Otology & Neurotology 34:961Y970 Ó 2013, Otology & Neurotology, Inc. A Comprehensive Network and Pathway Analysis of Human Deafness Genes *Georgios A. Stamatiou and †Konstantina M. Stankovic *Department of Otolaryngology, Hippokration General Hospital, University of Athens, Athens, Greece; and ÞDepartment of Otology and Laryngology, Harvard Medical School and Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, U.S.A. Objective: To perform comprehensive network and pathway factor beta1 (TGFB1) for Group 1, MAPK3/MAPK1 MAP kinase analyses of the genes known to cause genetic hearing loss. (ERK 1/2) and the G protein coupled receptors (GPCR) for Study Design: In silico analysis of deafness genes using inge- Group 2, and TGFB1 and hepatocyte nuclear factor 4 alpha (HNF4A) nuity pathway analysis (IPA). for Group 3. The nodal molecules included not only those known Methods: Genes relevant for hearing and deafness were iden- to be associated with deafness (GPCR), or with predisposition to tified through PubMed literature searches and the Hereditary otosclerosis (TGFB1), but also novel genes that have not been Hearing Loss Homepage. The genes were assembled into 3 groups: described in the cochlea (HNF4A) and signaling kinases (ERK 1/2). 63 genes that cause nonsyndromic deafness, 107 genes that cause Conclusion: A number of molecules that are likely to be key nonsyndromic or syndromic sensorineural deafness, and 112 genes mediators of genetic hearing loss were identified through three associated with otic capsule development and malformations. Each different network and pathway analyses. The molecules included group of genes was analyzed using IPA to discover the most new candidate genes for deafness. Therapies targeting these molecules interconnected, that is, ‘‘nodal’’ molecules, within the most statis- maybeusefultotreatdeafness.Key Words: Deafness genesVERK tically significant networks (p G 10j45). 1/2VGPCRVHNF4AVMAPK1VMolecular pathways analysisV Results: The number of networks that met our criterion for sig- TGFB1. nificance was 1 for Group 1 and 2 for Groups 2 and 3. Nodal molecules of these networks were as follows: transforming growth Otol Neurotol 34:961Y970, 2013. Hearing loss is the most common sensory deficit in the sensorineural and believed to account for 70% of cases world, affecting almost 600 million people (1), and the (4). The remaining 30% of hearing loss is syndromic most common congenital anomaly, affecting 2 to 6 per and can be sensorineural, conductive, or mixed. More 1,000 newborns (1,2). At least two-thirds of cases of than 400 syndromes include hearing loss as a part of prelingual deafness in the developed countries are due their phenotypic signature (3). Typically, genetic hear- to genetic factors; the remaining one-third is attributed ing loss is monogenic. Based on the mode of inheri- to environmental and unidentified genetic factors (3). tance, monogenic hearing loss is classified as autosomal In general, genetic hearing loss can be classified as recessive, accounting for 80% of cases; autosomal domi- syndromic or nonsyndromic, depending on whether other nant, accounting for almost 20% of cases; X-linked; and distinguishing physical features are present or absent, mitochondrial; the latter two account for less than 1% of respectively. Nonsyndromic hearing loss is typically cases (5). Hearing loss is one of the most genetically hetero- geneous disorders, with more than 100 mapped loci and Address correspondence and reprint requests to Konstantina Stankovic, more than 60 causally implicated genes within these loci M.D., Ph.D., Massachusetts Eye and Ear Infirmary, 243 Charles St, (6). Additional complexity exists because mutations in the Boston, MA 02114; E-mail: [email protected] Study conducted at: Department of Otolaryngology, Massachusetts same gene may cause syndromic or nonsyndromic hearing Eye and Ear Infirmary, Harvard Medical School, Boston, MA, USA. loss, and hearing loss may be oligogenic (7). Given the Source of Funding: NIDCD K08 DC010419 (K. M. S.) and the intricacy of normal hearing, which requires interaction of Bertarelli Foundation (K. M. S.). many diverse molecules, it is estimated that approximately The study was presented at the AOS Spring Meeting, April 21Y22, 2012, San Diego, CA. 1% of human genes play a role in hearing (8). The authors disclose no conflicts of interest. Although the shear number of genes involved in Supplemental digital content is available in the text. hearing and deafness may seem daunting, this complexity 961 Copyright © 2013 Otology & Neurotology, Inc. Unauthorized reproduction of this article is prohibited. 962 G. A. STAMATIOU AND K. M. STANKOVIC may be simplified by looking at well-characterized cell specified molecules. We refer to the most interconnected mol- signaling and metabolic pathways through which these ecule (or class of molecules) in a network as key or central node. genes are known to interact in various tissues. A common Networks of up to 140 molecules were studied to allow for the assumption of pathway analysis is that genes whose dys- possibility that all genes within a group belonged to the same function contributes to a disease phenotype tend to be network. Statistical analysis of networks and pathways was performed as part of the overall IPA using the right-tailed functionally related (9,10). Unraveling these pathways is Fisher’s exact test (11). The p value reflects the likelihood that essential to understanding biological mechanisms, disease the association between the input genes and a given pathway or states, and the function of drugs that affect them. We network is due to chance. Only networks with p G 10j45 were therefore undertook the first comprehensive pathway and considered significant. Less stringent criterion of p G 0.05 was network analysis of all known genes implicated in hu- used for pathway significance to facilitate discovery of man deafness. We used tools of bioinformatics to analyze potentially novel signaling pathways. interactions across multiple biological dimensions includ- Expression of select genes was validated by summarizing ing molecular interactions, cellular processes, and disease their reported expression in the mouse cochlear hair cells based processes. We focused on 3 groups of genes: those causing on RNASeq (12) or mouse spiral ganglion neurons based on nonsyndromic hearing loss, those causing nonsyndromic GeneChips (13). Both databases catalog changes in gene expres- sion during embryonic and early postnatal development. or syndromic sensorineural hearing loss, and those asso- ciated with malformation of the otic capsule. The third group of genes was augmented with genes implicated in RESULTS otic capsule development based on animal studies in mammals. Our analyses suggest new candidate genes for Network analysis of the nonsyndromic deafness deafness within the known deafness loci and highlight genes revealed 6 networks. The top network was very several genes as potential novel targets for diagnosis and highly significant (p =10j105) and included nearly treatment of genetic hearing loss. all (50 of 63) genes currently implicated in nonsyndro- mic HL, suggesting close coupling of deafness genes within the cacophony of human interactome. This network MATERIALS AND METHODS is shown in Figure S1, Supplemental Digital Content 2, A PubMed English search was performed to identify genes http://links.lww.com/MAO/A150, and all molecules of and molecules associated with human hearing loss and with the network are listed in Table S2, Supplemental Digital mammalian development and malformation of the otic capsule. Content 7, http://links.lww.com/MAO/A155. The remain- All genes listed on the Hereditary Hearing Loss Homepage en- ing 5 networks did not meet our criterion for significance tered our analyses. The pertinent genes were identified through (p =10j2). The central node of the top pathway was linkage analysis, immunohistochemistry, quantitative reverse transforming growth factor beta1 (TGFB1), having 35 con- transcription-polymerase chain reaction, or microarray ana- nections with other genes in the network (Fig. 1). The lyses. Only studies with relevant controls or appropriate sta- second and third central nodes were beta-estradiol and tistical analyses were included; studies without control groups or with unspecified statistical significance were excluded. filamentous actin (F-Actin), with 29 and 24 gene inter- Genes that met our criteria were classified based on their estab- connections, respectively (Table 1). Pathway analysis lished role in nonsyndromic or syndromic sensorineural hear- revealed 5 statistically significant pathways (Table 2), ing loss, or association with otic capsule development or highlighting the importance of actin cytoskeleton sig- malformation. The number of genes that entered our anal- naling, which is known to be critical for stereociliar and yses was 63 genes that cause nonsyndromic deafness, 107 genes hair cell function. The second ranked pathway was cell that cause nonsyndromic or syndromic sensorineural deaf- junction signaling (Table 2), consistent with the obser- ness, and 112 genes associated with otic capsule development vations that about 50% of cases of nonsyndromic HL are and malformations (Table S1, Supplemental Digital Content 1, due to mutations in GJB2 encoding a gap junction protein http://links.lww.com/MAO/A149, alphabetically lists genes used (14). The other top pathways point to previously under- in the