Clinical features and genetic spectrum in Chinese patients with recessive hereditary spastic paraplegia

Abstract

Background: Although many causative genes of hereditary spastic paraplegia (HSP) have been uncovered in recent years, there are still approximately 50% of HSP patients without genetically diagnosis, especially in autosomal recessive (AR) HSP patients. Rare studies have been performed to determine the genetic spectrum and clinical profiles of recessive HSP patients in the Chinese population.

Methods: In this study, we investigated 24 Chinese index AR/sporadic patients by targeted next-generation sequencing (NGS), Sanger sequencing and multiplex ligation-dependent probe amplification (MLPA). Further functional studies were performed to identify pathogenicity of those uncertain significance variants.

Results: We identified 11 mutations in HSP related genes including 7 novel mutations, including two (p.V1979_L1980delinsX, p.F2343 fs) in SPG11, two (p.T55 M, p.S308 T) in AP5Z1, one (p.S242 N) in ALDH18A1, one (p.D597fs) in GBA2, and one (p.Q486X) in ATP13A2 in 8 index patients and their family members. Mutations in ALDH18A1, AP5Z1, CAPN1 and ATP13A2 genes were firstly reported in the Chinese population. Furthermore, the clinical phenotypes of the patients carrying mutations were described in detail. The mutation (p.S242 N) in ALDH18A1 decreased enzyme activity of P5CS and mutations (p.T55 M, p.S308 T) in AP5Z1 induced lysosomal dysfunction.

Conclusion: Our results expanded the genetic spectrum and clinical profiles of AR-HSP patients and further demonstrated the efficiency and reliability of targeted NGS diagnosing suspected HSP patients.

Keywords: Autosomal recessive; Chinese; Genetic spectrum; Hereditary spastic paraplegia; Phenotype; Targeted next-generation sequencing.

Fig. 1

Chromatograms of 11 mutations identified in the present study. The upper chromatogram in each frame represents the reference sequence, and the lower one depicts the mutant sequence. The p.R112X in CYP7B1, p.S242 N in ALDH18A1, p.D597fs in GBA2, c.759 + 1G > A in CAPN1, and p.Q486XA in TP13A2 are homozygous

Fig. 2

Pedigrees of 8 HSP families (a-h) in our cohort. Squares indicate males; circles indicate females; the black symbols indicate affected individuals; arrows indicate the probands. Symbol with “+/+” indicate patient. Symbol with “+/−” or “+/−” indicate mutation carrier

Fig. 3

Brain MRI of case 1 and case 2. An ‘ears of the lynx’ appearance and thinning of corpus callosum were seen in case 1 (a) and case 2 (b)

Fig. 4

The novel homozygous variant (p.S242 N) in ALDH18A1 decreased enzyme activity of P5CS. a Hela cells were respectively transfected with WT and mutant plasmids (S242 N, V243 L). Mitochondria was visualized with MitoTracker red probe and no dramatic change in mitochondrial localization was found. (b, c) HEK293 cells were respectively transfected with WT and mutant plasmids (S242 N, V243 L). No significant difference of protein level was detected between WT P5CS and mutant P5CS. d The serum P5CS activity of case 4 decreased as compared with that of four gender matched healthy controls. Scale bar = 20 μm. Error bars represent SEM, *p < 0.05

Fig. 5

Functional analysis of both variants (p.T55 M and p.S308 T) in AP5Z1. (a and b) HEK293 cells were respectively transfected with WT and mutant plasmids (T55 M, S308 T). Western blot analysis revealed that both AP5Z1 variants decreased the level of AP-5 ζ protein. c ICH analysis revealed that Hela cells transfected with mutant plasmids (T55 M, S308 T) showed larger and brighter LAMP1-positive puncta as compared with cells transfected with the WT plasmid. Scale bar = 20 μm. (d and e) LAMP1 fluorescence intensity and area per cell were quantified in more than 100 cells quantified per visual field. Experiments were replicated three times. f Hela cells were respectively transfected with WT and mutant plasmids (T55 M, S308 T), then assessed for morphological changes by TEM. Ultrastructural analysis revealed that both AP5Z1 variants led to the accumulation of enlarged morphologically defined endocytic structures filled with aberrant storage material, including many intraluminal vesicles. Scale bar = 500 nm. Error bars represent SEM, *p < 0.05