RNA Sequencing in the Framingham Heart Study Third Generation Cohort Exam 2

RNA sequencing (RNA-seq) is a powerful tool to evaluate the transcriptome with incredible
depth and clarity. As compared to gene expression arrays, RNA-seq allows the identification
and quantification of a larger set of known transcripts (including long non-coding RNAs
[lncRNAs]), novel transcripts, alternative splicing events, and allele-specific expression
(including parent-of-origin allele-specific expression); all with a vastly higher
signal-to-noise ratio compared to gene expression profiling via microarrays. The relations of
these transcriptomic features to health and disease in very large population studies is
underexplored. It is our belief that this proposed project will identify new biomarkers of
disease risk and provide insights into disease pathogenesis. The Framingham Heart Study (FHS)
is uniquely suited to conduct RNA-seq because of the wealth of existing phenotype resources
in conjunction with whole genome sequence (WGS) data from TOPMed and methylomic data, data
and other omics data that can be leveraged at extremely low cost to maximize the impact of an
investment in RNA-seq.

The advent of high-throughput RNA-seq technology has revolutionized transcriptomic profiling
at an unprecedented scale, leading to the discovery of new RNA species and deepening our
understanding of transcriptomic dynamics. Compared to microarray-based RNA profiling, RNA-seq
is appreciated for its ability to reveal the complexity of the transcriptome, encompassing
previously unknown coding and lncRNA species, novel transcribed regions, alternative
splicing, allele-specific expression, and fusion genes This project proposes to build upon
and extend the work conducted using gene expression arrays in the FHS by examining complex
transcriptomic features that cannot be determined using microarray-based expression data.

In this proposal we focus on expression levels of protein-coding RNAs, lncRNAs, alternative
splicing, and allele-specific expression. There are ~18,000 mRNA transcripts at the
gene-level for protein-coding RNAs. Alternative splicing is a tightly regulated process that
produces different mRNA isoforms from genes that contain multiple exons. One major
application of RNA-seq is to detect even subtle differences in exon splicing. lncRNAs are
non-protein coding transcripts longer than 200 nucleotides and have been implicated in many
biological process. For example, some lncRNAs impact the expression of nearby protein-coding
genes, some can bind to enzymes regulating transcription patterns, and other lncRNAs are
precursors of small RNAs. A number of computational methods have been developed to detect
alternative splicing and lncRNAs from RNA-seq data. Identification of alternative splicing
and lncRNAs will be standardized across TOPMed studies and we will conduct analyses on
centrally called splice data as well as lncRNAs. Allele-specific expression (ASE), which
cannot be measured using microarrays, allows the differentiation between transcripts from the
two haplotypes of an individual at heterozygous sites. ASE enables a more granular
understanding of how a disease-related genotype affects gene expression. ASE has been linked
to human disease in small sample sets but has not been examined fully in large populations.
Standard

bioinformatics tools have been developed to study ASE. In addition, with TOPMed WGS data on
parents from the FHS Offspring cohort, it will be possible to study parent-of-origin ASE,
thus furthering our ability to dissect factors that contribute to the transgenerational
inheritance of cardiometabolic disease.

In this Application, we propose to extend the investigation of transcriptomics in FHS Third
Generation cohort exam 2 participants. The aims of conducting RNA-seq in the FHS Third
Generation cohort mirror and extend those of our original microarray-based gene expression
profiling. Specifically, we will examine the association of complex transcriptomic variation
to: 1) cardiometabolic disease outcomes, 2) genetic sequence variation, and 3) multiple
layers of omic data (Aims 1-3). With the proposed RNA-seq data, investigators as well as the
general scientific community (via dbGaP access) will have the ability to study
transcriptomics from different perspectives always leveraging existing resources to advance
the scientific value of this project. To maximize the return on investment, sequencing will
be performed by a designated TOPMed RNA-seq laboratory, and the aims of this project will be
coordinated with other

TOPMed studies that are conducting RNA-seq.

- INCLUSION CRITERIA:

To accomplish the Aims of this project we propose to conduct RNA-seq on FHS Third
Generation cohort participants with WGS as part of TOPMed. This can only be accomplished in
FHS Third Generation cohort participants who attended exam 2 when PaxGene tubes were
collected for RNA isolation. Therefore, we propose to conduct RNA-seq on FHS Third
Generation cohort exam 2 attendees with PaxGene tubes (total n=3300) and in whom we will
have direct or imputed WGS from TOPMed (n=1700).