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

Background and ObjectivesAn 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. MethodsWe carried out repeat number and repeat type estimation using flanking PCR and repeat primed PCR (AAAAG/AAAGG/AAGGG) in three 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 AIIMS. Further, 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 de novo and PHASE analysis respectively. ResultsGenetic screening of RFC1-TNR locus in 1998 uncharacterised cases (SCA12: 87; Uncharacterised 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)exp/(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. DiscussionApart from bi-allelic (AAGGG)exp, we report cases with a new pathogenic expansion of (AAAGG)exp/(AAGGG)exp in RFC1 and recessive risk haplotype, AAGA. We found different repeat motifs at RFC1 TNR locus, like, AAAAG, AAAGG, AAAGGG, AAAAGG, AAGAG, AACGG, AAGGC, AGAGG, 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 RFC1 locus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=156 SRC="FIGDIR/small/23290839v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@814cf9org.highwire.dtl.DTLVardef@e498b9org.highwire.dtl.DTLVardef@1e6c747org.highwire.dtl.DTLVardef@250a5e_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract:C_FLOATNO Genetic heterogenicity at RFC1-TNR locus. C_FIG