More than 100 cases of VLCAD deficiency have been documented in the literature with three different disease phenotypes. A severe infant-onset form is characterized by acute metabolic decompensation with hypoketotic hypoglycemia, dicarboxylic aciduria, liver dysfunction, and cardiomyopathy. A second form of the disease presents later in infancy or childhood but has a milder phenotype without cardiac involvement. The third form is of adolescent or adult onset and is dominated by muscle dysfunction that is often exercise-induced.

Not surprisingly, children with the severe phenotype tended to have null mutations (71 percent of identified alleles in these patients), while patients with the two milder forms of the disease were more likely to have missense mutations (82 percent and 93 percent of identified alleles for the milder childhood form and the adult form, respectively). Nevertheless, a few missense mutations were clearly associated with the severe phenotype. Although these data suggested that missense mutations in VLCAD might obviate clinical symptoms due to some degree of residual activity, no correlation was seen between the mutations identified and residual VLCAD activity in fibroblasts. Moreover, the function effects of few of the known VLCAD missense mutations have been directly characterized.

We have previously used prokaryotic expression systems to express, purify and characterize the biochemical properties of several ACD enzymes. Several of these have been crystallized and studied by X-ray diffraction, yielding informative three-dimensional models. The study of VLCAD, however, has been limited due to difficulties with prokaryotic expression. These difficulties may be related in part to physical properties that distinguish VLCAD from other ACD family members. Most of the ACDs share a common homotetrameric “dimer of dimers” structure and function in the mitochondrial matrix. In contrast, VLCAD is a homodimer with an extended 180 amino acid C-terminal domain of unknown function. Additionally, VLCAD is associated with the inner mitochondrial membrane, an interaction that has long been postulated without proof to be mediated by the C-terminus.

We have used our prokaryotic expression system to study six previously missense mutations described in VLCAD deficient patients (T220M, V243A, R429W, A450P, L462P, and R573W). T220M and V243A are the most frequently reported missense mutations in VLCAD deficient patients. R429W and R573W are among the few missense mutations believed to result in the severe clinical phenotype. A450P and L462P are located in the C-terminal domain unique to VLCAD and ACAD-9. Characterization of purified wild type, A450P, and L462P VLCAD proteins confirmed the long-held assumption that the C-terminus plays a key role in mitochondrial membrane association. The prokaryotic system developed will greatly facilitate investigation of VLCAD structure-function. 

Source(s) of Support

National Institutes of Health

Principal Investigator

Gerard Vockley, MD, PhD