A Defect in the Krebs Cycle in Retinitis Pigmentosa

Abstract

Purpose: : To identify novel genes causing recessive retinitis pigmentosa (RP) by searching for low mRNA expression levels in lymphoblasts.

Methods: : We created lymphoblast cell lines from 13 unrelated recessive RP families and 4 controls and isolated RNA that was hybridized to the Affymetrix gene-chip "U133Plus 2.0" containing probe sets for over 47,000 human mRNAs. After normalization, the hybridization intensities of individual transcripts were compared. Genes corresponding to mRNAs with significantly low expression levels were screened for mutations by direct DNA sequencing. Enzymes were assayed in extracts from lymphoblast cell lines.

Results: : The index patient from one family had a 64-91% reduction in hybridization measured by three probe sets that detected transcripts from the gene encoding the beta subunit of NAD-specific isocitrate dehydrogenase (IDH3B), an enzyme in the Krebs cycle. Sequencing of IDH3B in the index patient and her affected brother revealed that both were homozygous for a 1-basepair deletion (Ile197fs; c.589delA) leading to a frameshift and a premature stop codon that is likely to result in rapid mRNA degradation through nonsense-mediated decay. A subsequent search for IDH3B mutations in DNA from 546 additional, unrelated patients revealed a patient with simplex RP who was homozygous for the missense mutationLeu132Pro; c.395T>C. The index patients were in general good health except for RP. Enzyme assays showed a substantial reduction of NAD-specific IDH activity in lymphoblasts from both index patients, with an increase in the Km for NAD of almost 300 fold.

Conclusions: : Our results indicate that recessive mutations in IDH3B can be associated with recessive RP. That non-syndromic RP could be caused by mutations in IDH3B is unexpected since IDH3B plays a key role in the ubiquitous and essential Krebs cycle, also known as the citric acid cycle. The method we used for this work may be applicable for finding other recessive disease genes.

Keywords: retinal degenerations: hereditary • gene/expression • mutations