Uptake of CN-[57Co]Cbl and cbl coenzyme synthesis from CN-[57Co]Cbl was determined in intact fibroblasts as described earlier (21Fowler B. Jakobs C. Eur. J. Pediatr. 1998; 157: 88-93Crossref PubMed Google Scholar). CN-[57Co]Cbl was bound to transcobalamin in normal human serum by incubation for 30 min at 37 °C. Fibroblasts were incubated with medium containing this serum at a final concentration of 10% (v/v) for 4 days in the dark. Cells were harvested, disrupted by freezing and thawing, and cbl derivatives were extracted in hot ethanol and separated by high performance liquid chromatography. Specific Enzyme Assays—Fibroblasts were harvested by trypsinization and cell free lysates prepared for enzyme assay. MMA-CoA mutase activity was assayed by measuring the conversion of [14C]MMA-CoA to [14C]succinyl-CoA in the presence (total mutase) and absence (holomutase) of 50 μm AdoCbl as described earlier (22Baumgartner R. Hall C.A. The Cobalamins: Methods in Hematology. 10. Churchill Livingstone, Edinburgh, New York1983: 181-195Google Scholar). Methionine synthase activity was measured under high reducing conditions (dithiothreitol, 28 mm) by measuring the formation of [14C]methionine from [14C]methyltetrahydrofolate and l-homocysteine in the presence (total methionine synthase) and absence (holo-methionine synthase) of 50 μm MeCbl (modified from Ref. 23Mellman I. Willard H.F. Rosenberg L.E. J. Clin. Invest. 1978; 62: 952-960Crossref PubMed Scopus (39) Google Scholar). These two assays were performed in fibroblasts cultured in normal and OH-Cbl supplemented (1 mg/liter) medium. Cbl adenosyl transferase activity was determined by measuring the conversion of OH-[57Co]Cbl to Ado[57Co]Cbl in an H2 atmosphere as described (24Fenton W.A. Rosenberg L.E. Biochem. Biophys. Res. Commun. 1981; 98: 283-289Crossref PubMed Scopus (30) Google Scholar), except that the total OH-Cbl concentration in the assay mixture was 3.42 nm and OH-Cbl and AdoCbl were separated by high performance liquid chromatography as described for cbl coenzyme synthesis (see above). 5,10-Methylenetetrahydrofolate reductase (MTHFR) was assayed in its physiological forward direction in the presence of 75 μm flavin adenine dinucleotide as described earlier (18Suormala T. Gamse G. Fowler B. Clin. Chem. 2002; 48: 835-843Crossref PubMed Scopus (22) Google Scholar). Somatic Cell Complementation Analysis—Complementation analysis was performed by a modification of an earlier described method (25Zavadakova P. Fowler B. Zeman J. Suormala T. Pristoupilova K. Kozich V. J. Inherit. Metab. Dis. 2002; 25: 461-476Crossref PubMed Scopus (37) Google Scholar). Briefly, heterokaryons were produced in mixed fibroblast cultures by treatment with 40% (v/v) polyethylene glycol 1500 (Sigma P-7181) for 120 s. Three days later incorporation of [14C]propionate or formation of [14C]methionine from [14C]formate was measured as described above. Within each experiment mixed unfused cells, self fusions, and fusion of cells from different known complementation groups were included as background, negative, and positive controls, respectively. In addition, patients' cells were fused with cell lines belonging to at least two different complementation groups to control the quality of the cells in each individual experiment. Further, the presence of fused cells, indicated by a characteristic enlarged cell structure, was confirmed by microscopic examination. Molecular Genetic Studies—Genomic DNA and total RNA were extracted from cultured skin fibroblasts and cDNA was synthesized from RNA with standard protocols and kits (Invitrogen or Qiagen). The MTR (cblG) and MTRR (cblE) genes, and genes shown to be implicated in the reductive activation of methionine synthase (genes coding for NR1 and cytochrome b5) were analyzed in patients 1 and 2 with isolated homocystinuria. The MMAA (cblA) gene was analyzed in patient 3 with isolated MMA-uria. Entire coding regions of the genes were PCR-amplified from cDNA and sequenced. Each identified genetic variant was verified at the genomic DNA level using PCR-restriction fragment length polymorphism or a PCR amplification refractory mutation system. For analysis of the MTR gene cDNA containing the entire coding region (3798 bp) was amplified in four overlapping fragments (cDNA positions 65–1088/1027–2033/1921–2953/2887–3852). M13-specific sequence tags were attached to each primer in the 5′-position facilitating DNA sequencing using a subsequent PCR reaction. Automated sequence analysis was performed using an ABI Prism 3700 sequencer (Applied Biosystems) essentially as recommended by the manufacturer. The entire MTRR coding sequence with its flanking 5′- and 3′-untranslated regions was amplified in five overlapping segments employing cDNA and sequenced as described previously (25Zavadakova P. Fowler B. Zeman J. Suormala T. Pristoupilova K. Kozich V. J. Inherit. Metab. Dis. 2002; 25: 461-476Crossref PubMed Scopus (37) Google Scholar) except for a 5′-coding region (exons 1–5). This region contains alternative isoforms, and therefore exons 1–5 were analyzed by sequencing of PCR products derived from genomic DNA. The coding sequence of the MMAA gene was investigated by direct sequencing of PCR products derived from cDNA except for exons 6 and 7 (3′-coding region), which were analyzed by sequencing of PCR products derived from genomic DNA using published primers (8Dobson C.M. Wai T. Leclerc D. Wilson A. Wu X. Dorè C. Hudson T. Rosenblatt D.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15554-15559Crossref PubMed Scopus (118) Google Scholar). The coding region of human NR1 was investigated by direct sequencing of PCR products derived from two overlapping cDNA fragments using published primers (5Olteanu H. Banerjee R. J. Biol. Chem. 2003; 278: 38310-38314Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The coding region of human soluble cytochrome b5 gene was investigated by direct sequencing in both directions of PCR products derived from cDNA using primers containing the 15 nucleotides specific to the soluble isoform (26Giordano S.J. Steggles A.W. Biochem. Biophys. Res. Commun. 1991; 178: 38-44Crossref PubMed Scopus (35) Google Scholar). Enzyme Activities, cbl Uptake, and cbl Coenzyme Synthesis— Activities of enzymes related to MMA and homocysteine metabolism are shown in Tables I and II, respectively, and total cbl uptake and coenzyme synthesis results are given in Table III, in fibroblasts of the three patients, in reference cell lines belonging to different cbl complementation groups, and in control fibroblasts.Table IActivities of enzymes related to methylmalonic acid metabolism in cultured skin fibroblastsMethylmalonyl-CoA mutasePropionate incorporationHolo-enzyme (-AdoCbl)Total enzyme (+AdoCbl)Adenosyltransferasenmol/16 h/mg proteinpmol/min/mg proteinfmol/h/mg proteinOH-Cbl in medium →-+-+-+-Patient 18.4, 9.8, 118.4, 9.5, 1173, 93, 139102, 135, 281493, 753, 775465, 875, 11853.1, 2.5, 1.9Patient 26.2, 8.9, 9.46.4, 8.1, 1190, 110, 117104, 179, 192938, 998, 765919, 935, 7780.71, 0.76, 1.2Patient 31.3, 1.4, 1.916, 16, 1412, 13, 7846, 26, 231338, 2108, 554916, 1446, 3932.0, 2.5, 3.45 cblA cell lines1.3 (0.73-2.0)4.9 (2.8-7.4)31 (18-45)33 (13-69)989 (544-1762)247 (129-413)1.9 (1.5-2.4)3 cblB cell lines1.4 (0.62-1.9)3.6 (1.4-4.8)26 (11-45)34 (27-42)1334 (884-1615)845 (710-915)0 (0-0.03)3 cblB cell lines1.0 (0.71-1.5)1.2 (0.75-1.8)38 (4.4-94)36 (12-83)1188 (554-1826)786 (557-1086)0 (0-0.001)5 cblC cell lines1.1 (0.24-1.8)9.4 (2.5-19.6)43 (15-95)52 (30-127)1178 (676-1627)536 (150-813)2.3 (1.8-3.2)Original cblD1.44.7264013768282.89 cblE cell lines10 (3.7-22)10 (3.9-21)6 cblG cell lines11 (5.3-17)11 (5.8-18)Control cell lines12 (6.4-18)12 (5.2-21)53 (24-106)539 (265-1251)1029 (575-2057)1237 (585-2570)1.2 (0.2-2.3)n13132424242412 Open table in a new tab Table IIActivities of enzymes related to homocysteine metabolism in cultured skin fibroblastsMethionine synthaseHolo-enzyme (-MeCbl)Total enzyme (+MeCbl)Methionine formationMTHFRnmol/16 h/mg proteinpmol/min/mg proteinnmol/h/mgOH-Cbl in medium →-+-+-+-Patient 10.50, 0.58, 0.661.2, 1.9, 2.70.7, 3.4, 6.69.9, 28, 2822, 56, 7864, 66, 10119.9, 33.2, 38.6Patient 20.21, 0.43, 0.651.2, 1.2, 1.53.7, 4.7, 8.316, 19, 2426, 38, 3943, 44, 5813.5, 14.5, 23.3Patient 32.2, 2.2, 3.12.0, 2.6, 3.226, 29, 49189, 194, 220118, 130, 144338, 356, 38117.9, 21.6, 24.65 cblA cell lines3.2 (2.3-4.2)3.7 (3.0-4.3)6 cblB cell lines2.9 (2.4-3.4)3.4 (2.9-4.2)5 cblC cell lines0.07 (0.03-0.19)2.2 (1.3-3.6)1.1 (0-3.4)46 (10-141)48 (7.8-93)77 (40-195)23.6 (10.5-39.1)Original cblD0.380.848.120475323.69 cblE cell lines0.10 (0.04-0.23)0.14 (0.03-0.37)45 (21-77)163 (92-217)99 (51-136)230 (163-317)23.9 (17.8-42.9)5 cblG cell lines0.22 (0.03-0.29)0.23 (0.03-0.34)4.5 (2.1-7.1)13 (2.0-29)21 (1.4-53)41 (1.4-128)26.6 (19.8-49.6)1 cblG cell line0.63, 0.97, 1.02.2, 2.3, 2.43.217145423.9Control cell lines2.5 (1.4-4.3)2.9 (1.7-5.0)32 (13-56)223 (88-496)81 (33-159)348 (139-625)25.9 (14.5-54.6)n10101616161675 Open table in a new tab Table IIITotal uptake of CN-[57Co]cobalamin and cobalamin coenzyme synthesis in fibroblastsDistribution of cobalaminaCN-Cbl, cyanocobalamin; OH-Cbl, hydroxocobalamin; AdoCbl, adenosylcobalamin; MeCbl, methylcobalamin; n, number of different control cell lines.Total uptakeCN-CblOH-CblAdoCblMeCblOtherspg/mg protein% of totalPatient 134, 43, 5612, 20, 247.5, 11, 8.177, 58, 652.9, 5.1, 3.30, 6.4, 0Patient 262, 56, 5723, 35, 2416, 10, 5.855, 49, 655.6, 6.6, 5.00.6, 0, 0Patient 376, 98, 7617, 19, 207.8, 14, 9.28.2, 8.6, 5.367, 58, 660, 0, 05 cblA cell lines58 (44-87)20 (13-26)17 (13-28)7.7 (3.2-11)55 (49-64)0.2 (0-0.9)6 cblB cell lines64 (37-97)22 (11-35)18 (9.7-35)7.0 (4.1-13)53 (40-67)0.1 (0-0.6)5 cblC cell lines9.1 (3.5-17)73 (68-79)16 (13-24)6.4 (3.9-7.8)3.5 (0-7.0)1.0 (0-5.0)Original cblD174630101409 cblE cell lines54 (35-80)26 (16-39)34 (18-51)25 (14-34)12 (4.8-20)3.7 (0-13)6 cblG cell lines64 (53-85)13 (6.0-18)22 (12-35)58 (41-74)5.6 (3.2-8.8)1.8 (0-5.4)Controls (n = 23)62 (39-119)12 (6.0-17)14 (4.7-28)19 (14-26)54 (39-69)1.0 (0-5.6)a CN-Cbl, cyanocobalamin; OH-Cbl, hydroxocobalamin; AdoCbl, adenosylcobalamin; MeCbl, methylcobalamin; n, number of different control cell lines. Open table in a new tab Incorporation of propionate in intact fibroblasts measures integrity of the propionate to the succinate pathway, which is an indirect measure of MMA-CoA mutase activity. Similarly, formation of methionine from formate in intact fibroblasts indirectly measures the activity of methionine synthase. These a
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