Methods for comprehensive transcriptome analysis using next-generation sequencing and application in hypertrophic cardiomyopathy

Characterization of the RNA transcriptome by next-generation sequencing can produce an unprecedented yield of information that provides novel biologic insights. I describe four approaches for sequencing different aspects of the transcriptome and provide computational tools to analyze the resulting data. Methods that query the dynamic range of gene expression, low expressing transcripts, micro RNA levels, and start-site usage of transcripts are described.;Assessing changes in start-site usage can reveal major regulatory events that may be difficult to identify by standard gene expression analysis. By optimizing cDNA library construction steps to enhance for sequencing from 5' ends of transcripts, I developed a robust protocol to measure start-site usage of transcripts with high sensitivity. To identify genome-wide start-site usage changes between different biological specimens, I developed a computational approach that queries the distribution of reads at the 5' ends. This methodology is denoted as 5'RNAseq. Genome-wide 5'RNAseq of cardiac tissues from a mouse model of hypertrophic cardiomyopathy (HCM) identified Four-and-a-half LIM domains protein 1 (Fhl1) as the gene that exhibits the most marked change in start-site usage. Analysis of the specific cell populations in the heart revealed that the increased expression of Fhl1 and change in 5' regulation occurs in myocytes that become engulfed in fibrosis during disease progression. Further analyses of human ventricular specimens from subjects with a variety of cardiovascular pathologies revealed the identical Fhl1 start-site switch occurs in both primary and secondary cardiomyopathies.;Genetic ablation of Fhl1 in the mouse model of HCM resulted in markedly increased hypertrophy and histopathologic remodeling, indicating that Fhl1 acts as an adaptive modifier of HCM. This result contrasts with data from an earlier report that suggest that Fhl1 attenuates hypertrophy caused by pressure overload, and suggests that Fhl1 may be implicated in distinct responses to these different cardiac pathologies. As Fhl1 is encoded on chromosome X, the potentially protective role of Fhl1 in HCM may account for more severe clinical manifestations and outcomes in male patients with HCM and may provide novel therapeutic avenues to limit disease. More broadly, this work provides new tools that can be used to analyze transcriptomic information.