June 1, 2021  |  

Enrichment of unamplified DNA and long-read SMRT Sequencing in unlocking the underlying biological disease mechanisms of repeat expansion disorders

For many of the repeat expansion disorders, the disease gene has been discovered, however the underlying biological mechanisms have not yet been fully understood. This is mainly due to technological limitations that do not allow for the needed base-pair resolution of the long, repetitive genomic regions. We have developed a novel, amplification-free enrichment technique that uses the CRISPR/Cas9 system to target large repeat expansions. This method, in conjunction with PacBio’s long reads and uniform coverage, enables sequencing of these complex genomic regions. By using a PCR-free amplification method, we are able to access not only the repetitive elements and interruption sequences accurately, but also the epigenetic information.


June 1, 2021  |  

Alternative splicing in FMR1 premutations carriers

Over 40% of males and ~16% of female carriers of a FMR1 premutation allele (55-200 CGG repeats) are at risk for developing Fragile X-associated Tremor/Ataxia Syndrome (FXTAS), an adult onset neurodegenerative disorder while, about 20% of female carriers will develop Fragile X-associated Primary Ovarian Insufficiency (FXPOI), in addition to a number of adult-onset clinical problems (FMR1 associated disorders). Marked elevation in FMR1 mRNA levels have been observed with premutation alleles and the resulting RNA toxicity is believed to be the leading molecular mechanism proposed for these disorders. The FMR1 gene, as many housekeeping genes, undergoes alternative splicing. Using long-read isoform sequencing (SMRT) and qRT-PCR we have recently reported that, although the relative abundance of all FMR1 mRNA isoforms is significantly increased in the premutation group compared to controls, there is a disproportionate increase, relative to the overall increase in mRNA, in the abundance of isoforms spliced at both exons 12 and 14. In total, we confirmed the existence of 16 out of 24 predicted isoforms in our samples. However, it is unknown, which isoforms, when overexpressed, may contribute to the premutation pathology. To address this question we have further defined the transcriptional FMR1 isoforms distribution pattern in different tissues, including heart, muscle, brain and testis derived from FXTAS premutation carriers and age-matched controls. Preliminary data indicates the presence of a transcriptional signature of the FMR1 gene, which clusters more by individual than by tissue type. We identified additional isoforms than the 16 reported in our previous study, including a group with particular splice patterns that were observed only in premutations but not in controls. Our findings suggest that the characterization of expression levels of the different FMR1 isoforms is fundamental for understanding the regulation of the FMR1 gene as well as for elucidating the mechanism(s) by which “toxic gain of function” of the FMR1 mRNA may play a role in FXTAS and/or in the other FMR1-associated conditions. In addition to the elevated levels of FMR1 isoforms, the altered abundance/ratio of the corresponding FMRP isomers may affect the overall function of FMRP in premutations.


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