Identification And Function Analysis Of Hereditary Spastic Paraplegia Causative Gene SLC33A1 Mutation Site C.339T>G (p.Ser113Arg)

Hereditary spastic paraplegia (HSP or SPG) is a widely studied heterogeneous neurodegenerative disorder, both genetically and clinically. As a disease which is mainly featured by progressive muscle weakness and increased hypermyotonia of the symmetry lower limbs, the characterical pathological alteration of HSP presents is retrograde axonal degeneration in the corticospinal tracts. Up to now,76 SPG causative genes have been located, and 54 genes are fully identified. Among these, the SPG42 causative gene SLC33A1 was located and identified by our lab in a pedigree from Qingdao, Shandong Province. We found that all the patients from this pedigree carried c.339T>G (p.Ser113Arg) missense mutation in the SLC33A1 gene, while normal members and general populations are not. Via the study of zebrafish model, we confirmed that down-regulated slc33a1 level causes abnormal phenotypes such as defected tail development and abnormal axon growth of motor neurons. Therefore, via whole-exon sequencing, we determined that SPG42 is caused by c.339T>G (p.Ser113Arg) missense mutation in the SLC33A1 gene. Furthermore, we studied the function of SLC33A1 and its possible mechanism by which the c.339T>G (p.Ser113Arg) missense mutation implicate on HSP.Using whole-exome sequencing, we surveyed all the potential pathogenic variants in an SPG42 family and found five SNPs and four indels that are shared by two patients and lie in the mapped region. Two variants, SLC33A1 p.Ser113Arg and VEPH1 p.G1n433His, cosegregated with the disease. However, software analysis suggested that general function of VEPH1 could be sustained after p.Gln433His mutation occurred. Therefore, we believe that SLC33A1 c.339T>G (p.Ser113Arg) mutation is the most plausible causative variant in this SPG42 family.In order to deeply explore the SLC33A1 c.339T>G (p.Ser113Arg) mutation, we carried out a series of experiments in which we injected different ratio wild type and/or c.339T>G (p.Ser113Arg) mutation human SLC33A1 mRNA with/without slc33a1 morpholino antisense oligonucleotides (MO) into zebrafish embryo and then statistically analyzed their phenotypes. We found that abnormal zebrafish phenotypes caused by slc33al down-regulation can be rescued by wild type human SLC33A1 mRNA, while these effects is neutralized when wild type SLC33A1 mRNA is co-injected with mutation type. These results indicated that c.339T>G (p.Ser113Arg) mutation acted in a dominant negative manner.In addition, we further investigated the function and pathogenic mechanism of SPG42 causative gene SLC33A1. Using patient and normal human skin fibroblasts, we found that the level of bone morphogenetic protein receptor IA (BMPR1A) showed a significant elevation and BMP signaling pathway is abnormal activated in patients. The half-life test indicated the up-regulation of BMPR1A is caused by its abnormal degradation in cytoplasm.In conclusion, we further verified that SPG42 is caused by SLC33A1 gene c.339T>G (p.Ser113Arg) missense mutation which worked in a dominant negative manner. SLC33A1 mutation cause abnormal intracellular BMPR1A degradation, then up-regulated level of BMPR1A, and finally results in the abnormal activation of BMP signaling pathway. These findings would contribute to further comprehension of SLC33A1’s function and the molecular pathogenesis mechanism of SPG42.