A comprehensive catalogue of the coding and non-coding transcripts of the human inner ear

Highlights

• We performed RNA-Seq of the human cochlea, ampulla, saccule and utricle.

• We have built a comprehensive database of transcripts of the adult human inner ear.

• We describe a first detailed inventory of long non-coding RNA's in the inner ear.

• Our data supports potential tissue specificity of NMD in the inner ear.

• The expression of genes in deafness loci without gene identification is described.

Abstract

The mammalian inner ear consists of the cochlea and the vestibular labyrinth (utricle, saccule, and semicircular canals), which participate in both hearing and balance. Proper development and life-long function of these structures involves a highly complex coordinated system of spatial and temporal gene expression. The characterization of the inner ear transcriptome is likely important for the functional study of auditory and vestibular components, yet, primarily due to tissue unavailability, detailed expression catalogues of the human inner ear remain largely incomplete.

We report here, for the first time, comprehensive transcriptome characterization of the adult human cochlea, ampulla, saccule and utricle of the vestibule obtained from patients without hearing abnormalities. Using RNA-Seq, we measured the expression of >50,000 predicted genes corresponding to approximately 200,000 transcripts, in the adult inner ear and compared it to 32 other human tissues. First, we identified genes preferentially expressed in the inner ear, and unique either to the vestibule or cochlea. Next, we examined expression levels of specific groups of potentially interesting RNAs, such as genes implicated in hearing loss, long non-coding RNAs, pseudogenes and transcripts subject to nonsense mediated decay (NMD). We uncover the spatial specificity of expression of these RNAs in the hearing/balance system, and reveal evidence of tissue specific NMD. Lastly, we investigated the non-syndromic deafness loci to which no gene has been mapped, and narrow the list of potential candidates for each locus.

These data represent the first high-resolution transcriptome catalogue of the adult human inner ear. A comprehensive identification of coding and non-coding RNAs in the inner ear will enable pathways of auditory and vestibular function to be further defined in the study of hearing and balance. Expression data are freely accessible at https://www.tgen.org/home/research/research-divisions/neurogenomics/supplementary-data/inner-ear-transcriptome.aspx.

Introduction

The inner ear consists of auditory and vestibular components that involve a highly complex coordinated system of spatial and temporal gene expression to control the connectivity of neurons and circuits. The vestibular components are further divided into the utricle, saccule and three semicircular ducts each with a terminal enlargement called an osseous ampulla. The large number of genetic mutations and loci that have been implicated in hearing and balance disorders further illustrate structural and functional complexity of this organ. Yet, much of what is known about the genetics of hearing in humans comes from the study of Mendelian disorders. More than 100 loci were identified in familial studies of nonsyndromic hearing loss, with ∼30% of the studies failing to identify the specific gene or mutation implicated (http://hereditaryhearingloss.org/). The genetic basis for the more common and complex forms of hearing and balance disorders, such as presbycusis and disequilibrium and falls in the elderly, has been established (Fransen et al., 2014) but remains poorly understood.

Over the past decade, microarray technology has proven to be a useful tool for performing high throughput gene expression analyses of the inner ear. While initially used primarily to characterize the level of expression of mRNAs in the wild type tissue, it was later used to identify complex gene networks, decipher regulatory pathways and characterize response to various stimuli. Numerous studies have profiled the inner ear gene expression using microarray technology, focusing on model organisms such as mouse and zebrafish, both in normal and pathological conditions (Hertzano and Elkon, 2012, Scheffer et al., 2015). In contrast to the wealth of data available for model organisms, very little is known about gene expression in the human inner ear, mainly due to difficulties in obtaining the relevant tissues. In fact the only available genome wide profiling of the transcriptome in human inner ear was published in 1999 and 2002 by the laboratory of Dr. Cynthia Morton, using expressed sequence tags in human fetal cochlear tissue (Resendes et al., 2002, Skvorak et al., 1999). We believe that knowledge of the elementary expression patterns of the hearing/balance system in the functioning human ear will be a valuable tool for gaining systems-level understanding of basic biological processes during normal development, or development of pathological conditions and will allow significant progress in identification of new genes functionally important for human hearing.

Recent advances in whole genome expression analyses, namely RNA expression profiles produced by next-generation sequencing technology (RNA-Seq) allow comprehensive interrogation of transcribed sequences within cells or tissues, using very small amounts of total RNA. Advantages of this approach include an improved sensitivity to detect transcripts with low copy number, a significantly broader dynamic range resulting in enhanced sensitivity for detection of differential expression, detection of non-coding RNA's and isoform specific expression and identification of novel transcripts (Hertzano and Elkon, 2012, Scheffer et al., 2015). RNA expression profiles of tissues that are relevant to a particular trait are recognized as an important aspect of systematic genetic analysis of these traits. In addition, transcriptional data may be used in genetic mapping of functional genetic variants where large intervals that include multiple genes are identified, and often are difficult to narrow down to a specific gene of interest. Expression profiles of the specialized tissues were shown to be instrumental in narrowing the researchers' focus on genes that are functionally relevant to the trait (Vahedi et al., 2010). Last, regulatory non-coding RNAs display tissue-specific expression profiles and contribute to tissue patterning and to the control of different cellular programs, such as cell proliferation, differentiation, migration, or apoptosis. They have recently been implicated in the development of sensory organs and disorders (Conte et al., 2013). A recent paper focused on next generation sequencing of miRNAs in the mouse inner ear (Rudnicki et al., 2014), but a transcriptional inventory of long non-coding RNAs such as long intervening non-coding (lincRNAs) of the inner ear still remains incomplete. Thus, the lack of a well-characterized and in-depth analysis of the transcriptome of the human inner ear remains an impediment to progress in the field of hearing loss.

In this manuscript we describe, for the first time, RNA-Sequencing of the cochlea, ampulla, saccule and utricle of the adult human inner ear derived from fresh surgically resected tissue from four adult donor patients with normal hearing. We compare it to the transcriptome of 32 other human tissues, and focus on classes of RNAs potentially relevant to human hearing, such as specific mRNA isoforms and long non-coding RNAs. These data provide a rich, freely-accessible resource for understanding the inner ear and may prove useful as a resource for prioritization of candidate genes for the study of hearing loss.