Detecting AGG interruptions in male and female FMR1 premutation carriers by single-molecule sequencing

The FMR1 gene contains an unstable CGG repeat in its 5’ untranslated region. Premutation alleles ranging between 55-200 repeat units occur in the population with an estimated frequency of about 1 in 200 and are at risk for developing fragile X-associated tremor/ataxia syndrome or fragile X-associated primary ovarian insufficiency. Furthermore, the premutation allele often expands to a full mutation during female germline transmission giving rise to the fragile X syndrome. The risk for a premutation to expand depends on both size and AGG interruptions in the CGG repeat. Unfortunately, the detection of AGG interruptions is hampered by technical difficulties. Here, we demonstrate that single-molecule sequencing enables the determination of not only the repeat size, but also the complete repeat structure including AGG interruptions in male and female alleles with repeats ranging from 45 to 100 CGG units.