New phenotype of RTN2-related spectrum: Complicated form of spastic paraplegia-12

Abstract

Objective: Spastic paraplegia-12 (SPG12) is a subtype of hereditary spastic paraplegia caused by Reticulon-2 (RTN2) mutations. We described the clinical and genetic features of three SPG12 patients, functionally explored the potential pathogenic mechanism of RTN2 mutations, and reviewed RTN2-related cases worldwide.

Methods: The three patients were 31, 36, and 50 years old, respectively, with chronic progressive lower limb spasticity and walking difficulty. Physical examination showed elevated muscle tone, hyperreflexia and Babinski signs in the lower limbs. Patients 1 and 3 additionally had visual, urinary, and/or coordination dysfunctions. Patient 2 also had epileptic seizures. RTN2 mutations were identified by whole-exome sequencing, followed by Sanger sequencing, segregation analysis, and phenotypic reevaluation. Functional examination of identified mutations was further explored.

Results: Three variants in RTN2 were identified in Patient 1 (c.103C>T, p.R35X), Patient 2 (c.230G>A, p.G77D), and Patient 3 (c.337C>A, p.P113T) with SPG, respectively. Western blotting revealed the p.R35X with smaller molecular weight than WT and other two missense mutants. Immunostaining showed the wild type colocalized with endoplasmic reticulum (ER) in vitro. p.R35X mutant diffusely distributes in the cytoplasm, losing colocalization with ER. p.G77D and p.P113T co-localized with ER, which was abnormally aggregated in clumps.

Interpretation: In this study, we identified three cases with complicated SPG12 due to three novel RTN2 mutations, respectively, presenting various phenotypes: classic SPG symptoms with (1) visual abnormalities and sphincter disturbances or (2) seizures. The phenotypic heterogeneity might arise from the abnormal subcellular localization of mutant Reticulon-2 and improper ER morphogenesis, revealing the RTN2-related spectrum is still expanding.

Figure 1

(A) Pedigree of Patient 1’s family. Sequence chromatograms of RTN2 gene displays one heterozygous nonsense mutation of c.103C>T p.R35X (arrow) in the proband (II:1), which is negative in unaffected father (I:1) and brother (II:2). (B) Pedigree of Patient 2’s family. Sequence chromatograms of RTN2 gene displays one heterozygous missense mutation of c.230G>A p.G77D (arrow) in the proband (II:1), which is negative in the parents (I:1 and I:2). (C) Pedigree of Patient 3’s family. Sequence chromatograms of RTN2 gene displays one heterozygous missense mutation of c.337C>A p.P113T (arrow) in the proband (II:1), which is negative in unaffected bothers (II:2 and II:3). (A‐C) The mutations located in the highly conserved region of proteins are shown in the bottom half. (D) RTN2‐WT/Mut‐EGFP transfected HEK 293 T cells showing the presence of WT in cytoplasmic distribution colocalizing with CALR‐mcherry (ER), but R35X and R60fs are expressed in diffuse distribution in both nuclear and cytoplasm without specific colocalization with ER. G77D and P113T still colocalize with CALR‐mcherry (ER), with ER tending to form punctate aggregates of ER. The scale bar represents 10 μm. (E) Western blotting showed the signals at ~86 kDa from expressed HEK 293 T cells expressing RTN2‐WT‐EGFP, RTN2‐G77D‐EGFP, or RTN2‐P113T‐EGFP. The signals with relative smaller molecular weight were detected in R35X group (~32 kDa) and the pathogenic control R60fs group (~40 kDa). (F) The schematic diagram of RTN2 structure with all mutations documented. Full length of RTN2 (NM_005619) consists of 545 amino acids. RHD, reticulon homology domain (aa 345–545). Mutations identified with pure SPG12, complicated SPG12 and autism are in black, red, and purple, respectively. Mutations firstly identified in this paper are in bold font, c.103C>T (p.R35X), c.230G>A (p.G77D) and c.337C>A (p.P113T). [Colour figure can be viewed at wileyonlinelibrary.com]

Figure 2

The clinical features of SPG12 patients with RTN2 mutations. For each clinical manifestation, the proportion of patients is indicated. [Colour figure can be viewed at wileyonlinelibrary.com]