Abstract
An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcarnitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.
Similar content being viewed by others
Main
With the exception of β-ketothiolase deficiency (McKusick 203750), there are no reported human genetic abnormalities specific to L-isoleucine degradation (1). During routine metabolic work-up of a 4-mo-old male infant who presented with mild hypoglycemia, lethargy, and apnea, we detected increased plasma short-chain acylcarnitine (AC) using standard methods (2). Tandem mass spectrometric analysis of plasma AC revealed an elevated five-carbon species, representing the sum of isovaleryl- and/or 2-methylbutyrylcarnitine, isomers which are not differentiated by this methodology (3–5). Separation of isomers using a different method subsequently confirmed 2-MBC as the predominant species. Increased quantities of 2-MBG were routinely detected in the proband's urine, without other diagnostic abnormalities. These results suggested an isolated defect of 2-MBCDase (Fig. 1). Subsequent metabolic, enzymatic, immunoblot, molecular and prenatal diagnostic investigations verified 2-MBCDase deficiency.
The male proband was delivered at 39 wk gestation to a gravida 1, para 1 mother of Northern European descent. The father was Eritrean. There was no significant family history. Birth and delivery were uneventful. He was alert at 20 h, with weak suck and decreased tone. Discharge at 24 h revealed blood glucose at 50 mg/dL (2.8 mmol/L) (nl 55–100 mg/dL; 3.1–5.6 mmol/L). The patient was hospitalized at day 3 of life for poor feeding, lethargy, and hypothermia. Mother reported gagging without emesis in the preceding 24 h. At admission, weight was 2.42 kg, temperature 36.7°C, heart rate 206, blood glucose 10 mg/dL (0.6 mmol/L), and venous pH 7.31 (anion gap −5.0). Administration of 20 mL 10% dextrose improved glucose to 64 mg/dL (3.6 mmol/L); within 2 h, however, the value had dropped to 18 mg/dL (1 mmol/L), and he was continued on 10% dextrose at 18 mL/h to maintain blood glucose between 70–100 mg/dL (3.9–5.6 mmol/L). He was transferred to intensive care following two episodes of apnea, where he was described as hypothermic, tachycardic, lethargic, and significantly dehydrated with cool extremities and barely palpable peripheral pulse. EEG revealed mild attenuation of background activity and intermittent frequent bilateral sharp transients in the central and temporal regions, suggesting diffuse cerebral disturbance. MRI revealed altered signal intensities in the gray and white matter of the cerebral hemispheres, with some cortical enhancement in the post Gadolinium series mainly involving the parietal and occipital lobes bilaterally. Foci of slightly increased T1 signal intensity were observed in the lentiform nuclei bilaterally. These findings were consistent with global hypoxia. A full description of this patient will be presented elsewhere. Current therapeutic regimen includes protein restriction (1.4 g/kg/day) and L-carnitine supplementation (600 mg/day at 71 mg/kg, 3 times a day). All investigations were performed with informed consent and approval by the applicable Institutional Review Board.
METHODS
Carnitine in plasma and 2-MBG/2-MBC in urine were determined using established methods (3, 4, 6). Quantitation of 2-MBG in urine and amniotic fluid by stable-isotope dilution used D3-isovalerylglycine as internal standard with gas chromatography-mass spectrometric quantitation (7). The latter assumed roughly equal extraction efficiency for the different glycine conjugates. Quantitation of C5 and C3 AC in amniotic fluid were determined by MSMS (3–5). Oxidation of U-14C-isoleucine to 14CO2 in intact fibroblasts was performed as described (8). Intact cell oxidation of 13C6-leucine, 13C5-valine, and 13C6-isoleucine in fibroblasts and amniocytes, incubated in the presence of 0.4 mmol/L L-carnitine, followed established methods with determination of AC products by MSMS (9). Western blot analysis of 2-MBCDase in extracts of fibroblasts was performed as described (10).
Characterization of 2-MBCDase cDNA and genomic sequences was achieved using established molecular biologic methods (numbering refers to the published sequence, accession NM 001609.1). For reverse-transcription/polymerase chain reaction (RT-PCR) of cDNA, the following primers were used: nucleotides (nt) 90–113, 5′-TTGGAAGATTCCTCCTCATGTCTC-3′ forward primer 1; nt 844–822, 5′-CCAAGATATTGGCTTCTGGAACC-3′ back primer 1, producing a 755 nt product;nt 670–693, 5′-GTGATGGCAAATGTAGACCCTACC-3′ forward primer 2; nt 1744–1723, 5′-AATGAACCAGGCATGGTGGTGC-3′ back primer 2, producing a 1075 nt product. The total region amplified and sequenced spanned nt 90 to 1744; the first 90 bp could not be readily amplified using standard PCR methodology. For genomic analysis of the mutation found by RT-PCR, the following primers were used: 5′-TCTTAGTAGATCGTGATACTCCG-3′, nt 741–719 forward primer 3 and 5′-GGCAGGTGGAAGAAGCTCTG-3′, nt 799–780 back primer 3, producing an 80 bp fragment (apparently all within one exon); sequence analysis revealed a product of the expected sequence. The mutation detected in the proband was directly next to back primer 3, deleting a Dde I restriction site. Restriction digestion of the 80 bp amplified genomic fragment with Dde I produced a 22-bp product. In the region of the restriction site, wild-type sequence is GGGGcTCAGAGCT, while the same region in the patient was GGGGtTCAGAGCT (Dde I recognizes the sequence CTNAG, in which N represents any base; CAGAGCT is complimentary to the end of back primer 3). Expression of mutant and wild-type 2-MBCDase was performed in an E. coli system as previously described (11). Extracts from cells containing wild-type and mutant 2-MBCDase plasmids were tested for enzyme activity using the electron transferring flavoprotein assay previously described (11).
RESULTS
Short-chain AC in proband plasma were increased (range 8–22 μM, n = 7; nl, 3–10); free carnitine and the AC/free carnitine ratio were normal. MSMS analysis revealed elevated plasma C5-AC (1.4–2.4 μM; nl < 0.4), representing the sum of 2-methylbutyrylcarnitine and isovalerylcarnitine. Qualitative acyloxylactone analysis of urine AC by gas chromatography-mass spectrometry verified the presence of 2-MBC. Urine 2-MBG was consistently elevated (range 8–30 mg/g creatinine, n = 6; nl undetectable using routine methods;Fig. 2). Protein restriction and L-carnitine supplementation led to normalization of 2-MBG excretion in urine; urine 2-MBC was not routinely determined. Plasma amino acids were repeatedly normal.
In a subsequent pregnancy, the concentration of 2-MBG in amniotic fluid obtained at 15 wk gestation was 0.27 μmol/L (nl < 0.03, n = 4). C5-AC concentration determined by MSMS was significantly elevated at 1.93 μmol/L (mean control 0.37 ± 0.18 (SD), n = 27), and the ratio of C5:C3 AC (C3 = propionylcarnitine, normal in at-risk amniotic fluid) was elevated at 1.68 (mean control 0.43 ± 0.18 (SD), n = 27). During the second pregnancy, the mother was prescribed L-carnitine, and 2-MBC was detected in her urine using qualitative acyloxylactone analysis. Urine before L-carnitine supplementation was not available. The second pregnancy resulted in birth of a healthy female. Urine 2-MBG was increased in two urine samples (4–8 mg/g creatinine; nl undetected), and plasma C5-AC (again representing the sum of 2-methylbutyrylcarnitine and isovalerylcarnitine) was elevated at 1.8 μM (nl < 0.4).
Intact fibroblast conversion of U-14C-isoleucine to 14CO2 in cultured fibroblasts was: control (n = 7) 1,250 ± 427 dpm 14CO2/hr/mg protein, range 465–1,852, mean of duplicate determinations for each cell line); proband 322 (26% of control, > 2 SD below control mean); branched chain 2-oxo acid dehydrogenase deficiency, 11 (1% of mean control); β-ketothiolase deficient fibroblasts, 407 (33% of mean control) (see Fig. 1 for location of enzymes). The conversion of U-[13C]-branched chain amino acids to AC intermediates, in the presence of 0.4 mmol/L L-carnitine, was investigated in fibroblasts derived from proband and controls. The conversion of 13C6-leucine and 13C5-valine in proband fibroblasts revealed no abnormal accumulation of AC; conversely, there was a 10-fold accumulation of C5-AC (control 2.9 nmol 13C5 AC/mg protein/72 h incubation; patient, 29.8) when incubated with 13C6-isoleucine and L-carnitine. Comparable data were obtained using cultured amniocytes derived from the subsequent pregnancy. Oxidation of 13C6-leucine and 13C5-valine in intact at-risk amniocytes was normal; conversely, at-risk amniocytes accumulated 13C5-AC when incubated in the presence of 13C6-isoleucine (control, 2.4 and 6.9 (n = 2) nmol 13C5-AC/mg protein/96 h; at-risk amniocytes, 22.5). Western-blot analysis of proband fibroblasts, using antisera raised in rabbit against purified 2-MBCDase, revealed absence of 2-MBCDase cross-reactive material (11) (Fig. 3).
Screening of 2-MBCDase sequences amplified from cDNA synthesized from proband fibroblasts revealed a 778 C>T substitution in the coding region, leading to substitution of phenylalanine for leucine at amino acid 222 (L222F); the patient's mother also carried this mutation; analysis of 33 control genomic DNA samples (66 chromosomes) revealed a C at position 778. The patient appeared homozygous for this allele in RT-PCR cDNA analyses. However, he (and his mother) were clearly heterozygous in genomic PCR and PCR/restriction analyses. Lack of availability of the genomic structure of the 2-MBCDase has hampered our search for the paternal mutation, but these studies are in progress. The L222F protein is inactive when produced in an E. coli expression system, while wild-type enzyme activity is readily detected (61.9 ± 1.9 mU/mg protein using 2-methylbutyryl-CoA as substrate) (11). The L222F protein was still inactive upon addition of 50–100 times more protein to the assay. Immunoblotting of bacterial extracts showed that the mutant protein was efficiently expressed in this system, but was less soluble than the wild-type enzyme (data not shown).
DISCUSSION
Our results provide evidence for a defect in 2-MBCDase activity in our patient, a new inborn error of L-isoleucine metabolism. In contrast to patients with isovaleryl-CoA dehydrogenase deficiency, who manifest a defect at the corresponding step of L-leucine degradation, our patient did not demonstrate overwhelming metabolic decompensation nor ketosis, which may often accompany this disorder. One puzzling feature of our patient was the low levels of 2-MBG excreted, which contrasts with isovaleryl-CoA dehydrogenase deficient patients who may excrete hundreds of mg of isovalerylglycine per g of creatinine. Perhaps 2-methylbutyryl-CoA is a poor substrate for glycine conjugation, or much of the accumulated 2-methylbutyryl-CoA is hydrolyzed in lieu of conjugation. Our proband was also clinically and metabolically distinct from a patient with isolated isobutyryl-CoA dehydrogenase deficiency, which is the corresponding enzyme defect on the L-valine catabolic pathway. That patient manifested anemia, dilated cardiomyopathy and L-carnitine insufficiency (9). The clinical picture in our patient was that of neurologic disease, as the proband (currently 12 mo old) carries the diagnosis of athetoid cerebral palsy, and continues to manifest impaired visual, motor and cognitive skills but without persistent hypoglycemia. Brain lesions may have been due to early neonatal hypoglycemia or may be unrelated to the underlying enzyme deficiency.
Direct enzyme determination of 2-MBCDase in human fibroblasts is hampered by low levels of activity, and significant overlap with short-chain acyl CoA dehydrogenase (11). More optimal enzyme studies can be performed in biopsied liver or muscle, but this was not ethically possible in our proband. In lieu of such analyses, production of 2-MBCDase carrying the L222F mutation in bacteria verified that this alteration resulted in complete inactivation of enzyme activity. The identification of an affected female fetus in the subsequent pregnancy, who manifests the same metabolite pattern as her brother, in addition to healthy parents suggests that inheritance is autosomal recessive. We are currently assessing levels of 2-MBC in maternal urine (still on L-carnitine intervention) to determine whether this metabolite derived from the female fetus. Definitive demonstration of the autosomal recessive nature of the disease will require assay of 2-MBCDase using authentic CoA substrate in cells derived from the affected siblings and their parents. The presentation of patients with isolated deficiencies of 2-MBCDase, isovaleryl-CoA dehydrogenase and isobutyryl-CoA dehydrogenase indicates that separate enzymes are involved in L-leucine, L-valine and L-isoleucine metabolism in humans.
Abbreviations
- 2-MBCDase:
-
2-methylbutyryl-CoA dehydrogenase
- 2-MBG:
-
2-methylbutyrylglycine
- 2-MBC:
-
2-methylbutyrylcarnitine
- AC:
-
acylcarnitine
- MS:
-
mass spectrometry
- MSMS:
-
tandem mass spectrometry
References
Sweetman L, Williams JC 1995 Branched chain organic acidurias. In: Scriver CR, Beaudet AL, Sly WS, Valle D (eds) The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York, pp 1387–1449
McGarry JD, Foster DW 1976 An improved and simplified radioisotopic assay for the determination of free and esterified carnitine. J Lipid Res 17: 277–281
Millington DS, Norwood DL, Kodo N, Roe CR, Inoue F 1989 Application of fast atom bombardment with tandem mass spectrometry and liquid chromatography/mass spectrometry to the analysis of acylcarnitines in human urine, blood and tissue. Anal Biochem 180: 331–339
Shigematsu Y, Hata I, Nakai A, Kikawa Y, Sudo M, Tanaka Y, Yamaguchi S, Jakobs C 1996 Prenatal diagnosis of organic acidemias based on amniotic fluid levels of acylcarnitines. Pediatr Res 39: 680–684
Van Hove JLK, Chace DH, Kahler SG, Millington DS 1993 Acylcarnitines in amniotic fluid: application to the prenatal diagnosis of propionic acidemia. J Inherit Metab Dis 16: 361–367
Lowes S, Rose ME, Mills GA, Pollitt RJ 1992 Identification of urinary acylcarnitines using gas chromatography-mass spectrometry: preliminary clinical applications. J Chromatogr 577: 205–214
Shimizu N, Yamaguchi S, Orii T, Previs SF, Rinaldo P 1991 Mass spectrometric analysis of metabolite excretion in five Japanese patients with the late-onset form of glutaric aciduria type II. Biol Mass Spectrom 20: 479–483
Gibson KM, Lee CF, Kamali V, Johnston K, Beaudet AL, Craigen WJ, Powell BR, Schwartz R, Tsai MY, Tuchman M 1990 3-Hydroxy-3-methylglutaryl-CoA lyase deficiency as detected by radiochemical assay in cell extracts by thin-layer chromatography, and identification of three new cases. Clin Chem 36: 297–303
Roe CR, Cederbaum SD, Roe DS, Mardach R, Galindo A, Sweetman L 1998 Isolated isobutyryl-CoA dehydrogenase deficiency: an unrecognized defect in human valine metabolism. Molec Genet Metab 65: 264–271
Mohsen AW, Anderson BD, Volchenboum SL, Battaile KP, Tiffany K, Roberts D, Kim JJ, Vockley J 1998 Characterization of molecular defects in isovaleryl-CoA dehydrogenase in patients with isovaleric acidemia. Biochemistry 37: 10325–10335
Willard J, Vicanek C, Battaile KP, van Veldhoven PP, Fauq AH, Rozen R, Vockley J 1996 Cloning of a cDNA for short/branched chain acyl-coenzyme A dehydrogenase from rat and characterization of its tissue expression and substrate specificity. Arch Biochem Biophys 331: 127–133
Acknowledgements
The authors thank Dr. Kevin Battaile, Dr. Jone Sampson, and Patricia Himes for their assistance in development of this manuscript and genetic counseling of the family.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Gibson, K., Burlingame, T., Hogema, B. et al. 2-Methylbutyryl-Coenzyme A Dehydrogenase Deficiency: A New Inborn Error of L-Isoleucine Metabolism. Pediatr Res 47, 830–833 (2000). https://doi.org/10.1203/00006450-200006000-00025
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1203/00006450-200006000-00025
This article is cited by
-
Post-mortem tissue analyses in a patient with succinic semialdehyde dehydrogenase deficiency (SSADHD). I. Metabolomic outcomes
Metabolic Brain Disease (2020)
-
Clinical and biochemical characterization of four patients with mutations in ECHS1
Orphanet Journal of Rare Diseases (2015)
-
Advances and challenges in the treatment of branched‐chain amino/keto acid metabolic defects
Journal of Inherited Metabolic Disease (2012)
-
Enzymology of the branched‐chain amino acid oxidation disorders: the valine pathway
Journal of Inherited Metabolic Disease (2012)
-
Evidence that 2-methylacetoacetate induces oxidative stress in rat brain
Metabolic Brain Disease (2010)