中截断变量RFC1小脑性共济失调、神经病和前庭反射综合征

Nonsense-Mediated mRNA Decay and Haploinsufficiency of RFC1 (A) Real-Time qPCR showed a 40% reduction of RFC1 mRNA in 2 CANVAS cases carrying c.1267C> T/(AAGGG)_exp (n = 2) or c.2876del/(AAGGG)_exp (n = 1) mutations compared with healthy controls (n = 4, p value = 0.0305) and with biallelic (AAGGG)exp/(AAGGG)exp CANVAS cases (n = 4, p value <0.0001). No significant reduction was seen in control vs biallelic CANVAS cases (p = 0.154) (B) RNAseq confirmed a significant reduction in RFC1 transcript level in patients I-1 and I-2 compared with healthy controls (n = 6, p = 0.0086). (C) Binary Alignment Map (BAM) data from RNAseq and WGS performed in individuals from family 1 showed in I-1 and I-2 a lower number of reads containing the c.1267C>T mutation (green) compared reads mapping to the transcript derived from the second (brown), as opposed to an equal representation of the 2 alleles on WGS, supporting the presence of nonsense-mediated Decay (NMD) of the c.1267C>T mutant transcript. Schematic representation of the percentage of cytosine and thymine called at c.1267 of RFC1 in WGS and RNAseq for family 1. (D) Case II RFC1 gDNA and mRNA sequencing. The top left electropherogram show the presence on gDNA of the c.2876del frameshift variant, which is not evident on mRNA sequencing (bottom left) due to nonsense-mediated decay. Conversely, the cytosine allele at base 2,511, which is heterozygously expressed in gDNA and is part of the (AAGGG)_n expansion–containing haplotype, seems as “homozygous” in the mRNA sequencing due to nonsense-mediated decay of the second allele, which contains the c.2876del truncating variant. Together, the findings support the location in trans of (AAGG)_n repeat expansion and c.2876del variant in this case. (E) Western blotting revealed a 50% reduction of full-length RFC1 protein expression (red arrow) in CANVAS patients (I-1 and I-2) vs healthy control (control) and their unaffected sibling (I-3) (p = 0.0263) along with a reduction of RFC1 in HEK293 cells on siRFC1 transfection. Nontargeting siRNA-transfected cells were used as a control. RFC1 densitometric values were normalized to GAPDH. No significant reduction in RFC1 protein expression was seen between biallelic (AAGGG)exp/(AAGGG)exp CANVAS patient–derived fibroblasts (n = 5) and healthy control–derived fibroblasts (n = 5) (p = 0.94). Abbreviations: CANVAS = cerebellar ataxia, neuropathy, and vestibular areflexia syndrome; RFC1 = replication factor complex subunit 1.