CRISPR/Cas9 and SMRT Sequencing Yield New Phenotype Association for SCA10 Repeat Expansion Disorder

A compelling new paper from scientists at the Parkinson’s Institute and Clinical Center, Houston Methodist Research Institute, and several other organizations demonstrates the importance of fully sequencing repeat expansion regions for a clearer understanding of the underlying biology of the diseases they cause. This publication also offers a look at how CRISPR/Cas9 capture can be used in combination with SMRT Sequencing to access the expanded repetitive region at a base level resolution without any PCR bias.
“Parkinson’s disease associated with pure ATXN10 repeat expansion” comes from lead authors Birgitt Schüle and Karen McFarland, senior author Tetsuo Ashizawa, and collaborators. The study involved a Mexican family with one individual previously diagnosed with Parkinson’s disease and several members with spinocerebellar ataxia.
Clinical genetic testing had found an ataxia-associated pentanucleotide repeat expansion in the patient with Parkinson’s, and this team hoped to learn more. “To further genetically characterize the ATXN10 repeat expansion and to better understand the phenotypic differences of progressive cerebellar ataxia with seizures and parkinsonism,” they write, “we employed several advanced and novel molecular genetic techniques to dissect the genetic structure of the repeat expansion in this family.”
Among those techniques was a new method that combined the sequence-specific endonuclease activity of the CRISPR/Cas9 system with long-read SMRT Sequencing. The team reports that they were able to use this method to snip out genomic ATXN10 repeat expansion regions, some  spanning up to 7 kb in length, and sequence them “as one continuous fragment without prior amplification of the genomic DNA.” This was done for six family members, with results indicating that most affected family members had a string of 480 ATTCT repeats followed by about 920 ATTCC repeat interruptions. Strikingly, the family member with ataxia and parkinsonism had a different expansion: more than 1,300 ATTCT repeats but no ATTCC repeats. “We propose that the absence of repeat interruptions play a role in the underlying disease process acting as a genetic modifier and leading to the clinical presentation of L-Dopa responsive parkinsonism,” the scientists write, adding that the repeat interruptions may contribute to the development of epilepsy.
“Single molecule sequencing paired with SMRT/Cas9 capture approach allowed us to characterize the genetic composition of the complete repeat expansion which revealed a novel phenotype-genotype correlation for Parkinson’s disease and ATXN10,” the team adds, highlighting the importance of adding to existing knowledge of repeat expansion types and possible phenotypes. “We conclude that the underlying genetic architecture of ATXN10 repeat expansions is critical for presentation of clinical phenotypes and presumably also the underlying pathology.”