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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 69  |  Issue : 4  |  Page : 226-232

Polymorphic study of ataxin 3 gene in Eastern Uttar Pradesh population


1 Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
2 Department of Neurology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Date of Submission08-Aug-2019
Date of Acceptance24-Oct-2020
Date of Web Publication29-Dec-2020

Correspondence Address:
Prof. Royana Singh
Department of Anatomy, Institute of Medical Sciences, Banaras Hindu University, Varanasi - 221 005, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JASI.JASI_107_19

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  Abstract 


Introduction: Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a prevalent autosomal dominant-inherited disease that causes progressive problems with movement. Abnormal repetitive expansion of CAG trinucleotide in the ATXN3 gene results in SCA3. This study was done to review the corporation of CAG repeats and polymorphisms in definitive genes with the occurrence of SCA3 in the Indian community, especially in the eastern UP population. Material and Methods: The 40 Ataxia's patient and their parents were listed after obtaining written consent from the participant's attendant/guardians. Out of these, we have identified polymorphism in three patients. Results: In one patient, we have found a single base change, g.31483A>T in Exon 10, which changes the nucleotide from Adenine to Thymine (A31483T), while in the second patient, we have identified an intronic change at g.35690A>G in Exon 10, which changes the nucleotide from Adenine to Guanine (A35690G) and in the third patient DNA sequence analysis identified an intronic change at g.35587A>G Exon 10, which changes the nucleotide from Adenine to Guanine (A35587G). Discussion and Conclusion: Although the partial loss of ATXN3 function may also contribute, the disease mechanism in MJD is believed to be a toxic gain-of-function. Several pathogenic cascades have been reported to be triggered by mutant ATXN3, but the critical molecular events driving MJD pathogenesis stay unresolved. While significant developments in studies have enhanced our knowledge of MJD, there is presently a lack of preventive treatment. Results presented here also expand our knowledge about MJD found in the eastern UP population.

Keywords: ATXN3 gene, Machado-Joseph disease, polymerase chain reaction, polymorphism, Spinocerebellar ataxia type 3


How to cite this article:
Singh B, Bose P, Shamal S N, Joshi D, Singh R. Polymorphic study of ataxin 3 gene in Eastern Uttar Pradesh population. J Anat Soc India 2020;69:226-32

How to cite this URL:
Singh B, Bose P, Shamal S N, Joshi D, Singh R. Polymorphic study of ataxin 3 gene in Eastern Uttar Pradesh population. J Anat Soc India [serial online] 2020 [cited 2021 Jan 24];69:226-32. Available from: https://www.jasi.org.in/text.asp?2020/69/4/226/305370




  Introduction Top


Spinocerebellar ataxia type 3 (Machado-Joseph disease [MJD]/SCA3), also known as MJD is the prevalent autosomal dominant-inherited cerebellar ataxia.[1],[2] MJD is diagnosed with molecular genetic testing to detect an abnormal repetitive expansion of CAG trinucleotide in the ATXN3 gene. The gene ATXN3 is found on chromosome 14q32.1 and is suggested in people with progressive cerebellar ataxia and pyramidal signs as well as dystonia, ophthalmoplegia, action-induced facial and lingual fasciculation-like movements, and bulging eyes.[3],[4] Current therapy is symptomatic without inhibiting neuronal cell death or delaying the age of onset. Identifying molecular pathways of disease is, therefore, essential for unraveling future therapeutic goals. SCA3 is a polyglutamine (polyQ) neurodegenerative disorder. Normal alleles range from 11 to 44 repeats of CAG, while pathogenic expansions range from 61 to 87 CAGs.[5]

A range of clinical characteristics can be found in subjects with ≥52 CAG units.[6],[7],[8] The identification of founders with Portuguese-Azorean ancestry was noted in the first linkage studies.[9]

The trials find the interactions of ataxin-3 with two proteins, HHR23A and HHR23B, both homologs of the DNA repair protein Rad23.[10] Additional research disclosed ataxin-3 as a bona fide deubiquitinating (DUB) enzyme,[11],[12] giving vital insight into ataxin-3's normal function. More lately, ataxin-3 has been shown to communicate with the transcription factor FOXO4 in the oxidative stress reaction as a transcriptional coactivator.[13] Thus, while some very fundamental ataxin-3 features have been identified, these developments have produced nonintersecting inquiry lines and have not given an underlying mechanism for the pathogenesis of SCA3 disease. To determine the prevalence and distribution of MJD/SCA3 in the Cuba, a clinical and molecular genetic study of a cohort of SCA3 families was created. Also assessed were the ordinary variation of repeats of CAG and their volatile conduct.

Due to the low prevalence of intermediate alleles at this locus, SCA3 was not validated in the replication cohorts. The major symptoms are as gait ataxia as the disease's initial symptom. Other cerebellar characteristics were dysdiadochokinesia, dysmetria, cerebellar dysarthria, and kinetic tremor. Pathological nystagmus was the most significant oculomotor sign.


  Material and Methods Top


This study was done to review the corporation of CAG repeats and polymorphisms in definitive genes with the occurrence of SCA 3 in the Indian community, especially in the eastern UP population. The study will also help us in concluding the genetic explanation of these CAG/polyQ repeat expansion in the SCA3 gene. We looked for Atxn3 polymorphism in a subset of genomic DNA (DNA isolation from blood samples). The 40 Ataxia's patient and their parents were listed after obtaining written consent from the participant's attendant/guardians. A particular admission precedent was based on clinically recognizing cerebellar ataxia. It was determined by the study of medical records. Blood samples from the proband, mother and their various controls were collected from the OPD of the Department of Neurology (Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University [BHU], Varanasi) and evicted to Cytogenetic lab (Institute of Medical Sciences, BHU, Varanasi) for genetic studies in ethylenediaminetetraacetic acid vials and stored at 4°C under sterile condition till further scrutiny. Genomic DNA was isolated from blood using materials like heparinized blood.

After isolation of genomic DNA, quantification of DNA was measured for the further experiment using the Nanodrop-Spectrophotometer. After quantification, primers were designed for the coding region of ATXN3 gene including exon/intron boundaries. The ATXN3 gene contains 10 Exons. The primers were designed using Primer3 software version 0.4.0 (http://frodo.wi.mit.edu/primer3/).

Polymerase chain reaction

In molecular biology, the polymerase chain reaction (PCR) was used as a technique to amplify the single or a few copies of a piece of DNA across multiple order of magnitude, generating thousands to millions of copies of particular DNA sequence. The PCR was carried out on an AppliedBiosystem, 96 well Veriti Thermal Cycler (Applied Biosystem, USA).

During the study, molecular analysis was done for Exon 1-10. Primers were designed to amplify across each of the exons including Exon 10. Sequencing analysis was done to determine polymorphism in all the patients and control for Exon 1-10; however, polymorphism and intronic changes were observed only in Exon 10.

Gel electrophoresis

After running PCR, the PCR products were subjected to gel electrophoresis. We have done gel electrophoresis at temperature of 100°C and after that, with the help of the Gel doc system or gel imaging, we have measured and recorded the labeled nucleic acid or DNA. Gel results of amplified genomic DNA of Exon10 can be seen in [Figure 1]. We run PCR for all collected samples and after that, we had carried out the gel electrophoresis to see the amplification. In [Figure 1]a and [Figure 1]b, we can see that the Exon10 of the ATXN3 gene would get amplified at 643bp. After that all the samples were sent for sequencing. In sequencing, mutation was observed in Exon10. After that, the analysis was performed in dry lab using After that, the analysis was performed in dry lab using Mutation Taster.
Figure 1: (a and b) Showing gel pictures of amplified genomic DNA (Exon 10 of ATXN 3 Gene) of SCA3 samples

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The study was performed to determine the impact of the polymorphic site. None of the unaffected members of the family and none of the control individuals had this abnormal conformer. One polymorphism and one intronic changes have been found in the gene ATXN3.


  Results Top


Gel results of amplified genomic DNA of Exon 1 to Exon 10 as shown in [Figure 1]. We run PCR for all collected samples (40 patients and 20 Controls) as shown in [Table 1] and had carried out the gel electrophoresis to see the amplification. We can see that the Exon 10 of the Atxn 3 gene would get amplified at 638 bp.
Table 1: Showing polymorphism detected in 3 cases out of 40 families (3 familial and 37 sporadic cases)

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Phenotypic analysis

Three familial and sporadic patients ranging in age from 16 years to 65 years with clinically apparent ataxia presenting with nystagmus, decreased saccade velocity; amyotrophy fasciculations, sensory loss, and evaluated by history taking and physical examination, magnetic resonance imaging and computed tomography scan for SCA 3 were identified.

Molecular analysis

During the study, molecular analysis was done for Exon 1-10. Primers were designed to amplify across each of the Exons, including Exon 10. Sequencing analysis was done to determine polymorphism in all the patients and control for Exon 1-10; however, polymorphism was observed only in Exon 10. During the study, in silico analysis was done to determine the impact of the polymorphic site.

In the first case, a male aged 58 years whose ID is BHU/15/638 was detected with no family history (sporadic case) as shown in [Figure 2]. Chromatogram demonstrates a single base change, g.31483A>T in Exon 10, where adenine was replaced by Thymine (A31483T) (Ensembl transcript ID ENST00000545170), these alteration takes place in intron. With the help of MUTATION TASTER software, the polymorphic site was detected, wild type gDNA sequence compare to altered gDNA sequence, in these sequences A altered to T, Multiple sequence alignment was done to determine the single base exchange of A>T.
Figure 2: (a) Showing picture of SCA3 Patient, ID- BHU/15/638, (b) Pedigree of Patient BHU/15/638, (c) Sequence analysis of Patient BHU/15/638, (d) Sequence analysis demonstrates single nucleotide change at g.31483A>T position in Exon10 where Adenine was replaced by Thymine (A31483T), (e) MUTATION TASTER result showing polymorphic site

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In the second case, also a male aged 35 years whose ID is BHU/15/464 was detected with no familial history (as shown in [Figure 3]). Multiple sequence alignment was done to determine a single base exchange of A>G. Chromatogram demonstrates single nucleotide change at g.35690A>G in Exon 10, which changes the nucleotide from Adenine to Guanine (A35690G) (Ensembl transcript ID ENST00000398590), these alteration takes place in the intron. MUTATION TASTER result showing the polymorphic site as disease causing. Wild-type gDNA sequence compares to altered gDNA sequence, in this sequence A altered to G.
Figure 3: (a) Showing picture of SCA3 Patient, ID- BHU/15/464, (b) Pedigree of Patient BHU/15/464, (c) Sequence analysis of Patient BHU/15/464, (d) Sequence analysis demonstrates single nucleotide change at g.35690A>G in Exon 10, which changes the nucleotide from Adenine to Guanine (A35690G), (e) mutation taster result showing polymorphic site

Click here to view


In the third case, a male aged 40 years whose ID is BHU/17/28 was detected with no family history (sporadic case) as shown in [Figure 4]. Multiple sequence alignment was done to determine a single base exchange of A>G. (D) Chromatogram demonstrates single nucleotide change at g.35587A>G in Exon 10, which changes the nucleotide from Adenine to Guanine (A35587G), (Ensembl transcript ID ENST00000545170), MUTATION TASTER result showing the polymorphic site as disease causing. Wild type gDNA sequence compared to altered gDNA sequence, in this sequence A altered to G.
Figure 4: Pedigree of Patient BHU/17/28. (a) Picture of SCA3 patient, ID- BHU/17/28, (b) Pedigree of patient BHU/17/28, (c) Multiple sequence alignment was done to determine single base exchange of A > G. (d) Chromatogram demonstrates single nucleotide change at g. 35587A > G in Exon 10, which changes the nucleotide from adenine to guanine (A35587G), (e) MUTATIONTASTER result showing polymorphic site as disease causing. (f) Wild type gDNA sequence compared to altered gDNA sequence, in this sequence A altered to G

Click here to view



  Discussion Top


MJD, the most common dominantly inherited ataxia in the world, is triggered by an expansion of polyQ in the DUB enzyme ataxin-3. The polyQ repeat expansion is thought to give the various polyQ-encoding proteins a toxic gain-of-function, potentially supporting their misfolding and aggregation, but the accurate mechanism of disease pathogenesis remains unknown. The ATXN3 gene offers directions for the production of an enzyme called ataxin3, which is found in cells throughout the body. Ataxin3 is engaged in a mechanism called the ubiquitinproteasome system, which destroys surplus and damaged proteins and gets rid of them. The molecule ubiquitin attaches (binds) to unneeded proteins and marks them within cells to be broken down (degraded). Ataxin3 removes (cleaves) the ubiquitin from these unwanted proteins just before it is degraded to allow the ubiquitin to be reused. Ataxin3 is known as a DUB enzyme because of its function in slicing ubiquitin from proteins.

We observed polymorphism in three patients of SCA3 and those polymorphism and intronic changes are depicted in [Table 2] and [Figure 5]. SCA3 is a polyglutamine neurodegenerative disorder. Normal alleles range from 11 to 44 repeats of CAG, while pathogenic expansions range from 61 to 87 CAGs. A range of clinical features can be found in subjects carrying ≥52 CAG units. This has led us to speculate on the existence of common features in Ataxia type 1 disorder. While well characterized at the molecular level, the encoded protein function is not currently known.
Figure 5: Polymorphism detected in Exon 10 in all the three cases

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Table 2: Single base exchange variation identified in Atxn 3 in spinocerebellar ataxia patients with their position of nucleotide according to ensemble and position of amino acid

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Normally, ATXN3 helps to control the stability and activity of multiple proteins in various cellular pathways. Mutant (expanded) ATXN3 is susceptible to form insoluble aggregates and to undergo proteolysis that produces fragments containing polyQ that further encourage aggregation. Although the partial loss of the ATXN3 function may also contribute, the disease mechanism in MJD is believed to be a toxic gain-of-function. Several pathogenic cascades have been reported to be triggered by mutant ATXN3, but the critical molecular events driving MJD pathogenesis stay unresolved. In the pursuit of MJD treatment, several pathways have been targeted, but none have yet progressed to human clinical trials.

The single-nucleotide change in the first patient was at g.31483A>T position in Exon10, where Adenine was replaced by Thymine (A31483T) as shown. The change of nucleotide was in the intronic region. While, in the second patient, a single base change was observed at g.35690A>G in Exon 10, which changes the nucleotide from Adenine to Guanine (A35690G). It can be predicted as disease causing by altering the nucleotide variants as shown.

In the third patient, the nucleotide change occurred at site g.35587A>T position in Exon10, where Adenine was replaced by Guanine (A>G), as shown. This nucleotide change represents single base change. A study was done to screen polymorphism through mutation taster.


  Conclusion Top


Mutations in the ATXN3 gene mostly affect nerve cells and other types of brain cells. SCA3 is associated with cell death in the portion of the brain attached to the spinal cord (the brainstem), the part of the brain engaged in coordinating motions (the cerebellum), and other brain regions. This condition is also associated with the death of nerve cells in the spinal cord. Over time, the brain and spinal cord cell loss cause distinctive signs and symptoms of SCA3. While significant developments in studies have enhanced our knowledge of MJD, there is presently a lack of preventive treatment. Results presented here also expand our knowledge about MJD found in the eastern UP population. These findings deserve confirmation in a second population and more in-depth research in cellular and animal models.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Winborn BJ, Travis SM, Todi SV, Scaglione KM, Xu P, Williams AJ, et al. The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains. J Biol Chem 2008;283:26436-43.  Back to cited text no. 11
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2]



 

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