J. Inher. Metab. Dis. 14 (1991) 787-792 ~DSSIEM and KluwerAcademicPublishers. Printed in the Netherlands

JSSIEM Meeting

Maple Syrup Urine Disease: Clinical and Biochemical Significance of Gene Analysis Y. NOBUKUNI, H. MITSUBUCHI, I. AKABOSHI, Y. INDO, F. ENDO and I. MATSUDA* Department of Pediatrics, Kumamoto University Medical School, Kumamoto 860, Japan Branched-chain c~-ketoacid dehydrogenase (BCKDH) (EC 1.2.4.4) is a mitochondrial multienzyme complex catalysing the oxidative decarboxylation of branched-chain ~ketoacids derived from transamination of branched-chain amino acids such as valine, leucine, and isoleucine (reaction (1)): R--COCOOH + CoA-SH + NAD + TTP,Mg~R-Co~S-CoA + CO 2 + NADH + H +

(1)

The BCKDH complex consists of three catalytic components; branched chain c~ketoacid decarboxylase (El), dihydrotipoyl transacylase (E2), and dihydrolipoamide dehydrogenase (E3). E1 is further composed of two subunits, EV~ and E~/3. E~ and E 2 components are specific to BCKDH. The E3 component is common among the three ketoacid dehydrogenase complexes, BCKDH, pyruvate dehydrogenase and eketoglutarate dehydrogenase complexes. The BCKDH complex also contains two specific regulatory enzymes, a kinase and a phosphatase, compounds that are responsible for regulating the catalytic activity through phosphorylation and dephosphorylation. EIa is the catalytic subunit phosphorylated at two serine residues and is responsible for regulating the catalytic activity, by covalent modification. E 2 catalyses transfer of the acyl group from the lipoyl moiety to coenzyme A and forms the structural core of the enzyme complex and to this, El, E3, kinase and phosphatase are bound through non-covalent interactions. The function of E1/3 is unknown (Danner and Elsas t989; Yeaman, 1989). Impaired BCKDH activity leads to maple syrup urine disease (MSUD), an autosomal recessive inborn error of metabolism. MSUD is a heterogeneous disorder and the several different phenotypes heretofore identified include classical, intermediate, intermittent, and thiamine-responsive types as based on the clinical features (Danner and Elsas, 1989). To elucidate the molecular mechanisms of MSUD, we and other investigators isolated and characterized cDNAs encoding human BCKDH EV~ (Zhang et al., 1988; Fisher et al., 1989), E1/3 (Nobukuni et al., 1990), and E 2 (Hummel et at., 1988; Danner et al., 1989; Nobukuni et al., 1989). *Correspondence 787

788

Nobukuni et al.

tn two patients with classical MSUD we examined the enzyme activity, subunit structure, mRNA sequence and the genomic DNA of the affected enzymes; different mutations of E~c~ (Matsuda et aI., 1990) and E2 (Mitsubuchi et al., 1991) were identified in each case.

MATERIALS AND METHODS

Cell lines' and cell cultures: Lymphoblastoid celt lines were established by EpsteinBarr virus-mediated transformation of peripheral blood B lymphocytes from the proband (Matsuda et al., 1977). Cell lines, GM1099 and GM1655, were purchased from the human cell repository (Camden, N J, USA). Enzyme assay: The BCKDH activity was determined for disrupted lymphoblastoid cells by quantifying the COz released from c~-keto-[1-i4C]isovaleric acid, in the presence of co-factors, as described by Indo et al. (1987, 1988). Immunoblot analysis: Mitochondrial proteins were resolved by electrophoresis in a 10% polyacrylamide gel in the presence of sodium dodecyI sulphate and transferred to nitrocellulose membrane filters, using electroblotting. The resolved proteins were detected using affinity-purified rabbit antibovine BCKDH (El + E2) immunoglobulin (Indo et al., t987, 1988). Analysis o f c D N A : Total RNA was isolated from cells obtained from the patient, then cultured (Chomzynski et al., 1987). First-strand cDNAs were generated from total RNA, using Moloney leukaemia virus reverse transcriptase with specific antisense oligonucleotide primers (Newman et al., 1988). Two sets of sense/antisense oligonucleotides of each EI~, Elfl and E2 subunit were designed to cover the entire normal human cDNA sequences (Matsuda et al., 1990; Mitsubuchi et al., 1991). The cDNAs were then subjected to enzymatic amplification (Saiki et al., 1988). The specific amplified cDNAs were subcloned into the multicloning site of a plasmid vector pUC18, and five independent clones of each amplified cDNA segment were sequenced using a dideoxy chain termination method (Sanger et al., 1977). Analysis ofgenomic DNA: Genomic DNAs were purified from cultured cells from the patient (Kunkel et al., 1977). Genomic DNAs that encompass the region of the mutation detected in the mRNAs were amplified and then were subcloned and sequenced.

RESULTS AND DISCUSSION

Case 1. Missense mutation in the EI~ subunit (normal Ela subunit is essential for stability o f E l f l subunit): We examined the molecular defect in cases of Mennonite MSUD. The incidence of MSUD in the Mennonite population is 1 in 176 of live J. tnher. Metab. Dis. 14 (1991)

789

Gene Analysis in M S U D A

1

2

B

]GA

AT8

....H

E2

-

AATAA ^,

1

1

!

!

t350

T Ela-

A

EI,8-

4O t

1 I

I 394

Yyr ASh

Figure I (A) Immunoblot analysis of BCKDH proteins in disrupted lymphoblastoid cells from disease-free (lane 1) and MSUD Mennonite (lane 2). (B) Schematic representation of nucleotide and deduced amino acid sequence of the mutant Elc~ of BCKDH in MSUD Mennonite

births. In Mennonite msud, elfi was not detected and the amount of Elc~ was reduced (Figure 1A). We noted a T-to-A mutation in which tyrosine was replaced with asparagine at amino acid residue 394 of the Ele in the Mennonite MSUD patient (Figure 1B). This T-to-A mutation was identified in the last exon of the BCKDH Elc~ gene. The amino acid sequence of the Ea/~ subunit deduced from the cDNA of the patient was normal (Matsuda et at., 1990). It seems likely that the EI~ subunit is normally expressed, but is rapidly degraded because of failure to assemble in the stable E1 (~2fi2) as a result of the mutation of Elcc A region of the ElC~subunit seems to be necessary for stability. We also have data on a Japanese patient with classical MSUD in which the BCKDH E~fl subunit is absent and the E~c~subunit only weakly stained in Western blots, similar to findings in the Mennonite MSUD patient. A gene analysis revealed that in this patient there is an eleven-base-pair deletion in the first exon of the E~fi subunit gene but that the E~e subunit gene is normal (Nobukuni et al., 1991). In the light of all of these observations, the intact subunits of Ele and E1/~ seem obligatory for the stability of each E~fl and EI~ subunit, respectively, at the protein level. Case 2. A partial deletion in the inner Ez core domain (despite the E2 defect, EI~ and EI~ are as stable as E1 (~2/~2) : The MSUD Kumamoto was the progeny of secondcousin parents. The BCKDH activity of the patient was ~4.5% of the control level. Immunoblot analysis revealed that the E 2 subunit of BCKDH (52000) was absent and another protein band with Mr 49 000 was present (Figure 2A). A 78-bp deletion was identified in the region encoding a part of the inner E 2 c o r e domain in the E 2 cDNA. The molecular size of the translation products, as deduced from the abnormal mRNA sequence, was compatible with size of the abnormal protein band (Mr 49 000) detected in the patient's cells by immunoblot analysis. Analysis of genomic DNA of

J. Inher. Metab. Dis. 14 (1991)

790

Nobukuni et al.

A

B

1 l: 2

2

5~

NORMAL

PAT||NT

--

ElaEIB-

C S£gnal LipOy i Pepti~e Bearinq Domain

! I.HS}

E B~ndinq Do~n

InrteE

...........

K

mm ]/

V !

I

.

CoA-SH

Core

Binding

Dou~

SAte n ,

26 ~ a l n o D~letion

I Acids

H:3-q ....

K

Figure 2 (A) Immunoblot analysis of BCKDH proteins in disrupted lymphoblastoid cells from disease-free (lane 1) and MSUD Kumamoto (lane 2). (B) Sequence analysis of amplified genomic DNAs obtained from cultured cells from the patient and a control. Note the single base deletion of G at the 5' splice donor site. The arrow indicates the exon-intron splice junction. (C) Schematic comparison of domain structures of the normal and of the mutant E2 of BCKDH in MSUD Kumamoto

B C K D H E 2 revealed that this 78-bp deletion in the mRNA was caused by exon skipping due to a single base deletion in the Y-splice donor site (Figure 2B). As a result of this mutation, part of the inner E2 core domain was omitted (Figure 2C) (Mitsubuchi et at., 1991). It seems that despite the E 2 defect, E l e and Elfl are as stable as the E~ (~2/~2). CONCLUSIONS The aetiology of M S U D is heterogeneous, as mutations in different regions of any of the B C K D H proteins could lead to decreased function of the entire complex. To clarify the related mechanisms in MSUD, measurements of the enzyme activity (Dancis et aI., 1972; Jinno et al., 1984a), complementation analysis (Lyons et al., 1973;

J. Inher. Metab. Dis. 14 (1991)

Gene Analysis in M S U D

791

Jinno et al., 1984b) and i m m u n o b l o t analysis (Danner et al., 1985; Indo et al., 1987, t988; Fisher et al., 1989) have been done. In some cases it was difficult to identify which c o m p o n e n t of the B C K D H complex was primarily affected. We studied two cases of classical type M S U D by gene analysis and identified different mutations of Elc~ and E2, as described above. It became clear the molecular mechanisms of M S U D included not only the function of each subunit but also stability of the subunit and p r o t e i n - p r o t e i n interactions between each subunit. These studies shed light on structural and functional relationships of c o m p o n e n t s of the B C K D H complex.

ACKNOWLEDGEMENTS

We are grateful to M. Ohara for critical comments and M. Hayashi and M. Tsutsui for secretarial services. This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (01480553) and a Grant for Pediatric Research from the Ministry of Health and Welfare of Japan (63A-01).

REFERENCES

Chomczynski, P. and Sacchi, N. Single-step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162 (1987) 156-159 Dancis, J., Hutzler, J., Snyderman, S. E. and Cox, R. P. Enzyme activity in classical and variant forms of maple syrup urine disease. J. Pediatr. 81 (1972) 312 320 Danner, D. J. and Elsas, II. L. J. Disorders of branched chain amino acid and keto acid metabolism. In Scriver, C. R., Beaudet, A. L., Sly, W. S. and Valle, D. (eds.) The Metabolic Basis of Inherited Disease, McGraw-Hill, New York, 1989, pp. 671-692 Danner, D. J., Armstrong, N., Heffelfinger, S. C., Sewell, E. T., Priest, J. H. and Elsas, L. J. Absence of branched chain aminotransferase as a cause of maple syrup urine disease. J. Ctin. Invest. 75 (1985) 858-860 Danner, D. J., Litwer, S., Herring, W. J. and Pruckler, J. Construction and nucleotide sequence of a cDNA encoding the full-length preprotein for human branched chain acyltransferase. J. Biol. Chem. 264 (1989) 7742 7746 Fischer, C. W., Chuang, J. L., Griffin, T. A., Lau, K. S., Cox, R. P. and Chaung, D. T. Molecular phenotypes in cultured maple syrup urine disease cells. J. Biol. Chem. 264 (1989) 3448 3453 Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J. and Yeaman, S. J. Nucleotide sequence of a cDNA for branched chain acyttransferase with analysis of the deduced protein structure. J. Biol. Chem. 263 (1988) 6165 6168 Indo, Y., Kitano, A., Endo, F., Akaboshi, I. and Matsuda, I. Altered kinetic properties of the branched-chain ~-keto acid dehydrogenase complex due to mutation of the /Lsubunit of the branched-chain e-keto acid decarboxylase (El) component in lymphoblastoid cells derived from patients with maple syrup urine disease. J. Clin. Invest. 80 (1987) 63-70 Indo, Y., Akaboshi, I., Nobukuni, Y., Endo, F. and Matsuda, I. Maple syrup urine disease: a possible biochemical basis for the clinical heterogeneity. Hum. Genet. 80 (1988) 6-10 Jinno, Y., Akaboshi, I., Katsuki, T. and Matsuda, I. Study on established lymphoid cells in maple syrup urine disease. Correlation with clinical heterogeneity. Hum. Genet. 65 (1984a) 358-361 Jinno, Y., Akaboshi, I. and Matsuda, I. Complementation analysis in lymphoid cells from five patients with different forms of maple syrup urine disease. Hum. Genet. 68 (1984b) 54-56

J. Inher. Metab. Dis. 14 (1991)

792

Nobukuni et al.

Kunkel, L. M., Smith, K. D., Boyer, S. H., Borgaonkar, D. S., Wachtel, S. S., Miller, O. J., Breg, W. R., Jones, H, W. Jr. and Rary, J. M. Analysis of human Y-chromosome-specific reiterated DNA in chromosome variants. Proc. Natl Acad. Sci. USA. 74 (1977) 1245 1249 Lyons, L. B., Cox, R. P. and Dancis, J. Complementation analysis of maple syrup urine disease in heterokaryons derived from cultured human fibroblasts. Nature (London) 243 (1973) 533-535 Matsuda, I., Yamamoto, J., Nagata, N., Ninomiya, N., Akaboshi, I., Ohtsuka, H. and Katsuki, I. Lysosomal enzyme activities in cultured lymphoid cell lines. CIin. Chim. Acta 80 (1977) 483-486 Matsuda, I., Nobukuni, Y., Mitsubuchi, H., Indo, Y., Endo, F., Asaka, J. and Harada, A. A T-to-A substitution in the E lc~ subunit gene of the branched-chain e-ketoacid dehydrogenase complex in two cell lines derived from Mennonite maple syrup urine disease patients. Biochem, Biophys. Res. Commun. 172 (1990) 646-651 Mitsubuchi, H., Nobukuni, Y., Akaboshi, I., Indo, Y., Endo, F. and Matsuda, I. Maple syrup urine disease caused by a partial deletion in the inner E 2 core domain of the branched chain c~-keto acid dehydrogenase complex due to aberrant splicing: a single base deletion at a Y-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region. J. Clin. Invest. 87 (1991) 1207 1211) Newman, P. J., Gorski, J., White, II. G. C., Gidwitz, S., Cretney, C. J. and Aster, R. H. Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction. J. Clin. Invest. 82 (1988) 739-743 Nobukuni, Y., Mitsubuchi, H., Endo, F. and Matsuda, I. Complete primary structure of the transacylase (E2b) subunit of the human branched chain c~-keto acid dehydrogenase complex. Biochem. Biophys. Res, Commun. 161 (1989) 10351041 Nobukuni, Y., Mitsubuchi, H., Endo, F., Akaboshi, I., Asaka, J. and Matsuda, I. Maple syrup urine disease. Complete primary structure of the Elfi subunit of human branched chain eketoacid dehydrogenase complex deduced from the nucleotide sequence and a gene analysis of patients with this disease. J. Clin. Invest. 86 (1990) 242-247 Nobukuni, Y., Mitsubuchi, H., Akaboshi, I., Indo, Y, Endo, F., Yoshioka, A. and Matsuda, I. Maple syrup urine disease. Complete defect of the Elfi subunit of the branched chain c~ketoacid dehydrogenase complex due to a deletion of an ll-bp repeat sequence which encodes a mitochondrial targetting leader peptide in a family with the disease. J. CIin. Invest. 87 (1991) 1862-1866 Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 (1988) 487-491 Sanger, F., Nicken, S. and Coulson, A. R. DNA sequencing with chain-terminating inhibitors. Proc. Natl Acad. Sci. USA. 74 (1977) 5463-5467 Yeaman, S. J. The 2-oxo acid dehydrogenase complexes; recent advances. Biochem. J. 257 (1989) 625--632 Zhang, B., Crabb, D. W. and Harris, R. A. Nucleotide and deduced amino acid sequence of the Ele subunit of human liver branched-chain ~-ketoacid dehydrogenase. Gene 69 (1988) 159-164

J. Inher. Metab. Dis. 14 (1991)