A novel autosomal dominant ERLIN2 variant activates endoplasmic reticulum stress in a Chinese HSP family

Abstract

Objective: Hereditary spastic paraplegia (HSP) has been reported rarely because of a monoallelic variant in ERLIN2. The present study aimed at describing a novel autosomal dominant ERLIN2 pedigree in a Chinese family and exploring the possible mechanism of HSP caused by ERLIN2 variants.

Methods: The proband and his family underwent a comprehensive medical history inquiry and neurological examinations. Whole-exome sequencing was performed on the proband, and Sanger sequencing was performed on some family members. HeLa cell lines and mouse primary cortical neurons were used for immunofluorescence (IF) and reverse transcription-PCR (RT-PCR).

Results: Seven patients were clinically diagnosed with pure spastic paraplegia in four consecutive generations with the autosomal dominant inheritance model. All patients presented juvenile-adolescent onset and gradually worsening pure HSP phenotype. Whole-exome sequencing of the proband and Sanger sequencing of all available family members identified a novel heterozygous c.212 T>C (p.V71A) variant in exon 8 of the ERLIN2 gene. The c.212 T>C demonstrated a high pathogenic effect score through functional prediction. RT-PCR and IF analysis of overexpressed V71A revealed an altered ER morphology and increased XBP-1S mRNA levels, suggesting the activation of ER stress. Overexpression of V71A in primary cultured cortical neurons promoted axon growth.

Interpretation: The novel c.212 T>C heterozygous variant in human ERLIN2 caused pure HSP. Moreover, c.212 T>C heterozygous variant in ERLIN2 increased ER stress and affected axonal development.

Figure 1

Pedigree of affected family, ERLILN2 variants, and conservation among species. (A) Family pedigree. Black filled symbol, affected; white symbol, unaffected; and black arrows, proband. Sanger sequencing was performed in some subjects (+/+: normal, M/+: heterozygous), demonstrating complete segregation of the ERLIN2 missense variant c.212 T>C, p.V71A with the disease. (B) The partial nucleotide sequences of exon 7 of ERLIN2 show the c.212 T>C variant in the affected or unaffected family members (II‐3, III‐5, IV‐3, and IV‐2). (C) Schematic representation of the basic structure and domain organization of the ERLIN2 protein. The figure was generated using the Illustrator for Biological Sciences (IBS). The observed variants were labeled. The site of the novel variant p.V71A identified in this study is indicated by red color. (D) Sequence alignment of ERLIN2 proteins from various species. The arrows indicate the amino acid changes in this study. Emboldened amino acids are conserved.

Figure 2

ERLIN2‐V71A overexpression alters ER morphology and increases XBP‐1 splicing. (A) The expression levels of ERLIN2‐WT‐HA, ERLIN2‐V71A‐HA, and IP3R were detected through WB. (B) HeLa cells were cultured with or without TM or transfected with ERLIN2‐WT‐HA or ERLIN2‐V71A‐HA and then immunostained for HA (green) and CANX (red) to detect ER. Merged images are to the right. Bars, 10 μm. (C–E) HeLa cells were cultured with or without TM or transfected with ERLIN2‐WT‐HA or ERLIN2‐V71A‐HA, and then, the relative mRNA level of XBP‐1S/Actin and XBP‐1S/XBP‐1U was detected using RT‐PCR. One‐way ANOVA test with Dunnett's test or unpaired t‐test; values are presented as mean n ± SEM. *p < 0.05, ****p < 0.0001.

Figure 3

Overexpression of ERLIN2‐V71A facilitates axon outgrowth. (A) Representative images of primary cortical neurons at DIV 5. Neurons were transfected with pCAGGS‐IRES‐GFP (CAGGS), ERLIN2‐WT, or ERLIN2‐V71A at DIV2. Bars, 10 μm. (B and C) Quantification of total neurites length or the axon length. One‐way ANOVA test with Dunnett's test; values for more than 60 neurons from three independent experiments are presented as mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001, unpaired t‐test.