IARS2 mutations lead to Leigh syndrome with a combined oxidative phosphorylation deficiency

Abstract

Background: Leigh syndrome (LS) is a common mitochondrial disease caused by mutations in both mitochondrial and nuclear genes. Isoleucyl-tRNA synthetase 2 (IARS2) encodes mitochondrial isoleucine-tRNA synthetase, and variants in IARS2 have been reported to cause LS. However, the pathogenic mechanism of IARS2 variants is still unclear.

Methods: Two unrelated patients, a 4-year-old boy and a 5-year-old boy diagnosed with LS, were recruited, and detailed clinical data were collected. The DNA of the patients and their parents was isolated from the peripheral blood for the identification of pathogenic variants using next-generation sequencing and Sanger sequencing. The ClustalW program, allele frequency analysis databases (gnomAD and ExAc), and pathogenicity prediction databases (Clinvar, Mutation Taster and PolyPhen2) were used to predict the conservation and pathogenicity of the variants. The gene expression level, oxygen consumption rate (OCR), respiratory chain complex activity, cellular adenosine triphosphate (ATP) production, mitochondrial membrane potential (MMP) and mitochondrial reactive oxygen species (ROS) levels were measured in patient-derived lymphocytes and IARS2-knockdown HEK293T cells to evaluate the pathogenicity of the variants.

Results: We reported 2 unrelated Chinese patients manifested with LS who carried biallelic IARS2 variants (c.1_390del and c.2450G > A from a 4-year-old boy, and c.2090G > A and c.2122G > A from a 5-year-old boy), of which c.1_390del and c.2090G > A were novel. Functional studies revealed that the patient-derived lymphocytes carrying c.1_390del and c.2450G > A variants exhibited impaired mitochondrial function due to severe mitochondrial complexes I and III deficiencies, which was also found in IARS2-knockdown HEK293T cells. The compensatory experiments in vitro cell models confirmed the pathogenicity of IARS2 variants since re-expression of wild-type IARS2 rather than mutant IARS2 could rescue complexes I and III deficiency, oxygen consumption, and cellular ATP content in IARS2 knockdown cells.

Conclusion: Our results not only expand the gene mutation spectrum of LS, but also reveal for the first time the pathogenic mechanism of IARS2 variants due to a combined deficiency of mitochondrial complexes I and III, which is helpful for the clinical diagnosis of IARS2 mutation-related diseases.

Keywords: IARS2; Leigh syndrome; Mitochondrial disease; OXPHOS.

Fig. 1

Family pedigree analysis, brain MRI images and variant analysis of IARS2. A. Pedigree analysis of patient 1 and patient 2 from two unrelated Chinese families. Rectangles indicated males, circles female, solid circle rectangled the affected individuals and probands were pointed out by arrows. B. Brain MRI images of patient 1 and 2. The patient 1 on the left showed lesions in the thalamus, periaqueductal, cerebellum and pons when he was 4 years old. The arrow pointed to the high signal were diseased areas. The patient 2 on the right showed long T1 and T2 signals in the lateral caudate nucleus and putamen, reduced cerebral white matter, bilateral basal ganglia symmetry damage, and cerebral dysplasia. The circled pointed to the symmetrical patchy were diseased areas. C. Sequencing chromatograms of IARS2 in patients and their parents. The arrows indicated the mutation sites. D. Quantitative analysis results of IARS2 in patient 2 and his parents

Fig. 2

Conservative and pathogenicity analysis of IARS2 variants. A. The conservation of amino acid of the mutation sites. B. Bioinformatic analysis of allele frequency and pathogenicity prediction about three variants (c.2450G > A, c.2122G > A and c.2090G > A)

Fig. 3

IARS2 expression and mitochondrial functional validation in patient-derived lymphocytes with c.1_390del and c.2450G > A variants. A. Immunoblot and quantification analysis of IARS2 protein in healthy age-matched control and patient-derived lymphocytes. β‐actin was used as control. B. BN-PAGE/immunoblot and quantification analysis for OXPHOS complexes in healthy age-matched control and patient-derived lymphocytes. TOM70 was used as control. C. Oxygen consumption rate in healthy age-matched control and patient-derived lymphocytes by using Oxygraph‐2k. Basal, basal respiration; Oligo, ATP-linked mitochondrial respiration of the cells added with 2 µg/mL oligomycin. D-E. Relative cellular ATP (D) and MMP (E) content in healthy age-matched control and patient-derived lymphocytes. N.S. P > 0.05, * P < 0.05, *** P < 0.001

Fig. 4

Mitochondrial function was impaired in IARS2 knockdown cells. A. Immunoblot and quantification analysis of IARS2 protein in control and IARS2 knockdown cells. β-actin was used as control. B. BN-PAGE/immunoblot analysis and quantification for OXPHOS complexes in control and IARS2 knockdown cells. TOM70 was used as control. C. Oxygen respiration rate in control and IARS2 knockdown cells by using Oxygraph-2k. Basal, basal respiration; Oligo, ATP-linked mitochondrial respiration of the cells added with 2 µg/mL oligomycin. D-E. Relative cellular ATP content (D) and mitochondrial ROS level (E) of control and IARS2 knockdown cells. N.S. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001

Fig. 5

IARS2 variants at c.2090G > A, c.2122G > A and c.2450G > A led to mitochondrial dysfunction. A-B. Immunoblot (A) and quantification (B) analysis of IARS2 protein in IARS2 knockdown HEK293T cells with re-expression of wild-type IARS2 or mutant IARS2. β‐actin was used as control. C-D. BN-PAGE/immunoblot (C) and quantification (D) analysis for OXPHOS complexes in IARS2 knockdown HEK293T cells with re-expression of re-expressed wild-type IARS2 or mutant IARS2. TOM70 was used as control. E. Oxygen respiration rate of IARS2 knockdown HEK293T cells with re-expression of wild-type IARS2 or mutant IARS2. Basal, basal respiration; Oligo, ATP-linked mitochondrial respiration of the cells added with 2 µg/mL oligomycin. F. Relative cellular ATP content of with re-expression of wild-type IARS2 or mutant IARS2 carrying c.2090G > A, c.2122G > A or c.2450G > A in IARS2 knockdown HEK293T cells. N.S. P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001