Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Patients and Their Intragenic SMN1 Mutations
2.2. Outline of the Experiments
2.3. Plasmid Construction
2.4. Transfection into HeLa Cells
2.5. RNA Analysis by RT-PCR
2.6. SMN1 Protein Quantification by Western Blotting
2.7. Pull-Down Assay with Immunoprecipitation
2.8. Statistics
3. Results
3.1. Plasmid Construction and Transfection
3.1.1. Plasmid Construct Carrying Exogenous SMN cDNA
3.1.2. Transfection of the Plasmid Containing SMN1 cDNA
3.2. SMN Transcript Analysis
3.2.1. Expression of Exogenous SMN Transcript in HeLa Cells
3.2.2. Expression of Endogenous SMN Transcript in HeLa Cells
3.2.3. Quantification of Exogenous and Endogenous SMN Transcripts by RT-PCR
3.3. SMN1 Protein Analysis
3.3.1. Western Blotting Analysis with Anti-SMN Antibody
3.3.2. Pull-Down Assay with Immunoprecipitation
4. Discussion
4.1. SMA Patients with an Intragenic Mutation in the Retaining SMN1 Allele
4.2. Stability of the Mutated SMN1 Proteins
4.3. Oligomerization of SMN Proteins
4.4. Clinical Phenotype of the Patients with an Intragenic Mutation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Patient | Mutation Name | Clinical Subtype | SMN1 Copy Number | SMN2 Copy Number | Mutation Type | Nucleotide Change | Amino Acid Change | Exon Domain | Ref. |
---|---|---|---|---|---|---|---|---|---|
01 02 | W92S | 1 | 1 | 3 | Missense | c.275 G > C | p.Trp92Ser | Ex3 Tudor | [20,22] |
03 | E134K | 2 | 1 | 2 | Missense | c.400 G > A | p.Glu134Lys | Ex3 Tudor | [27,30] |
04 | Y276H | 2 | 1 | 2 | Missense | c.826 T > C | p.Tyr276His | Ex6 C-term. * | [28] |
05 | Y277C | 2/3 | 1 | 1 | Missense | c.830 A > G | p.Tyr277Cys | Ex6 C-term. * | [19] |
06 | T274Y fsX32 | 1 | 1 | 2 | Frame shift | c.819_820 insT | p.Thr274TyrfsX32 | Ex6 C-term. * | [19,22,29] |
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Niba, E.T.E.; Nishio, H.; Wijaya, Y.O.S.; Ar Rochmah, M.; Takarada, T.; Takeuchi, A.; Kimizu, T.; Okamoto, K.; Saito, T.; Awano, H.; et al. Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy. Genes 2022, 13, 205. https://doi.org/10.3390/genes13020205
Niba ETE, Nishio H, Wijaya YOS, Ar Rochmah M, Takarada T, Takeuchi A, Kimizu T, Okamoto K, Saito T, Awano H, et al. Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy. Genes. 2022; 13(2):205. https://doi.org/10.3390/genes13020205
Chicago/Turabian StyleNiba, Emma Tabe Eko, Hisahide Nishio, Yogik Onky Silvana Wijaya, Mawaddah Ar Rochmah, Toru Takarada, Atsuko Takeuchi, Tomokazu Kimizu, Kentaro Okamoto, Toshio Saito, Hiroyuki Awano, and et al. 2022. "Stability and Oligomerization of Mutated SMN Protein Determine Clinical Severity of Spinal Muscular Atrophy" Genes 13, no. 2: 205. https://doi.org/10.3390/genes13020205