HETEROGENEITY OF MUTATIONS IN MAPLE-SYRUP-URINE-DISEASE (MSUD) - SCREENING AND IDENTIFICATION OF AFFECTED E1-ALPHA AND E1-BETA SUBUNITS OF THE BRANCHED-CHAIN ALPHA-KETO-ACID DEHYDROGENASE MULTIENZYME COMPLEX

Maple syrup urine disease (MSUD) is an autosomal recessive disease cau sed by a deficiency in subunits of the branched-chain alpha-keto-acid dehydrogenase complex (BCKDH). To characterize the mutations present i n five patients with MSUD (four classic and one intermediate), three-s tep analyses were established: (1), identification of the involved sub unit by complementation analysis using three different cell lines deri ved from homozygotes having E1 alpha, E1 beta or the E2 mutant gene; ( 2), screening for a mutation site in cDNA of the corresponding subunit by RT-PCR-SSCP and (3), mutant analysis by sequencing the amplified c DNA fragment. Four single-base missense mutations, R115W, Q156K, A209T and I282T, were detected in the E1 alpha subunit. A single-base misse nse mutation H156R and three frame-shift mutations to generate stop co dons downstream, including an 11-bp deletion of the tandem repeat in e xon 1, a single-base (T) deletion and a single-base (G) insertion, wer e identified in the E1 beta subunit gene. All except one (11-bp deleti on in E1 beta (Nobukuni, Y., Mitsubuchi, H., Akaboshi, I., Indo, Y., E ndo, F., Yoshioka, A. and Matsuda, I. (1991) J. Clin. Invest. 87, 1862 -1866)) were novel mutations. The sites of amino-acid substitution wer e all conserved in other species. Thus, mutations causing MSUD are het erogenous.