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Investigation of RFC1 tandem nucleotide repeat locus in diverse neurodegenerative outcomes in an Indian cohort

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Abstract

An intronic bi-allelic pentanucleotide repeat expansion mutation, (AAGGG)400–2000, at AAAAG repeat locus in RFC1 gene, is known as underlying genetic cause in cases with cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) and late-onset sporadic ataxia. Biallelic positive cases carry a common recessive risk haplotype, “AAGA,” spanning RFC1 gene. In this study, our aim is to find prevalence of bi-allelic (AAGGG)exp in Indian ataxia and other neurological disorders and investigate the complexity of RFC1 repeat locus and its potential association with neurodegenerative diseases in Indian population-based cohorts. We carried out repeat number and repeat type estimation using flanking PCR and repeat primed PCR (AAAAG/AAAGG/AAGGG) in four Indian disease cohorts and healthy controls. Haplotype assessment of suspected cases was done by genotyping and confirmed by Sanger sequencing. Blood samples and consent of all the cases and detailed clinical details of positive cases were collected in collaboration with A.I.I.M.S. Furthermore, comprehension of RFC1 repeat locus and risk haplotype analysis in Indian background was performed on the NGS data of Indian healthy controls by ExpansionHunter, ExpansionHunter Denovo, and PHASE analysis, respectively. Genetic screening of RFC1-TNR locus in 1998 uncharacterized cases (SCA12: 87; uncharacterized ataxia: 1818, CMT: 93) and 564 heterogenous controls showed that the frequency of subjects with bi-allelic (AAGGG)exp are 1.15%, < 0.05%, 2.15%, and 0% respectively. Two RFC1 positive sporadic late-onset ataxia cases, one bi-allelic (AAGGG)exp and another, (AAAGG)~700/(AAGGG)exp, had recessive risk haplotype and CANVAS symptoms. Long normal alleles, 15–27, are significantly rare in ataxia cohort. In IndiGen control population (IndiGen; N = 1029), long normal repeat range, 15–27, is significantly associated with A3G3 and some rare repeat motifs, AGAGG, AACGG, AAGAG, and AAGGC. Risk-associated “AAGA” haplotype of the original pathogenic expansion of A2G3 was found associated with the A3G3 representing alleles in background population. Apart from bi-allelic (AAGGG)exp, we report cases with a new pathogenic expansion of (AAAGG)exp/(AAGGG)exp in RFC1 and recessive risk haplotype. We found different repeat motifs at RFC1 TNR locus, like AAAAG, AAAGG, AAAGGG, AAAAGG, AAGAG, AACGG, AAGGC, AGAGG, and AAGGG, in Indian background population except ACAGG and (AAAGG)n/(AAGGG)n. Our findings will help in further understanding the role of long normal repeat size and different repeat motifs, specifically AAAGG, AAAGGG, and other rare repeat motifs, at the RFC1 locus.

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Abbreviations

CANVAS:

Cerebellar ataxia, neuropathy, and vestibular areflexia syndrome

f-PCR:

Flanking PCR

RP-PCR:

Repeat primed PCR

LR-PCR:

Long range PCR

EHDn:

ExpansionHunter Denovo

IndiGen:

Indian genomes

ADL:

Activities of daily living

ROS:

Reactive oxygen species

STR:

Short tandem repeats

TNR:

Tandem nucleotide repeats

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Acknowledgements

We acknowledge Prof. Mitali Mukerji’s initial direction towards this work. We sincerely appreciate everyone who has provided constant support during this study, including lab assistants Subhash Gurjar, Usha Rawat, and Suman Mudila. We are thankful for the participation of the patients and their families.

Funding

Funding support from MLP1802, OLP1120 CSIR funded project. M.F. thanks C.S.I.R.-I.G.I.B. for research grant. N.T. is supported by Union Grant Commission (U.G.C.). P.S. has been supported by ICMR-SRF. Additionally, S.S.’s Department of Biotechnology (DBT-JRF) fellowship is acknowledged. V.A.’s Indian Council of Medical Research (ICMR-SRF) fellowship is also acknowledged.

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Authors and Affiliations

  1. Genomics and Molecular Medicine Division, CSIR - Institute of Genomics and Integrative Biology, New Delhi, 110007, India

    Nishu Tyagi, Bharathram Uppili, Pooja Sharma, Shaista Parveen, Sheeba Saifi, Abhinav Jain, Istaq Ahmed, Uzma Shamim, Avni Anand, Vivekanand Asokachandran, Sridhar Sivasubbu, Vinod Scaria & Mohammed Faruq

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India

    Nishu Tyagi, Bharathram Uppili, Pooja Sharma, Abhinav Jain, Vivekanand Asokachandran & Mohammed Faruq

  3. Department of Neurology, Neuroscience Centre, All India Institute of Medical Sciences (AIIMS), 110608, New Delhi, India

    Akhilesh Sonakar, Shweta Sahni, Varun Suroliya & Achal Srivastava

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  1. Nishu Tyagi
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Tyagi, N., Uppili, B., Sharma, P. et al. Investigation of RFC1 tandem nucleotide repeat locus in diverse neurodegenerative outcomes in an Indian cohort. Neurogenetics 25, 13–25 (2024). https://doi.org/10.1007/s10048-023-00736-6

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