Transfection screening for primary defects in the pyruvate dehydrogenase E1alpha subunit gene

Abstract

In a significant number of patients with biochemical evidence of a defect in the E1 (pyruvate dehydrogenase) component of the pyruvate dehydrogenase complex, it has not proved possible to identify a mutation in the gene coding regions. To assess the need for more extensive genetic analysis in these patients and to establish a test system in which to study the biochemical consequences of mutations in the E1alpha subunit gene (which is responsible for the great majority of defined cases of pyruvate dehydrogenase deficiency), we have developed a method to screen for E1alpha gene defects based on complementation of the enzyme deficiency in transformed fibroblast cell lines following transfection and expression of the normal cDNA. Using this system, cell lines from patients with a variety of different defined mutations in the E1alpha gene show restoration of enzyme activity. A number of patients have been identified in whom deficient enzyme activity is not restored by expression of the normal cDNA indicating that an alternative explanation for the enzyme defect must be sought.

A putative exonic splicing enhancer in exon 7 of thePDHA1 gene affects splicing of adjacent exons

“…Immunocytochemical staining of the cells was performed with monoclonal antibodies to the E1α and E2 subunits of the PDH complex (Lib, et al, 2002). Clones of patient fibroblasts expressing either the normal or mutant X chromosome were isolated after transformation of the primary cultures as previously described (Brown, et al, 1997).…”

Arginine 302 mutations in the pyruvate dehydrogenase E1α subunit gene: Identification of further patients and in vitro demonstration of pathogenicity

“…Primary fibroblasts were routinely transformed using a plasmid (T22) containing SV40 DNA sequences (provided by Dr. P.H.Gallimore, University of Birmingham) as described previously [Brown et al, 1997]. In females heterozygous for PDH E1α mutations, normal and deficient clones are obtained in proportion to the X-inactivation pattern in the primary culture [Brown et al, 1997].…”

“…In females heterozygous for PDH E1α mutations, normal and deficient clones are obtained in proportion to the X-inactivation pattern in the primary culture [Brown et al, 1997]. This provides a useful approach to the analysis of female patients with suspected PDH deficiency.…”

“…PDH E1α protein was demonstrated using an affinity purified E1α-specific antibody as previously described [Brown et al, 1989]. PDH E1α mRNA was analysed in preparations of total cytoplasmic RNA from freshly harvested fibroblasts following Northern blotting and hybridisation with a digoxygenin-labelled riboprobe transcribed from a vector containing the normal PDH E1α cDNA as previously described [Brown et al, 1997]. A probe to 2-oxoglutarate dehydrogenase (OGDH) was included as an internal control.…”

“…As very few PDH E1α mutations have been demonstrated to be pathogenic, we have developed an in vitro system to study the consequences of specific mutations and have applied this to the R302C mutation. The mutant cDNA has been inserted into an expression vector and the properties of the expressed protein studied following transfection of the construct into a PDH E1α null cell line [Brown et al, 1997]. This patient was born at term with birth weight 2.9 kg.…”