Fig. 3 Friedreich’s Ataxia a) locus on Chr 9q b) mechanism of reduced frataxin expression c) measurement of triplet repeat size in carrier parents and their affected child d) hypermethylation in the region
Friedreich’s Ataxia is an autosomal recessive neurodegenerative disorder, affecting approximately one in 50,000 individuals, with symptoms including progressive limb ataxia and dysarthia. The disorder is caused by reduced levels of frataxin, a protein encoded by the FXN gene on Chromosome 9q (Fig. 3a) that is involved in the assembly of iron-sulphur clusters in mitochondria. The first intron of the gene contains a GAA repeat, which reaches 70–1,000 copies in both alleles of patients, compared to 5–30 copies in unaffected individuals. The longer the repeat, the more severe the symptoms and the earlier the age of onset. Repeat expansion is thought to alter the structure of the DNA at the locus and to lead to hypermethylation. This, in turn, suppresses production of frataxin, leading to disease symptoms (Fig. 3b). The presence of a large repeat and the involvement of methylation mean that PCR is best avoided when investigating the genetics of the disease. We enriched the region from carrier parents and their affected child using Cas9. We aligned reads to hg38 using minimap2 and haplotyped with MarginPhase. We counted repeat length in base-space (Fig. 3c). Sequencing results agree well with the values derived from Southern blotting. A high level of somatic instability has been reported in expanded FXN alleles, which may account for the difference in repeat size between the father and child. Next we called CpG methylation with Nanopolish. In the parent samples, reads from the repeat-expanded haplotype were hypermethylated in the ~2 kb region between exon 1 and the repeat expansion. In the patient sample, both haplotypes were expanded, and both were hypermethylated. The haplotype with the largest expansion has a slightly higher level of methylation (Fig. 3d).
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