Category: Ataxia
Objective: 1. To study the miRNA binding site variations by direct sequencing of 3’ UTR of ATXN-2gene.
2. To check the mRNA expression by using qRT-PCR in SCA2 cases in comparison to healthy controls.
Background: Expansion of CAG repeats in exon-1 of the ataxin-2 gene causes polyglutamine stretch expansion in the ubiquitous ataxin-2 protein, resulting in spinocerebellar ataxia type-2 (SCA-2). 3′-untranslated regions are noncoding sections of mRNAs (UTRs). A stop codon (UAA, UAG, or UGA) separates the 3′-UTR from the 5′ end, while a poly (A) tail separates the 3′ end from the 5′ end. Mutations in the AU-rich regions induce atypical mRNA stability and diseases. Nothing is known about SCA2 in this regard, hence the goal of this work is to figure out how 3′UTR alterations play a role in the neurodegenerative condition SCA-2.
Method: For 3′-UTR sequencing of ATXN2 gene of, SCA2 patients (N=24) & controls (N=26) were involved in Sanger sequencing on ABI-3500 sequencer. DNA was isolated from cases and controls blood samples by using salting out method. The 3′-UTR Sequencing data analysis was done by using SeqMan software and variations were checked by online tool Mutation Taster. For to check mRNA expression, total RNA was isolated from peripheral blood mononuclear cells (PBMCs) of blood using Trizol method and qualified using Bioanalyzer (Agilent Technologies, USA). High quality RNA was used in further experiments. Checked the expression of SCA2 mRNA (N=20) & controls (N=20) using qRT-PCR SYBR green method on Roche Light Cycler 480 version. Data was analyzed at 1.5 fold change expression level through delta delta ct method.
Results: Three interesting variations in the sequence were noticed (chr12:111890146T/A, chr12:111890076T/A and chr12:111890076T/C). The position 111890076 showed changes in alleles T/A and T/C”. Interestingly, the shortening of polyA nucleotide sequence was also significantly noticed. This shortening of two nucleotides (21 nucleotides in controls as compared to 19/20 poly A sequences in SCA2 Cases) could affect the stability of ATXN2 mRNA which were found significantly down regulated in this study. qRT-PCR results showed significant downregulated mRNA levels of SCA2 patients as compared to healthy controls (P<0.05).
Conclusion: Our findings from 3’UTR sequencing in SCA2 patients and controls imply that potentially pathogenic mutations and shortening of poly’A’ repeats may change mRNA stability.
References: 1.Almaguer-Mederos, L. E., Falcon, N. S., Almira, Y. R., Zaldivar, Y. G., Almarales, D. C.,
Gongora, E. M., Herrera, M. P., Batallan, K. E., Arminan, R. R., Manresa, M. V., Cruz,
G. S., Laffita-Mesa, J., Cyuz, T. M., Chang, V., Auburger, G., Gispert, S., Perez, L.
V. Estimation of the age at onset in spinocerebellar ataxia type 2 Cuban patients by
survival analysis. Clin. Genet. 78: 169-174, 2010.
2. Auburger, G., Orozco Diaz, G., Capote, R. F., Sanchez, S. G., Perez, M. P., Estrada del
Cueto, M., Meneses, M. G., Farrall, M., Williamson, R., Chamberlain, S., Baute, L.
H. Autosomal dominant ataxia: genetic evidence for locus heterogeneity from a Cuban
founder-effect population. Am. J. Hum. Genet. 46: 1163-1177, 1990.
3. Babovic-Vuksanovic, D., Snow, K., Patterson, M. C., Michels, V. V. Spinocerebellar
ataxia type 2 (SCA 2) in an infant with extreme CAG repeat expansion. Am. J. Med. Genet.
79: 383-387, 1998.
4. Christine Mayr “What Are 3′ UTRs Doing?” Cold Spring Harb Perspect Biol. 2019 Oct
1;11(10).
5. Dafne Campigli Di Giammartino, Kensei Nishida, and James L. Manley “Mechanisms and
consequences of alternative polyadenylation” Mol Cell. 2011 Sep 16; 43(6): 853–866.
6. Zeynep Özözen Ayas and Gülgün Uncu “A Rare Coexistence of Paraneoplastic Cerebellar
Degeneration: Papillary Thyroid Carcinoma” Curr Oncol. 2021 Feb; 28(1): 560–564.
7. Ugur Sahin, Katalin Karikó, and Özlem Türeci “mRNA-based therapeutics — developing
a new class of drugs” Nature Reviews Drug Discovery volume 13, pages759–780 (2014).
8. Flavio Mignone, Carmela Gissi, Sabino Liuni, and Graziano Pesole “Untranslated regions
of mRNAs” Genome Biol. 2002; 3(3).
9. Carine Barreau, Luc Paillard, and H. Beverley Osborne “AU-rich elements and associated
factors: are there unifying principles?” Nucleic Acids Res. 2005; 33(22): 7138–7150.
10. Anders Rehfeld, Mireya Plass, Anders Krogh, and Lennart Friis-Hansen “Alterations in
Polyadenylation and Its Implications for Endocrine Disease” Front Endocrinol
(Lausanne). 2013; 4: 53.
11. Ana Curinha, Sandra Oliveira Braz, Isabel Pereira-Castro, Andrea Cruz, and Alexandra
Moreira “Implications of polyadenylation in health and disease” Nucleus. 2014; 5(6): 508–
519.
12. Elisa Mariella, Federico Marotta, Elena Grassi, Stefano Gilotto and Paolo Provero “The
Length of the Expressed 3′ UTR Is an Intermediate Molecular Phenotype Linking Genetic
Variants to Complex Diseases” Front. Genet., doi: 10.3389/fgene.2019.00714.
13. Helen Budworth and Cynthia T. McMurray” A Brief History of Triplet Repeat Diseases”
Methods Mol Biol. 2013; 1010: 3–17.
14. Laura P.W. Ranum1 and Thomas A. Cooper “RNA-Mediated Neuromuscular Disorders”
Annu. Rev. Neurosci. 2006. 29:259–77.
15. L A Balmer 1, D J Beveridge, J A Jazayeri, A M Thomson, C E Walker, P J Leedman
“Identification of a novel AU-Rich element in the 3′ untranslated region of epidermal
growth factor receptor mRNA that is the target for regulated RNA-binding proteins” Mol
Cell Biol. 2001 Mar;21(6):2070-84.
16. Hiroshi Otsuka, Akira Fukao, Yoshinori Funakami, Kent E. Duncan and Toshinobu
Fujiwara “Emerging Evidence of Translational Control by AU-Rich Element-Binding
Proteins” Front. Genet., 02 May 2019 /fgene.2019.00332.162.
17. Jacob O’Brien, Heyam Hayder, Yara Zayed and Chun Peng “Overview of MicroRNA
Biogenesis, Mechanisms of Actions, and Circulation” Front. Endocrinol., 03 August 2018
/fendo.2018.00402.
18. Ayla Valinezhad Orang, Reza Safaralizadeh,* and Mina Kazemzadeh-Bavili
“Mechanisms of miRNA-Mediated Gene Regulation from Common Downregulation to
mRNA-Specific Upregulation” Int J Genomics. 2014; 2014: 970607.
19.Kramer MF. Stem-loop RT-qPCR for miRNAs. Curr Protoc Mol Biol. 2011;Chapter
15:Unit15.10-15.10. doi:10.1002/0471142727.mb1510s95.
20. Caifu Chen, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL,
Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ: Real-time quantification
of microRNAs by stem-loop RT-PCR. Nucleic Acids Res 2005, 33:e179.
21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402-8.
doi: 10.1006/meth.2001.1262. PMID: 11846609.
To cite this abstract in AMA style:
R. Singh, V. Swarup, M. Faruq, A. Srivastava. 3′ UTR variations may alter the mRNA expression in spinocerebellar ataxia type-2. [abstract]. Mov Disord. 2022; 37 (suppl 2). https://www.mdsabstracts.org/abstract/3%e2%80%b2-utr-variations-may-alter-the-mrna-expression-in-spinocerebellar-ataxia-type-2/. Accessed September 7, 2024.« Back to 2022 International Congress
MDS Abstracts - https://www.mdsabstracts.org/abstract/3%e2%80%b2-utr-variations-may-alter-the-mrna-expression-in-spinocerebellar-ataxia-type-2/