Fig. 4: Nuclease activity of FAN1 variants identified in individuals with HD correlates with residual age at onset of motor symptoms.
a, Lymphoblastoid cell lines derived from patients carrying a heterozygous R507H FAN1 variant are significantly more sensitive to mitomycin C than age-matched and pure CAG-length-matched control HD lines with homozygous wild-type FAN1 (**P = 1.3 × 10−2, two-tailed t-test). Four independent lines for each genotype, mean of three independent experiments shown for each line (dots), as well as mean ± s.e.m. for each genotype (horizontal lines). b, Representative gel showing nuclease activity of FAN1 variants on 5′ flap DNA. FAN1 protein (10 nM) was incubated with fluorescently labeled 5′ flap DNA substrate (5 nM) for 10 minutes at 37 °C in the presence of MnCl2. Reactions were denatured and analyzed using 15% TBE-urea gel electrophoresis. All experiments were repeated at least three times. c, FAN1 variants identified in individuals with HD have significantly reduced nuclease activity compared to wild-type FAN1. Variants associated with early/more severe phenotypes (orange) had less nuclease activity than variants associated with late/less severe phenotypes (green). The nuclease-inactive D981A R982A FAN1 variant was used as a negative control. Activities of variants are normalized to wild-type FAN1 nuclease activity. (n = 3 independent experiments, **P < 1 × 10−2 and ****P < 1 × 10−4; one-way ANOVA; mean ± s.d. shown). d, Graph of mean age at motor onset residual (using pure CAG length from sequencing) against FAN1 nuclease activity for six variants, normalized to wild-type FAN1 activity: R377W n = 10; R507H n = 18; D702E n = 1; K794R n = 1; R982C n = 1; C1004G n = 1. There was a significant correlation between average motor onset residual and in vitro nuclease activity (P = 2.7 × 10−2). Mean ± s.d. nuclease activity is shown for each variant (n = 3 independent experiments). Three individuals had two FAN1 variants: C1004G and R507H; R982C and N621S; and R377W and R507H. For analyses, these individuals were included in the groups of the most damaging of the two variants they carried. See also Supplementary Fig. 3. e, Graph of age at motor onset against CAG length for the continuous phenotype group (n = 558), highlighting those individuals carrying damaging FAN1 variants assayed for nuclease activity. FAN1 nuclease activity: very low (<20% of wild-type), low (20–50% of wild-type) and moderate (50–80% of wild-type). nt, nucleotide; WT, wild-type.
