Whole exome sequencing reveals mutations in NARS2 and PARS2, encoding the mitochondrial asparaginyl-tRNA synthetase and prolyl-tRNA synthetase, in patients with Alpers syndrome

Abstract

Alpers syndrome is a progressive neurodegenerative disorder that presents in infancy or early childhood and is characterized by diffuse degeneration of cerebral gray matter. While mutations in POLG1, the gene encoding the gamma subunit of the mitochondrial DNA polymerase, have been associated with Alpers syndrome with liver failure (Alpers-Huttenlocher syndrome), the genetic cause of Alpers syndrome in most patients remains unidentified. With whole exome sequencing we have identified mutations in NARS2 and PARS2, the genes encoding the mitochondrial asparaginyl-and prolyl-tRNA synthetases, in two patients with Alpers syndrome. One of the patients was homozygous for a missense mutation (c.641C>T, p.P214L) in NARS2. The affected residue is predicted to be located in the stem of a loop that participates in dimer interaction. The other patient was compound heterozygous for a one base insertion (c.1130dupC, p.K378 fs*1) that creates a premature stop codon and a missense mutation (c.836C>T, p.S279L) located in a conserved motif of unknown function in PARS2. This report links for the first time mutations in these genes to human disease in general and to Alpers syndrome in particular.

Keywords: Alpers syndrome; NARS2; PARS2; whole exome sequencing.

Figure 1

(A, B) Degeneration of the cerebral cortex with vacuolization in the middle layers, marked loss of nerve cells and prominent gliosis in patient I. The images illustrate the entire cortex from the white matter (left) to the pial surface (right). (A) Luxol fast blue/Cresyl violet, 50×. (B) Immunostaining of glial acidic fibrillary protein (GFAP, 50×). (C) Representative kidney section with occasional focal segmental glomerulosclerosis (FSGS, arrow and upper inset). There was a prominent nodular hyalinosis with a perihilar distribution in several glomeruli (arrowhead and lower inset). The parenchyma was fairly normal except for occasional small cysts and autolysis (periodic acid and Schiff (PAS) 15×; insets 100×). Courtesy Johan Mölne.

Figure 2

(A, B) Degeneration with loss of nerve cells, gliosis, and capillary proliferation in the brain cortex of patient II. The images illustrate the cortex adjacent to a sulcus of the brain cortex (A) overview 15×. (B) Detail demonstrating extreme loss of nerve cells and glial cell proliferation (Luxol fast blue/Cresyl violet, 150×). (C) Necrosis of the putamen in patient II with loss of nerve cells and infiltration of macrophages, many of them exhibiting features of foam cells (Luxol fast blue/Cresyl violet, 150×). (D) Myocardium of patient II demonstrating marked interstitial fibrosis (hematoxylin/van Gieson, 150×).

Figure 3

Sequencing chromatograms confirming the whole exome sequencing findings. (A) The arrow shows the homozygous c.641 C>T transition in NARS2 in patient I. (B) Patient II presented a heterozygous c.836 C>T transition inherited from the mother and a heterozygous c.1130dupC mutation from the father in PARS2.

Figure 4

Asparaginyl-tRNA synthetase (AsnRS) and prolyl-tRNA synthetase (ProRS) abundance in control and patients cultured fibroblasts assessed by Western blotting. AsnRS (A) and ProRS (B) protein abundance in controls versus patient. Data are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) abundance and presented as mean ± SE. Controls n = 3–4, with 1–5 independent repeats each. Patient n = 1, with four independent repeats each.

Figure 5

(A) Primary structure and principal domains of human NARS2. ACDB: anticodon-binding domain. (B) The mutated proline at position 214 is located in close vicinity to three residues that participate in monomer–monomer interaction (highlighted in yellow). (C) Detail of the crystal structure of asparaginyl-tRNA synthetase of P. horokoshii showing the loop from where the side chains of the three amino acids protrude. The mutated proline in the human protein is located at the step of the loop, in the position occupied by Y182 in P. horokoshii. The image was obtained with the Molsoft MolBrowser 3.7-2a browser.

Figure 6

(A) Primary structure and principal domains of human PARS2. ACDB: anticodon-binding domain. (B) 34 residue domain specific for prolyl-tRNA synthetases. Highlighted in yellow is the conserved motif where the S279L mutation is located. This motif is flanked by CXXC motifs, a characteristic of functional domains inserted into larger proteins.