Sequencing the Unsequenceable: Expanded CGG Repeats in the Human FMR1 Gene

Abstract

Alleles of the FMR1 gene with more than 200 CGG repeats generally undergo methylation-coupled gene silencing, resulting in fragile X syndrome, the leading heritable form of cognitive impairment. Smaller expansions (55-200 CGG repeats) result in elevated levels of FMR1 mRNA, which is directly responsible for the late-onset neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS). Despite the importance of this gene, no existing DNA sequencing method is capable of sequencing through more than ∼100 CGG repeats, thus limiting the ability to precisely characterize the disease-causing alleles.The recent development of single molecule, real-time sequencing represents a novel approach to DNA sequencing that couples the intrinsic processivity of DNA polymerase with the ability to read polymerase activity on a single-molecule basis. Further, the accuracy of the method is improved through the use of circular templates, such that each molecule can be read multiple times to produce a circular consensus sequence (CCS). We have succeeded in generating CCS reads representing multiple passes through both strands of repeat tracts exceeding 700 CGGs (>2 kb of 100 percent CG) flanked by native FMR1 sequence, with single-molecule readlengths exceeding 12 kb. This sequencing approach thus enables us to fully characterize the previously intractable CGG-repeat sequence, leading to a better understanding of the distinct associated molecular pathologies. The method will enable us to study details of allele-expansion mosaicism and repeat instability in a manner not previously possible. Real-time kinetic data also provides insight into the activity of DNA polymerase inside this unique sequence.The methodology should be widely applicable for studies of the molecular pathogenesis of an increasing number of repeat expansion-associated neurodegenerative and neurodevelopmental disorders, and for the efficient identification of such disorders in the clinical setting.