Repeat conformation heterogeneity in cerebellar ataxia, neuropathy, vestibular areflexia syndrome

Abstract

Cerebellar ataxia, neuropathy, vestibular areflexia syndrome (CANVAS) is a late-onset, slow-progressing multisystem neurodegenerative disorder. Biallelic AAGGG repeat expansion in RFC1 has been identified as causative of this disease, and repeat conformation heterogeneity (ACAGG repeat) was also recently implied. To molecularly characterize this disease in Japanese patients with adult-onset ataxia, we accumulated and screened 212 candidate families by an integrated approach consisting of flanking PCR, repeat-primed PCR, Southern blotting and long-read sequencing using Sequel II, GridION or PromethION. We identified 16 patients from 11 families, of whom seven had ACAGG expansions [(ACAGG)exp/(ACAGG)exp] (ACAGG homozygotes), two had ACAGG and AAGGG expansions [(ACAGG)exp/(AAGGG)exp] (ACAGG/AAGGG compound heterozygotes) and seven had AAGGG expansions [(AAGGG)exp/(AAGGG)exp] (AAGGG homozygotes). The overall detection rate was 5.2% (11/212 families including one family having two expansion genotypes). Long-read sequencers revealed the entire sequence of both AAGGG and ACAGG repeat expansions at the nucleotide level of resolution. Clinical assessment and neuropathology results suggested that patients with ACAGG expansions have similar clinical features to previously reported patients with homozygous AAGGG expansions, although motor neuron involvement was more notable in patients with ACAGG expansions (even if one allele was involved). Furthermore, a later age of onset and slower clinical progression were implied in patients with ACAGG/AAGGG compound heterozygous expansions compared with either ACAGG or AAGGG homozygotes in our very limited cohort. Our study clearly shows the occurrence of repeat conformation heterogeneity, with possible different impacts on the affected nervous systems. The difference in disease onset and progression between compound heterozygotes and homozygotes might also be suspected but with very limited certainty due to the small sample number of cases in our study. Studies of additional patients are needed to confirm this.

Genetic and clinical features of cerebellar ataxia with RFC1 biallelic repeat expansions in Japan

“…All the 15 patients shared a core haplotype (<17.8 kb), ranging from rs11939718 (chr4:39337634) to rs13142220 (chr4:39355501). This result was consistent with the results of another report on Japanese CANVAS cases ( 10 ). In that report rs368936934 was found to distinguish the (ACAGG)exp allele from the (AAGGG)exp allele with the C allele being associated with the former and the T with the latter.…”

“…We conducted haplotype analysis using SNPs enrolled by a previous Japanese study ( 10 ). All the 15 patients shared a core haplotype (<17.8 kb), ranging from rs11939718 (chr4:39337634) to rs13142220 (chr4:39355501).…”

“…At this locus of RFC1 , in addition to the reference allele (AAAAG) 11 , other nonpathogenic repeat motifs, (AAAAG)exp and (AAAGG)exp, have been reported ( 1 ). Regarding the pathogenic repeat motifs, other than (AAGGG)exp, (ACAGG)exp has been identified in the Asia-Pacific region ( 4 , 9 , 10 ) and (AAAGG) 10−25 (AAGGG)exp has been detected in Māori individuals ( 11 ).…”

Comprehensive Genetic Analyses of Inherited Peripheral Neuropathies in Japan: Making Early Diagnosis Possible

“…Bi-allelic (AAGGG)exp in RFC1 was found to be associated to Caucasian patients with CANVAS in 2019. Thereafter, a new pathogenic genotype, (ACAGG)exp, was reported in Japan, Indonesia, and Niue [ 13 , 32 , 33 ], and [(AAAGG)10–25(AAGGG)exp] was found only in Māori tribes [ 34 ]. Analysis that targets (ACAGG)exp should be added in the Asia-Pacific region, unlike other regions.…”

“…RFC1 -related neuropathy is often accompanied by chronic cough and autonomic disturbance [ 6 , 35 ], which are suggestive signs of RFC1 -related disorders among IPNs. To date, hyperCKemia and muscle cramps have been reported only in patients carrying (ACAGG)exp [ 13 , 33 ]. However, our study found it as common among various genotypes.…”