The online version of this article (https://doi.org/10.1186/s13024-018-0269-1) contains supplementary material, which is available to authorized users.
En-Lin Dong, Chong Wang and Shuang Wu contributed equally to this work.
Hereditary spastic paraplegias (HSP) is a heterogeneous group of rare neurodegenerative disorders affecting the corticospinal tracts. To date, more than 78 HSP loci have been mapped to cause HSP. However, both the clinical and mutational spectrum of Chinese patients with HSP remained unclear. In this study, we aim to perform a comprehensive analysis of clinical phenotypes and genetic distributions in a large cohort of Chinese HSP patients, and to elucidate the primary pathogenesis in this population.
We firstly performed next-generation sequencing targeting 149 genes correlated with HSP in 99 index cases of our cohort. Multiplex ligation-dependent probe amplification testing was further carried out among those patients without known disease-causing gene mutations. We simultaneously performed a retrospective study on the reported patients exhibiting HSP in other Chinese cohorts. All clinical and molecular characterization from above two groups of Chinese HSP patients were analyzed and summarized. Eventually, we further validated the cellular changes in fibroblasts of two major spastic paraplegia (SPG) patients (SPG4 and SPG11) in vitro.
Most patients of ADHSP (94%) are pure forms, whereas most patients of ARHSP (78%) tend to be complicated forms. In ADHSP, we found that SPG4 (79%) was the most prevalent, followed by SPG3A (11%), SPG6 (4%) and SPG33 (2%). Subtle mutations were the common genetic cause for SPG4 patients and most of them located in AAA cassette domain of spastin protein. In ARHSP, the most common subtype was SPG11 (53%), followed by SPG5 (32%), SPG35 (6%) and SPG46 (3%). Moreover, haplotype analysis showed a unique haplotype was shared in 14 families carrying c.334C > T (p.R112*) mutation in CYP7B1 gene, suggesting the founder effect. Functionally, we observed significantly different patterns of mitochondrial dynamics and network, decreased mitochondrial membrane potential (Δψm), increased reactive oxygen species and reduced ATP content in SPG4 fibroblasts. Moreover, we also found the enlargement of LAMP1-positive organelles and abnormal accumulation of autolysosomes in SPG11 fibroblasts.
Our study present a comprehensive clinical spectrum and genetic landscape for HSP in China. We have also provided additional evidences for mitochondrial and autolysosomal-mediated pathways in the pathogenesis of HSP.
Additional file 1: Summary information for mutational spectrum, targeted sequencing genes, amplification primers, antibodies, clinical features and haplotype analysis. Table S1. A reanalysis of the mutational spectrum in 41 reported studies exhibiting HSP in China. Table S2. HSP related genes included in the targeted sequencing panel. Table S3. Primers for amplification in the experimental procedures. Table S4. List of antibodies, related to immunocytochemistry (ICH) and western blotting (WB) in the experimental procedures. Table S5. Summary of clinical features of SPG4 patients in China (n=273). Table S6. Summary of clinical features of SPG11 patients in China (n=52). Table S7. Summary of clinical features of SPG5 patients in China (n=28). Table S8. Haplotypes of tagSNPs linkage to CYP7B1 gene in the 16 unrelated SPG5 families. (XLSX 69 kb)
Additional file 2: Pedigrees, sequencing chromatograms of disease-causing gene related to HSP families in our cohort. Figure S1. Pedigree, sequencing chromatograms of SPAST gene detected in 16 SPG4 families in our cohort. Figure S2. Western blot analysis of novel mutations of SPAST gene in HEK 293T cells. Figure S3. Pedigree, sequencing chromatograms of 6 ADHSP families in our cohort. Figure S4. Pedigree, sequencing chromatograms of CYP7B1 gene detected in 16 unrelated SPG5 families in our cohort. Figure S5. Pedigree, sequencing chromatograms of 5 ARHSP families in our cohort. (DOCX 9157 kb)
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