Biallelic DDHD2 mutations in patients with adult-onset complex hereditary spastic paraplegia

Abstract

Objective: Hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative disorders characterized by slowly progressive lower limb spasticity and weakness. HSP type 54 (SPG54) is autosomal recessively inherited and caused by mutations in the DDHD2 gene. This study investigated the clinical characteristics and molecular features of DDHD2 mutations in a cohort of Taiwanese patients with HSP.

Methods: Mutational analysis of DDHD2 was performed for 242 unrelated Taiwanese patients with HSP. The clinical, neuroimaging, and genetic features of the patients with biallelic DDHD2 mutations were characterized. A cell-based study was performed to assess the effects of the DDHD2 mutations on protein expression.

Results: SPG54 was diagnosed in three patients. Among them, two patients carried compound heterozygous DDHD2 mutations, p.[R112Q];[Y606*] and p.[R112Q];[p.D660H], and the other one was homozygous for the DDHD2 p.R112Q mutation. DDHD2 p.Y606* is a novel mutation, whereas DDHD2 p.D660H and p.R112Q have been reported in the literature. All three patients manifested adult onset complex HSP with additional cerebellar ataxia, polyneuropathy, or cognitive impairment. Brain proton magnetic resonance spectroscopy revealed an abnormal lipid peak in thalamus of all three patients. In vitro studies demonstrated that all the three DDHD2 mutations were associated with a considerably lower DDHD2 protein level.

Interpretation: SPG54 was detected in approximately 1.2% (3 of 242) of the Taiwanese HSP cohort. This study expands the known mutational spectrum of DDHD2, provides molecular evidence of the pathogenicity of the DDHD2 mutations, and underlines the importance of considering SPG54 as a potential diagnosis of adult-onset HSP.

Figure 1

Genetic analysis of the patients with spastic paraplegia type 54 (SPG54). (A–C) Pedigrees of the three SPG54 families and the Sanger sequencing traces of DDHD2 mutations including DDHD2 c.[335G>A];[1978G>C] (p.[R112Q];[D660H]) and DDHD2 c.[335G>A];[1818C>A] (p.[R112Q];[Y606*]) in compound heterozygous form and DDHD2 c.335G>A (p.R112Q) in homozygous form in families A, B, and C, respectively. The probands are indicated by arrows. The “M” represents a mutant DDHD2 allele, and the “W” refer to a wild‐type allele. The squares and circles denote for males and females, separately. The filled and open symbols represent affected and unaffected members, respectively. Dotted symbols indicate obligate carriers. The altered amino acid residues are labeled in red. (D) Scheme of the DDHD2 protein and its domains (WWE, lipase, sterile‐alpha‐motif (SAM), and DDHD), labeling with SPG54‐related mutations identified in the literatures (marked in black) and the present study (the novel mutation marked in red, and the recurred mutations marked in green).

Figure 2

Proton magnetic resonance spectroscopy obtained from the patient A‐II‐2 (A), patient B‐II‐3 (B), patient C‐II‐6 (C) and healthy control (D) at a magnetic field of 3‐Tesla (echo time 35 ms). The arrows indicate the pathologic lipid peak at 1.3 ppm detected at bilateral thalami. Cho, choline; Cr, creatine; NAA, N‐acetylaspartate.

Figure 3

In vitro functional study of the identified DDHD2 variants in HEK293T cells. (A) Representative reverse‐transcription polymerase chain reaction analysis of DDHD2 mRNA levels in HEK293T cells transfected with different DDHD2 constructs. GAPDH mRNA was used as a loading control. Densitometric quantification is shown below. The error bars indicate standard error of the mean (SEM) from three independent experiments. (B) Representative western blot analysis for steady‐state expression of DDHD2 proteins in HEK293T cells transfected with different DDHD2 constructs. The band of the truncated DDHD2‐Y606* protein was labeled by a red arrow. β‐actin was used as a loading control. (C) Relative DDHD2 mutant protein expression at steady‐state was normalized to β‐actin and calculated in reference to DDHD2‐WT/β‐actin. Data were obtained from four independent experiments. Statistical analysis was performed by one‐way ANOVA followed by post hoc analysis (n = 4). (D) HEK293 cells were transfected with either DDHD2‐WT, R112Q, or D660H constructs for 24 h and subsequently subjected to cycloheximide (CHX)‐chase assays. Data were obtained from three independent experiments. Representative western blot analysis is shown. (E) The quantified results of the CHX‐chase assays. Two‐way ANOVA and post hoc Tukey tests were performed at every time point (n = 3). All data are presented as mean ± SEM. *P < 0.05, **P < 0.01 and ***P < 0.001 for DDHD2 mutants versus Wild‐type groups.