Characterization of new ACADSB gene sequence mutations and clinical implications in patients with 2-methylbutyrylglycinuria identified by newborn screening

Abstract

Short/branched chain acyl-CoA dehydrogenase (SBCAD) deficiency, also known as 2-methylbutyryl-CoA dehydrogenase deficiency, is a recently described autosomal recessive disorder of isoleucine metabolism. Most patients reported thus far have originated from a founder mutation in the Hmong Chinese population. While the first reported patients had severe disease, most of the affected Hmong have remained asymptomatic. In this study, we describe 11 asymptomatic non-Hmong patients brought to medical attention by elevated C5-carnitine found by newborn screening and one discovered because of clinical symptoms. The diagnosis of SBCAD deficiency was determined by metabolite analysis of blood, urine, and fibroblast samples. PCR and bidirectional sequencing were performed on genomic DNA from five of the patients covering the entire SBCAD (ACADSB) gene sequence of 11 exons. Sequence analysis of genomic DNA from each patient identified variations in the SBCAD gene not previously reported. Escherichia coli expression studies revealed that the missense mutations identified lead to inactivation or instability of the mutant SBCAD enzymes. These findings confirm that SBCAD deficiency can be identified through newborn screening by acylcarnitine analysis. Our patients have been well without treatment and call for careful follow-up studies to learn the true clinical impact of this disorder.

Figure 1

Ribbon representation of point mutations identified in SBCAD gene. A. The tetrameric SBCAD crystal structure, PDB code 2JIF, is shown with each subunit colored a different color. FAD is shown as yellow and bound substrate as green. The amino acid substitutions identified in this and other studies are represented as yellow balls with precursor protein numbering given. Leu255Phe and Ser368Pro have previously been reported in Korman, et al, 2005). The former is likely to be important in stabilizing the interaction between two adjacent alpha helices. Ser368 likes in an alpha helix that is likely to have its trajectory altered by substitution of a proline. B. Residues at the 148 and 387 positions (precursor numbering) are illustrated. The Thr148 hydroxlate group lies in a hydrophilic pocket, but does not directly interact with other residues side chains. The carboxylate oxygen molecules of Glu387 lie between the Lys384 side chain amino group and the hydroxyl group of Thr411’ of the second subunit forming a hydrogen bonding conduit. In addition, the Glu387 backbone oxygen is directly involved in FAD binding through hydrogen bonding.

Figure 1

Ribbon representation of point mutations identified in SBCAD gene. A. The tetrameric SBCAD crystal structure, PDB code 2JIF, is shown with each subunit colored a different color. FAD is shown as yellow and bound substrate as green. The amino acid substitutions identified in this and other studies are represented as yellow balls with precursor protein numbering given. Leu255Phe and Ser368Pro have previously been reported in Korman, et al, 2005). The former is likely to be important in stabilizing the interaction between two adjacent alpha helices. Ser368 likes in an alpha helix that is likely to have its trajectory altered by substitution of a proline. B. Residues at the 148 and 387 positions (precursor numbering) are illustrated. The Thr148 hydroxlate group lies in a hydrophilic pocket, but does not directly interact with other residues side chains. The carboxylate oxygen molecules of Glu387 lie between the Lys384 side chain amino group and the hydroxyl group of Thr411’ of the second subunit forming a hydrogen bonding conduit. In addition, the Glu387 backbone oxygen is directly involved in FAD binding through hydrogen bonding.

Figure 2

Western blotting of an SDS-PAGE gel of expressed SBCAD patient point mutations. The arrow shows the migration position of purified recombinant SBCAD. Wild type SBCAD expressed in E. coli yields a high level of SBCAD antigen in crude cell extracts. The Glu387Lys and Thr148Ilu are minimally or not detectable.