B cell dysfunction in thalamus and brainstem involvement and high lactate caused by novel mutation of EARS2 gene

Abstract

Purpose: The EARS2 gene, a member of the mt-aaRS family, encodes mitochondrial glutamyl-tRNA synthetase (GluRS), which is involved in the synthesis of mitochondrial proteins. Pathogenic defects in EARS2 may cause mitochondrial OXPHOS deficiency, which is associated with a rare autosomal-recessive mitochondrial disease, leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL).

Methods: In this study, clinical features were obtained, and whole-exome sequencing was conducted on a patient with LTBL. B- and T-cell immunophenotyping and protein expression were analyzed using flow cytometry, and B-cell metabolism was investigated using confocal microscopy.

Results: The patient with LTBL exhibited typical neurological manifestations, recurrent respiratory tract infections, and humoral immune disorders. Molecular analysis revealed a compound heterozygous novel mutation in c.1304T > A (p.L435Q) and a previously reported c.319 C > T (p.R107C) mutation of EARS2. The mutations led to protein structural modifications of EARS2. The patient also exhibited disrupted peripheral B-cell differentiation and B-cell receptor signal transduction. The EARS2 mutation led to decreased expression of CD38 and dysfunction of mitochondrial metabolism, with elevated reactive oxygen species levels in B cells.

Conclusion: We identified a novel mutation of the EARS2 gene in a patient with LTBL, expanding the mutation database. The mutation of EARS2 modified protein structure and impaired B-cell function, decreased CD38 expression, and led to dysfunction of mitochondrial metabolism, all of which may account for the recurrent respiratory tract infections and humoral immune disorders observed in LTBL.

Keywords: B cell; BCR signal; EARS2; Gene mutation; LTBL.

Fig. 1

EARS2 mutation leads to neurological alterations. Magnetic resonance imaging (MRI) images of our patient acquired at 7 months of age (a, c) and 10 months of age (b, d), showing bilateral and symmetrical abnormal long signals on T2-weighted images of the dorsal thalamus, head of caudate nucleus, brain stem, cerebellar hemispheres, and frontal, parietal, and occipital white matter areas. Hypogenesis of the corpus callosum is shown (c, d). An elevated lactate peak is observed in the short TE MR spectrum (e). Diffuse background slow waves (1.5–3 Hz) are shown on electroencephalography (f)

Fig. 2

Pedigree of the families and mutation analysis of c.319 C > T (p.R107C)/c.1304T > A (p.L435Q) mutations in the EARS2.(a) Family pedigree. (b) Father of the proband with the heterozygous p.R107C mutation and mother of the proband with the heterozygous p.L435Q mutation. Probands with p.R107C and p.L435Q mutations. (c) Conservation analysis revealed that arginine at position 107 and leucine at position 435 are highly conserved in different animal species and human EARS2 proteins. Arrow indicates the mutation location. (d) Superimposed structure of wild-type EARS2 protein (green) and R107C/L435Q variant (white color). (e-h) Changes in local environment and polar residue distance of EARS2 protein caused by the R107C and L435Q mutations. The residual structures of wild-type R107 (e) and L435 (g) protein are shown. The R107C (f) and L435Q (h) variants are shown. Green dotted lines, hydrogen bonds

Fig. 3

EARS2 mutation leads to B cell alterations. (a, b) Analysis of CD19 + B cell subsets from human peripheral blood mononuclear cells (PBMCs) from the patient with EARS2 mutation and healthy control. Memory B cells (CD27 + IgD-), naïve B cells (CD27-IgD+), transitional B cells (CD24 + CD38+), and PBCs (CD24-CD38+) were gated from CD19 + B cells. (c, d) Analysis of the MFI of Annexin V and Ki67 of B-cell subsets. (e, f) Analysis of the MFI of BAFFR and CD79a of B-cell subsets. (g, h) The levels of pSYK, pY, pCD19, pBtk, CD86, pPI3K, pmTOR, F-actin, pWASP, and CD38 of CD19 + B-cells

Fig. 4

EARS2 mutation alters B-cell metabolism. (a) The EARS2 level of PBMCs was analyzed by western blot. (b–e) The levels of ROS and mitochondrial mass of B cells were analyzed by confocal. (f) MFI of mitochondria of B cell subsets were analyzed by FCM

Fig. 5

EARS2 mutation alters T cells. (a, b) Analysis of CD4 + and CD8 + T cell subsets from PBMCs in the patient with EARS2 mutation and healthy control. Central Memory T cells (TCM) (CCR7 + CD45RA-), Naive T cells (TN) (CCR7 + CD45RA+), Effective Memory T cells (TEM) (CCR7-CD45RA-), and effector memory T cells re-expressing CD45RA (TEMRA) (CCR7-CD45RA+) were gated from CD4 + and CD8 + T cells. (c) The MFI of CD38 of CD4 + T-cell subsets were analyzed. (d) The MFI of CD38 of CD8 + T-cell subsets were analyzed