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. 2023 Oct 27;11(5):101152.
doi: 10.1016/j.gendis.2023.101152. eCollection 2024 Sep.

Dominant negative effect as a novel mechanism of SPAST gene mutation in a large family with hereditary spastic paraplegia

Ke Deng  1   2 , Haibo Ruan  3 , Feifei Yu  4 , Zhenle Pei  1   2 , Congjian Xu  1   2   5 , Shuo Zhang  1   2
Affiliations

Dominant negative effect as a novel mechanism of SPAST gene mutation in a large family with hereditary spastic paraplegia

Ke Deng et al. Genes Dis. .
No abstract available

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Figures

Fig. 1
Figure 1
Dominant negative effect as a novel mechanism of SPAST gene mutation in a large family with hereditary spastic paraparesis (HSP). (A) Pedigrees of the HSP-affected families are shown. The affected male and female individuals are indicated with filled squares and circles, respectively. Normal individuals are shown as empty symbols. The proband is indicated with an arrow. Patients included I-2, II-3, II-7, III-2, III-3, III-5, and III-7, all of whom had similar symptoms of lower extremity spasticity. (B) Photograph of the lower extremities of one affected individual (III:3) in the family. The patient had leg and foot deformities manifested by pes cavus and atrophy of lower limb muscles. (C) The novel mutation was confirmed by Sanger sequencing. Affected family members (II-7, III-3, III-5, and III-7) and one unaffected individual (IV-2) underwent singer sequencing. DNA sequencing results of an unaffected family member (upper panel) and an affected family member (lower panel) are shown. (D) RT-PCR using RNA isolated from the blood of the proband and one healthy control. The band of the wide type is named “a”, and the bands of the proband are named “a” and “b”. A longer amplicon of approximately 528 bp detected in the control subject corresponded to the expected band size. III-2 had two bands, indicating heterozygosity. The b-band is shorter than the a-band. (E) RT-PCR product sequencing results. Sequencing analysis of normal and short-sized bands confirmed the deletion of 75 nucleotides of exon 8 in the shorter fragment. (F) Schematic diagram of the primer design and the c.1173 + 1_1173+2dup variant related abnormal splicing. The asterisk indicates the location of the c.1173 + 1_1173+2dup variant. (G) The minigene build strategy for pcDAN3.1-SPAST-wt/mut was to insert partial exon 7 (94 bp)–partial intron 7 (495 bp)–exon 8 (75 bp)–intron 8 (1385 bp)–exon 9 (72 bp) into pcDNA3.1. PcDAN3.1-SPAST-wt and pcDAN3.1-SPAST-mut plasmids were transiently transfected into HeLa and 293T cells. RT-PCR of total RNA obtained from cells transfected with pcDAN3.1-SPAST-wt produced a 350-bp band presenting correct mRNA splicing, and a shorter band was observed in cells transfected with pcDAN3.1-SPAST-mut. (H) Mini-gene product sequencing results: (i) The wild-type mini-gene (pcDNA3.1-SPAST-wt) formed normal mRNA composed of exons 7, 8, and 9; (ii) The mutant mini-gene (pcDNA3.1-SPAST-mut) caused a splicing abnormality, resulting in the deletion of exon 8. (I) Sequencing results showed that the mutation c.1173 + 1_1173+2dup was successfully introduced. (J) The constructed recombinant eukaryotic expression vectors, phage-SPAST-wt/mut and pEGFP-C1-SPAST-wt/mut, were transiently transfected into 293T cells. The expression level of WT or mut mRNA in transfected 293T cells was detected by qPCR. There was no significant difference in mRNA expression. (K) The protein expression of wt-spastin and mut-spastin in 293T cells. Western blot analysis showed that the protein levels of the mut were similar to those of the wt. (L) Effects of mutation on microtubule stability. Tubulin acetylation of transfected cells was analyzed by Western blotting with the indicated antibodies. The expression of acetylated α-tubulin increased in the mCherry-SPAST-wt and pEGFP-SPAST-mut co-transfected groups compared with that in the mCherry-SPAST-wt and pEGFP co-transfected groups. (M) Effects of mutation on spastin microtubule-severing activity. Representative immunofluorescence images for mt-spastin (green), wt-spastin (red), α-tubulin (orange), and nuclei (blue) were shown. In the mCherry-SPAST-wt and pEGFP co-transfected groups, microtubule protein was severed, whereas, in the mCherry-SPAST-wt and pEGFP-SPAST-mut co-transfected groups, the microtubule-severing activity was significantly reduced. Spastin-labeled filaments in pEGFP-SPAST-mut-transfected cells colocalized with tubulin.
None
Figure S1 Results of the pcDNA3.1 vector mini-gene splicing assay. (A) Sequencing results of the target fragment of pcDNA3.1-SPAST-wt (upper panel) and mut (lower panel) mini-gene. (B) Schematic diagram of mini-gene construction and the c.1173 + 1_1173+2dup variant related abnormal splicing. The asterisk indicates the location of the c.1173 + 1_1173+2dup variant.
None
Figure S2 Results of the pcMINI vector mini-gene splicing assay. (A) Gel electrophoresis of RT-PCR products: the band of the wild-type was bigger than the mutant. (B) Schematic diagram of mini-gene construction and the c.1173 + 1_1173+2dup variant related abnormal splicing. The asterisk indicates the location of the c.1173 + 1_1173+2dup variant. (C) Mini-gene product sequencing results: (i) The wild-type mini-gene (pcMINI-SPAST-wt) formed normal mRNA composed of exons A, 8, and B; (ii) The mutant mini-gene (pcMINI-SPAST-mut) caused a splicing abnormality, resulting in the deletion of exon 8.
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