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.

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