Expression signature of the Leigh syndrome French-Canadian type

Abstract

As a result of a founder effect, a Leigh syndrome variant called Leigh syndrome, French-Canadian type (LSFC, MIM / 220,111) is more frequent in Saguenay-Lac-Saint-Jean (SLSJ), a geographically isolated region on northeastern Quebec, Canada. LSFC is a rare autosomal recessive mitochondrial neurodegenerative disorder due to damage in mitochondrial energy production. LSFC is caused by pathogenic variants in the nuclear gene leucine-rich pentatricopeptide repeat-containing (LRPPRC). Despite progress understanding the molecular mode of action of LRPPRC gene, there is no treatment for this disease. The present study aims to identify the biological pathways altered in the LSFC disorder through microarray-based transcriptomic profile analysis of twelve LSFC cell lines compared to twelve healthy ones, followed by gene ontology (GO) and pathway analyses. A set of 84 significantly differentially expressed genes were obtained (p ≥ 0.05; Fold change (Flc) ≥ 1.5). 45 genes were more expressed (53.57%) in LSFC cell lines compared to controls and 39 (46.43%) had lower expression levels. Gene ontology analysis highlighted altered expression of genes involved in the mitochondrial respiratory chain and energy production, glucose and lipids metabolism, oncogenesis, inflammation and immune response, cell growth and apoptosis, transcription, and signal transduction. Considering the metabolic nature of LSFC disease, genes included in the mitochondrial respiratory chain and energy production cluster stood out as the most important ones to be involved in LSFC mitochondrial disorder. In addition, the protein-protein interaction network indicated a strong interaction between the genes included in this cluster. The mitochondrial gene NDUFA4L2 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2), with higher expression in LSFC cells, represents a target for functional studies to explain the role of this gene in LSFC disease. This work provides, for the first time, the LSFC gene expression profile in fibroblasts isolated from affected individuals. This represents a valuable resource to understand the pathogenic basis and consequences of LRPPRC dysfunction.

Keywords: ATP, adénosine-5'-triphosphate; COPD, chronic obstructive pulmonary disease; COX, cytochrome c-oxidase; Cytochrome c oxidase; DMEM, Dubelcco’s Modified Essential Medium; ETC, electron transport chain; Flc, fold change; GO, gene ontology; Gene expression; HES1, hairy and enhancer of split 1; HIF-1, hypoxia inducible factor-1; LRPPRC; LRPPRC, leucine-rich pentatricopeptide repeat-containing; LSFC, Leigh syndrome, French-Canadian type; Leigh syndrome; Leigh syndrome French-Canadian type (LSFC); Microarrays; Mitochondrial chain respiration; NAFLD, non-alcoholic fatty liver disease; ND6, NADH dehydrogenase, subunit 6; NDUFA4L2; NDUFA4L2, NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2; OXPHOS, oxidative phosphorylation; PFKFB4, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4; PPI, protein‐protein interaction; RMA, robust multi-array analysis; ROS, reactive oxygen species; RPL13A, ribosomal protein L13a; SLIRP, stem-loop interacting protein; SLSJ, Saguenay–Lac-Saint-Jean; SRA, steroid receptor RNA activator; qRT-PCR, Real-time PCR; rare diseases.

Fig. 1

Differentially expressed genes clusters according to their molecular function Comparison of gene expression profile of twelve paired LSFC and controls cell lines (fibroblasts) by microarrays showed a set of 84 significant differentially expressed genes (Flc ≥ 1.5 and p ≤ 0.05). Based on the molecular function of these genes, they were classified on seven clusters: mitochondrial respiratory chain and energy production (5), glucose and lipids metabolism (7), oncogenesis (9), immune response (10), cell growth and apoptosis (15), transcription (5), signal transduction (6), and 27 genes with other not yet known function.

Fig. 2

Expression of the four selected genes using real-time PCR (qRT-PCR). NADH dehydrogenase, subunit 6 (complex I) (ND6), NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4-like 2 (NDUFA4L2), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), and hairy and enhancer of split 1, (Drosophila) (HES1) mRNA was extracted from skin fibroblasts of LSFC (gray bars) and paired controls (white bars) individuals. Measure of the mRNA expression by real-time RT-PCR was done twice in triplicate with negative control and normalized to RPL13A expression using two-standard curves method. Data are expressed as mean + SEM values. NDUFA4L2, PFKFB4, and HES1 mRNA level are significantly (p < 0.05) higher in LSFC skin fibroblasts participants compared with controls.

Fig. 3

Protein protein interactions network. Network analysis of dysregulated genes was performed using STRING database, considering a combined interaction score > 0.4 cut off for significant interaction. A strong interaction between the differentially expressed genes of the mitochondrial and energy production cluster was observed.

Fig. 4

Depiction of the respiratory chain defects in LSFC patients. The five mitochondrial complexes are shown embedded in the inner mitochondrial membrane and called I, II, III, IV, and V. Loss of LRPPRC decreases the activity of the mitochondrial complex IV that results in accumulation of reactive oxygen species (ROS) in the mitochondria. As an adaptative mechanism, cells switch away from mitochondrial ATP production toward glycolysis, a necessary adaptation to the loss of mitochondrial respiratory capacity in LSFC cells leading to increasing level of blood lactic acid. This will cause hypoxia condition that increases the expression of the NDUFA4L2 gene. NDUFA4L2 decreases oxygen consumption by inhibiting the electron transport chain activity.