Whole-exome sequencing identifies novel variants in PNPT1 causing oxidative phosphorylation defects and severe multisystem disease

Abstract

Recent advances in next-generation sequencing strategies have led to the discovery of many novel disease genes. We describe here a non-consanguineous family with two affected boys presenting with early onset of severe axonal neuropathy, optic atrophy, intellectual disability, auditory neuropathy and chronic respiratory and gut disturbances. Whole-exome sequencing (WES) was performed on all family members and we identified compound heterozygous variants (c.[760C>A];[1528G>C];p.[(Gln254Lys);(Ala510Pro)] in the polyribonucleotide nucleotidyltransferase 1 (PNPT1) gene in both affected individuals. PNPT1 encodes the polynucleotide phosphorylase (PNPase) protein, which is involved in the transport of small RNAs into the mitochondria. These RNAs are involved in the mitochondrial translation machinery, responsible for the synthesis of mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) complexes. Both PNPT1 variants are within highly conserved regions and predicted to be damaging. These variants resulted in quaternary defects in the PNPase protein and a clear reduction in protein and mRNA expression of PNPT1 in patient fibroblasts compared with control cells. Protein analysis of the OXPHOS complexes showed a significant reduction in complex I (CI), complex III (CIII) and complex IV (CIV). Enzyme activity of CI and CIV was clearly reduced in patient fibroblasts compared with controls along with a 33% reduction in total mitochondrial protein synthesis. In vitro rescue experiments, using exogenous expression of wild-type PNPT1 in patient fibroblasts, ameliorated the deficiencies in the OXPHOS complex protein expression, supporting the likely pathogenicity of these variants and the importance of WES in efficiently identifying rare genetic disease genes.

Figure 1

Assay of PNPT1 mRNA expression levels, protein levels, mitochondrial protein synthesis and respiratory chain enzyme activity in the fibroblasts and blood of control and patient. (a) Relative mRNA expression of PNPT1 in mRNA extracted from fibroblasts and blood of proband (S1), affected brother (S2), unaffected father (S3) and unaffected mother (S4). The expression level was normalized relative to GAPDH. (b) The PNPase protein level in subject S1 and control fibroblasts using whole-cell lysates and mitochondrial lysates. GAPDH was used as a loading control. (c) PNPase protein levels in control and all family members using protein extracts from white blood cells. GAPDH was used as a loading control. (d) Non-denaturing gel electrophoresis analysis was performed to examine the assembly of PNPase in S1 and control fibroblasts. GAPDH was used as a loading control. (e) Analysis of mitochondrial translation in fibroblasts from S1 and two controls using a [³⁵S]methionine pulse-chase labeling experiment. Phosphorimage in the upper panel and corresponding immunoblot in the lower panel. (f) Protein levels of OXPHOS complex subunits (CI NDUFB8, CII 30 kDa, CIII Core 2, CIV MT-CO2, CVα) in subject S1 and control fibroblasts using OXPHOS cocktail antibodies. Porin was used as a loading control. (g) CI and CIV enzyme activity dipstick assay in S1 and control fibroblasts.

Figure 2

Rescue experiments for CI and CIV enzyme activity. Fibroblasts from S1 and a control were transfected with PNPT1^wt (wild-type), PNPT1^p.Gln254Lys (p.(Gln254Lys)), PNPT1^p.Ala510Pro (p.(Ala510Pro)) and empty vector constructs. Untransfected cells from the control and S1 were included. Data are means±SEM from three independent transfection experiments, each analysed in triplicate (n=9). *P<0.05, **P<0.01 and ***P<0.001 significantly different from untransfected control cells. ^##P<0.01 and ^###P<0.001 are significantly different from untransfected S1 cells.

Figure 3

Rescue experiments for CI, CII, CIII, CIV and CIV protein expression. Fibroblasts from S1 and a normal control were transfected with PNPT1^wt (wild-type), PNPT1^p.Gln254Lys (p.(Gln254Lys)), PNPT1^p.Ala510Pro (p.(Ala510Pro)) and empty vector constructs. Porin was used as a loading control. This is a representative figure, and experiments were performed independently six times (n=6).