Proceedings of the Pakistan Academy of Sciences: Pakistan Academy of Sciences B. Life and Environmental Sciences 57 (2): 15-26 (2020) Copyright © Pakistan Academy of Sciences ISSN: 2518-4261 (print), ISSN 2518-427X (online) Research Article

An Overview of Pathways Network Analysis of Pendred Syndromic

Mirza J. Hasnain1, Muhammad U. Z. Khan2, Khizra Maqsood1, Tahera Aslam1, Masroor E. Babar3, Shunli Yang2, Huma Sohail1, Muhammad T. Pervez1*, and Jianping Cai2

1Department of Bioinformatics & Computational Biology, Virtual University of Pakistan, Pakistan 2State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, 730046, PR, China 3Department of Genetics, Virtual University of Pakistan, Pakistan

Abstract: Pendred syndrome, mainly labeled as sensorineural hearing loss and goiter, is an autosomal recessive disorder that forms an association to worsen the disease severity. It is the most known and prevalent form of the audiological disease. Some studies showed that the disorder may account for almost 10% of inherited deafness. Like all other disorders, a set of genes are known to characterize the Pendred Syndrome, where each one of them is segregated with the disease according to the families they belong to. The proposed study focusing on exploring the extent of segregation of each that is playing a role in the disorder. SLC26, a family of ion transporters, is known to play a significant role in this accord. The mutations in the genes mainly SLC26A4 underlie the major anomalies and malfunctioning that characterize the Pendred Syndrome. The biological pathway analysis using different online tools and databases like Ensemble decision analysis, DAVID (Database for Annotation, Visualization, and Integrated Discovery), IPA (Ingenuity Pathway Analysis software ) was performed to see Cellular Enrichment Components and Biological Enrichment Processes followed by gene network analysis, which determines the candidate gene interactions including the ion transporter family genes that are present on the plasma membrane, out of which two genes belonged to the focused protein family SLC26. The proposed study has highlighted the central role of the SLC26 protein family in hearing impairment and hearing loss.

Keywords: Pendred syndrome, Hearing impairment, PDS gene, SLC26A4, Gene network, Ingenuity Pathway Analysis software (IPA).

1. INTRODUCTION and its pathogenesis involves dozens of genes. The approach of genetic diagnosis of Hearing Loss is The inner ear of vertebrates acts as a sensory of incredible significance for patients with hearing organ with complex sensitivity along with the anomalies and plays a key role in evaluating the structure [1]. The recurring utilization of pathways danger of recurring in families [5]. HL influences of common signaling along with the roles for one in 500–1000 newborn babies [6] and around transmembrane is one of the noteworthy half of these cases have a hidden hereditary reason mechanisms that is involved in the development for their abnormality. More than 400 hereditary and arrangement of the inner ear that arises from disorders are associated with HL, and practically the otic placode [2-4]. Hearing loss (HL) is the most 80% of disorders linked to familial Hearing Loss occurring and well-known sensorineural disorder are Non-Syndromic Hearing Loss (NSHL) [7, 8].

———————————————— Received: February 2020; Accepted: June 2020 *Corresponding Author: Muhammad T. Pervez 16 Mirza J. Hasnain et al

Despite huge advancements in the identification of The proposed study aimed to investigate the deafness-related genes, the hereditary reasons for biological pathways that help in the analysis of acquired NSHL regularly stay indistinct because the trend of PDS related genes in human deafness of extraordinary ethnicity-explicit variety and by looking into the behavior of candidate genes in constrained phenotypic variance [9, 10]. disease states and the effects of drugs by constructing their network. Various bioinformatics tools and In the current era, the Next-Generation databases were used to investigate the interactions Sequencing (NGS) technologies have empowered among candidate genes in human deafness and the specialists to recognize the obscure damaging sensory perception of sound across multiple variations among the noteworthy count of biological aspects including disease processes, Hearing Loss (HL) patients [11]. The merging molecular interactions, and cellular processes. of initial linkage analysis and NGS techniques The biological behavior of most common protein for narrowing down the chromosomal regions families that are involved in sensory perception of related to variations has proved to be a new sound and highlighted several genes as potential strategy for examining deafness-related genes targets of genetic hearing loss for diagnosis and [12]. Genome-wide association studies (GWAS) treatment purposes was also explored. measuring a huge number of Single Nucleotide Polymorphisms (SNPs) from thousands of subjects 2. MATERIALS AND METHODS have massively recognized several relationships of genetic variations underlying 80 or more diseases 2.1 Gene Mining associated with Hearing approximately [13]. Impairment

Pendred Syndrome (PDS), the extensively Gene mining is a supervised learning process for recognized reason for syndromic deafness is a blend the identification of contributed genes in a specific of cochlear formative abnormalities, sensorineural biological pathway. Gene mining can also be hearing misfortune, vestibular dysfunction, and employed to figure out the gene associated with any goiter representing 10% of all reported genetic syndrome. To acquire the data of genes allied with hearing loss [14]. Mutations in the SLC26 gene Hearing Impairment (HI), an exhaustive literature family root both DFNB4 and Pendred disorder, survey was performed to find out the genes which are two autosomal recessive disorders, share exclusively associated with Pendred Syndrome. hearing misfortune as an emblematic characteristic Only (PDSO) using the analytical method based on [15]. The PDS gene (SLC26A4) which encodes Pearson`s correlation matrix by ensemble decision Pendrin protein is positioned on chromosomal analysis of all 80 genes. region 7q31 and was recognized by Bioinformatics positional cloning in 1997 [16]. More than 50 2.1.1 Cellular Enrichment Components and autonomous gene variations have been considered Biological Enrichment Process for instigating PDS and non-syndromic deafness, which was re-affirmed by a discharge test The behavior of the candidate genes in biological featuring perchlorate. PDS mutations are dispersed pathways and the labeling of the associated genes throughout the coding region, being recognized in were predicted by analyzing the , 18 of the 21 exons. Most transformations insertions, cellular enrichment components, and biological deletions, missense, splice site, and frameshift enrichment process of the PDSO gene using DAVID mutations [17, 18]. The infrequent permutation [20], a Database for Annotation, Visualization of goiter and deafness experienced in Pendred and Integrated Discovery database (https://david. disorder brings up some intriguing issues that how ncifcrf.gov/ ). flaws with regards to a similar protein (Pendrin) prime to the impacts that are tissue-explicit [17]. 2.1.2 Gene Network To pick up the answers to these intriguingly arising questions, the expression of PDS and localization The output of gene ontology predicting the biological of Pendrin at subcellular locations in several tissues pathways is the broad range of information that is were examined [19]. necessary to be narrowed down to find out the larger Pathways Network Analysis of Pendred Syndromic Genes 17 gene families that are responsible for the hearing network were referred to as a central node. The impairment, especially in Pendred syndrome. For network of each group was studied to estimate this purpose, the confidence score of <=0.05 was the likelihood that all genes of a group fit into the used for the prediction of the interactions among same network. Statistical analysis of network and associated genes for top-ranked protein family pathways was performed in IPA using the right- based on the well-characterized cell signaling tailed Fisher`s exact test to filter pathways by using and metabolic pathways including molecular the cut off value of (p<10-27) to find the whole drug- and biochemical studies, a network analysis was able statistical background. performed using Ingenuity Pathway Analysis software (IPA; Ingenuity Systems, Redwood City, 3. RESULTS CA, USA) [21], software connected to world`s largest knowledge-based biological network Based on an exhaustive literature review, we system. The most interconnectedPathways Pathwaysmolecules Network Network Analysisin a Analysis offind Pendred out of Pendred80 Syndromic genes Syndromic and Genes after Genesapplying the filtration

Table 1. CompleteTable 1 .information Complete informationabout Pendred about syndrome Pendred-associated syndrome genes-associated and their genes phenotypic and their characteristics. phenotypic characteristics. The fifth column shows relevantThe fifth literature. column shows relevant literature.

HearingHearing Impairment Impairment in in Pendred Pendred Syndrome Syndrome GenesGenes ChromosomeChromosome SyndromesSyndromes PhenotypicalPhenotypical Effect ReferencesReferences GJB3GJB3 1 1 VestibularVestibular aqueductaqueduct syndromesyndrome HearingHearing loss loss [34] [34] (EVAS),(EVAS), PendredPendred syndromesyndrome (PDS)(PDS) PJVKPJVK 2 2 NonsyndromicNonsyndromic hearinghearing loss,loss, PendredHearing Hearing loss loss [35] [35] syndrome.Pendred syndrome. GRXCR1GRXCR1 4 4 UsherUsher syndrome,syndrome, PendredPendred syndrome,Otoacoustic Otoacoustic loss loss(OAE (OAE loss), [36] [36] syndrome, Hearingloss), loss Hearing loss MARVELD2MARVELD2 5 AlportAlport andand HearingHearing loss, loss, Sensorineural [37] [37] CHARGECHARGE syndromes,syndromes, hearingSensorineural loss (SNHL) hearing WaardenburgWaardenburg syndrome,syndrome, phenotypeloss (SNHL) phenotype PendredPendred syndrome,syndrome, typetype 2 2 BaraitserBaraitser--WinterWinter syndrome DIAPH1 5 Pendredsyndrome syndrome, Hearing Loss Hearing Loss age of [38] DIAPH1 (HL)Pendred syndromes syndrome, Hearing Hearingonset, Loss severity, age of onset, [38] Loss (HL) syndromes severity,audiogram audiogram shape shape FOXI1FOXI1 PendredPendred syndromesyndrome PendredPendred syndrome syndrome (PDS) [39] [39] phenotype, deafness (PDS) phenotype, phenotype deafness phenotype GRXCR2 Waardenburg syndrome, Hearing loss (HL), hearing [40] GRXCR2 Waardenburg syndrome, auditory- Hearing loss (HL), [40] auditory-pigmentary syndrome, and speech disorders pigmentary syndrome, hearing and speech multiple neoplasia syndrome, multiple neoplasia syndrome, disorders Stickler syndrome, Stickler syndrome, Usher syndrome, Pendred Usher syndrome, Pendred syndrome, syndrome,Jervell and Lange-Nielsen Jervellsyndrome and Lange-Nielsen syndrome EYA4EYA4 6 WaardenburgWaardenburg syndrome,syndrome, deafness congenitalcongenital Hearing Hearing Loss [41] [41] 6 syndromedeafness syndrome (EVA Syndrome), (EVA (HL),Loss (HL), UsherSyndrome), and Pendred/Enlarged pigmentationpigmentation anom anomaliesalies in vestibular Usher and aqueduct Pendred/Enlarged syndrome eyes,in skin, eyes, and skin, hair and hair vestibular aqueduct syndrome DFNA5DFNA5 7 MuckleMuckle--WellsWells syndrome,syndrome, Pendred audiometricaudiometric phenotype, phenotype, [42] [42] syndrome,Pendred syndrome, Usher syndrome, Usher (unilateral(unilateral Hearing Hearing Waardenburgsyndrome, syndrome Impairment)Impairment) SLC26A3 7 Bartter'sWaardenburg syndrome, syndrome Pendred Deafness [43] SLC26A3 SyndromeBartter's syndrome, (PDS) Pendred Deafness [43] SLC26A4 PendredSyndrome syndrome (PDS) (PDS), Hereditary deafness [44] SLC26A5SLC26A4 PendredPendred syndromesyndrome (PDS),(PDS), Hereditaryhearing deafness loss [45] [44] SLC26A5 nonsyndromicPendred syndrome recessive (PDS), hearing hearing loss [45] impairmentnonsyndromic recessive hearing LRTOMT 11 Pendredimpairment syndrome, Clouston Age at onset, [46] LRTOMT syndrome,Pendred syndrome, usher syndrome. Clouston AgePenetrance, at onset, Penetrance, [46] syndrome, usher syndrome. ExpressivityExpressivity key keyfeatures, features, Audiological features.Audiological features. RDX RDX 11 clinicallyclinically distinctdistinct AgeAge at onset, at onset, Penetrance, [47] [47] syndromes,syndromes, UsherUsher syndrome,syndrome, ExpressivityPenetrance, PendredPendred syndromesyndrome key Expressivityfeatures, Audiological CloustonClouston syndromesyndrome features,key features, TECTA Usher syndrome, Pendred deafness,Audiological hearing lossfeatures, [7] TECTA Ushersyndrome syndrome, and Pendred syndromephenotype deafness, hearing loss [7] andJer vell and Lange-Nielsen phenotype Jervellsyndrome, and LangeWolfram-Nielsen syndrome, syndrome, Wolfram syndrome, Keratitis-Ichthyosis-Deafness (KID) 5 5

Pathways Network Analysis of Pendred Syndromic Genes

Table 1. Complete information about Pendred syndrome-associated genes and their phenotypic characteristics. The fifth column shows relevant literature.

Hearing Impairment in Pendred Syndrome Genes Syndromes Phenotypical Effect References GJB3 1 Vestibular aqueduct syndrome Hearing loss [34] (EVAS), Pendred syndrome (PDS) PJVK 2 Nonsyndromic hearing loss, Pendred Hearing loss [35] syndrome. GRXCR1 4 Usher syndrome, Pendred syndrome, Otoacoustic loss (OAE [36] loss), Hearing loss MARVELD2 5 Alport and Hearing loss, [37] CHARGE syndromes, Sensorineural hearing Waardenburg syndrome, loss (SNHL) phenotype Pendred syndrome, type 2 Baraitser-Winter syndrome DIAPH1 Pendred syndrome, Hearing Loss Hearing Loss age of [38] (HL) syndromes onset, severity, audiogram shape FOXI1 Pendred syndrome Pendred syndrome [39] (PDS) phenotype, deafness phenotype GRXCR2 Waardenburg syndrome, auditory- Hearing loss (HL), [40] pigmentary syndrome, hearing and speech multiple neoplasia syndrome, disorders Stickler syndrome, Usher syndrome, Pendred syndrome, Jervell and Lange-Nielsen syndrome EYA4 6 Waardenburg syndrome, deafness congenital Hearing [41] syndromePathways (EVA Network Syndrome), Analysis of PendredLoss Syndromic(HL), Genes Usher and Pendred/Enlarged pigmentation anomalies vestibular aqueduct syndrome in eyes, skin, and hair 18 Table 1. Complete information aboutMirza Pendred J. Hasnain syndrome et al -associated genes and their phenotypic characteristics. DFNA5 7 The fifth columnMuckle shows-Wells relevant syndrome, literature. Pendred audiometric phenotype, [42] syndrome, Usher syndrome, (unilateral Hearing HearingWaardenburg Impairment syndrome in Pendred SyndromeImpairment) SLC26A3 Bartter's syndrome, Pendred Deafness [43] Genes Chromosome Syndromes Phenotypical Effect References Syndrome (PDS) SLC26A4GJB3 1 PendredVestibular syndrome aqueduct (PDS), syndrome HereditaryHearing loss deafness [44][34] (EVAS), SLC26A5 Pendred syndrome (PDS), hearing loss [45] Pendred syndrome (PDS) nonsyndromic recessive hearing PJVK 2 Nonsyndromic hearing loss, Pendred Hearing loss [35] impairment syndrome. LRTOMT 11 Pendred syndrome, Clouston Age at onset, [46] GRXCR1 4 Usher syndrome, Pendred syndrome, Otoacoustic loss (OAE [36] syndrome, usher syndrome. Penetrance, loss), Hearing loss Expressivity MARVELD2 5 Alport and Hearing loss, [37] key features, CHARGE syndromes, Sensorineural hearing Audiological features. Waardenburg syndrome, loss (SNHL) phenotype RDX clinically distinct Age at onset, [47] Pendred syndrome, syndromes, Usher syndrome, Penetrance, type 2 Baraitser-Winter syndrome Pendred syndrome Expressivity DIAPH1 Pendred syndrome, Hearing Loss Hearing Loss age of [38] Clouston syndrome key features, (HL) syndromes onset, severity, Audiological features, audiogram shape TECTA Usher syndrome, Pendred syndrome deafness, hearing loss [7] FOXI1 Pendred syndrome Pendred syndrome [39] and phenotype (PDS) phenotype, JervellPathways and Lange Network-Nielsen Analysis of Pendred Syndromic Genes deafness phenotype syndrome, Wolfram syndrome, GRXCR2 Waardenburg syndrome, auditory- Hearing loss (HL), [40] Keratitis-Ichthyosis-Deafness (KID) pigmentarysyndrome syndrome, hearing and speech multiple neoplasia syndrome, disorders P2RX2 12 Waardenburg syndrome, unquestionable [47] Stickler syndrome, 5 auditory-pigmentary syndrome, phenotypic Usher syndrome, Pendred syndrome,multiple neo plasia syndrome, manifestations, JervellSTL syndrome, and Lange -Nielsen syndrome deafness, EYA4 6 WaardenburgUsher syndrome, syndrome, deafness Hearingcongenital Loss, Hearing [41] syndrome (EVA Syndrome), hearingLoss (HL), impairment Pendred syndrome (PDS) Usher and Pendred/Enlarged pigmentation anomalies STRC 15 auditory-pigmentary syndrome, hearing loss, [48] vestibular aqueduct syndrome in eyes, skin, and hair multiple neoplasia syndrome, malformations of the

Stickler syndrome, Pendred external ear DFNA5 7 Muckle-Wells syndrome, Pendred audiometric phenotype, [42] syndrome (PDS), progressive deafness syndrome, Usher syndrome, (unilateral Hearing Jervell and Lange-Nielsen Waardenburg syndrome Impairment) syndrome, Alport syndrome, SLC26A3 Bartter's syndrome, Pendred Deafness [43] Mohr-Tranebjaerg syndrome CRYM 6 SyndromePendred syndrome (PDS) (PDS), Age at onset, [49] SLC26A4 Pendrednonsyndromic syndrome hearing (PDS), loss, Clouston Penetrance,Hereditary deafness [44] SLC26A5 Pendredsyndrome syndrome (PDS), Expressivityhearing loss key [45] nonsyndromic recessive hearing features, Audiological impairment features, LRTOMT 11 Pendred syndrome, Clouston SyndromicAge at onset, features, [46] syndrome, usher syndrome. hearingPenetrance, loss. DeafnessExpressivity TBC1D24 Usher syndrome, Pendred syndrome prelingualkey features, profound [50] (PDS) hearingAudiological loss features. RDXLOXHD1 18 clinicallyusher syndrome distinct type 1B (USH1B) retinalAge at phenotypes, onset, [51][47] syndromes,and USH3, UsherUsher syndromesyndrome, 1D Deafness,Penetrance, hearing loss Pendred (USH1D), syndrome Oto-Spondylo -Mega- Expressivity CloustonEpiphyseal syndrome Dysplasia (OSMED) key features, syndrome, syndrome type If Audiological features, TECTA Usher(USH1F), syndrome, Pendred syndrome deafness, hearing loss [7] andPendred syndrome (PDS), Smith- phenotype JervellMagenis and syndrome Lange-Nielsen GIPC3 19 syndrome,Usher syndrome, Wolfram Pendred syndrome, syndrome prelingual profound [52] Keratitis(PDS) -Ichthyosis-Deafness (KID) hearing loss

3.1. Cellular Enrichment Components 5 analysis shown in Table 2, the whole genome and Biological Enrichment Process statistical background was assumed. A group of a maximum of 15 genes with Pathways ID GO.0007605 was observed to be involved in David database was used to assess the signal the sensory perception of sound with an FDR transmission in the cell. The results (False Discovery Rate) value of 2.59x10-25. It highlighted the cellular enrichment was also predicted that the SLC26 family components including the pathway description genes were a member of each pathway of each cluster of genes. The Pendrin involved in the overall biological enrichment SLC264A gene was found in the largest group processes in Gene Ontology (Table 3). IPA of 9 genes with Pathways ID GO.0042995 database supported the same results as involved in the cell projection, a type of predicted from the DAVID Database, shown cellular protrusion. For the enrichment in Fig 1.

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Pathways Network Analysis of Pendred Syndromic Genes 19 through ensemble decision analysis the number the cellular enrichment components including the reduced to 21, all having the association with pathway description of each cluster of genes. The Pendred Syndrome with hearing loss [22]. Table 1 Pendrin SLC264A gene was found in the largest depicts the overall chromosome wise distribution group of 9 genes with Pathways ID GO.0042995 of the 21 genes associated with the syndrome involved in the cell projection, a type of cellular and the phenotypic effect caused by a mutation protrusion. For the enrichment analysis shown in in each gene. The results showed that Chr. Table 2, the whole genome statistical background 1,2,4,6,12,15,18,19 have only one PDS associated was assumed. A group of a maximum of 15 genes gene, Chr.16 and 11 have 2 and 3 PDS associated with Pathways ID GO.0007605 was observed to genes respectively and 5 and 7 have be involved in the sensory perception of sound 4 genes. It was also observed that some genes were with an FDR (False Discovery Rate) value of responsible not only for hearing loss but also for the 2.59x10-25. It was also predicted that the SLC26 nonfunctioning of the audiological features. These family genes were a member of each pathway genes were DIAPH1, LRTOMT, RDX, P2RX2, involved in the overall biological enrichment STRC, and CRYM. Some genes like GRXCR2 processes in Gene Ontology (Table 3). IPA database and EYA4 showed different behavior, for example, supported the same results as predicted from the it was noted that due to the malfunctioning of the DAVID Database, shown in Fig 1. GRXCR2 gene hearing and speech disorders came into account while a mutation in EYA4 may cause 3.2. Gene Network pigmentation anomalies in the eye, skin, and hair. Network analysis of genes including audiological Pathways Network Analysis of Pendred Syndromic Genes 3.1 Cellular Enrichment Components and features such as hearing and speech disorder, Biological Enrichment Process malformation of the external ear, and pigmentation anomalies in eye, skin, and hair was studied David database was used toPathways assess Network the signal Analysis ofwhich Pendred revealed Syndromic 3 Genesnetworks. The network with the transmission in the cell. The results highlighted maximum number of 35 molecules with 10 focused

Fig 1. FigGene 1. GeneOntology Ontology behavior: behavior: disease disease-associated-associated biological biological expressionalexpressional analysis analysis keeping keeping the wholethe whole genome genome as a statisticalas a statistical background background for forthe theidentification identification of of Pendred Pendred syndrome: Hearing Hearing Loss Loss (HL). (HL).

Table Table2. Gene 2. Gene Ontology Ontology behavior: behavior: cellular cellular enrichment enrichment analysis keepingkeeping the the whole whole genome genome as a astatisticals a statistical backgroundbackground (DAVID (DAVID Database) Database)

Cellular Enrichment Components (GO) Cellular Enrichment Components (GO) Pathway ID Pathway Observed False discovery Matching proteins clusters (labels) Pathway ID Pathwaydescription Observedgene count False ratediscovery Matching proteins clusters (labels) GO.0032420 descriptionstereo gene count5 9.95Erate-08 GRXCR1, GRXCR2, LOXHD1, GO.0032420 stereo cilium 5 9.95E-08 GRXCR1,RDX, GRXCR2, STRC LOXHD1, GO.0032421 stereo cilium 5 2.74E-07 GRXCR1, GRXCR2,RDX, STRC LOXHD1, GO.0032421 stereo ciliumbundle 5 2.74E-07 GRXCR1,RDX, GRXCR2, STRC LOXHD1, bundle RDX, STRC

GO.0005902 5 6.93E-06 GRXCR1, GRXCR2, LOXHD1, RDX, STRC GO.0005902GO.0042995 microvilluscell 5 9 6.93E0.00238-06 DIAPH1,GRXCR1, GRXCR1, GRXCR2, GRXCR2, LOXHD1, projection LOXHD1,RDX, P2RX2, STRC RDX, GO.0042995 cell 9 0.00238 SLC26A4,DIAPH1, STRC, GRXCR1, TBC1D24 GRXCR2, GO.0060091 projectionKino cilium 2 0.00445 LOXHD1,GRXCR1, P2RX2,STRC RDX, SLC26A4, STRC, TBC1D24 GO.0060091 Kino cilium 2 0.00445 GRXCR1, STRC

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Pathways Network Analysis of Pendred Syndromic Genes Pathways Network Analysis of Pendred Syndromic Genes

20 Mirza J. Hasnain et al Fig 1. Gene Ontology behavior: disease-associated biological expressional analysis keeping the whole genome asFig a 1.statistical Gene Ontology background behavior: for the d iseaseidentification-associated of Pendred biological syndrome: expressional Hearing analysis Loss keeping (HL). the whole genome as a statistical background for the identification of Pendred syndrome: Hearing Loss (HL). Table 2. Gene Ontology behavior: cellular enrichment analysis keeping the whole genome as a statistical backgroundTable 2. Gene (DAVID Ontology Database) behavior: cellular enrichment analysis keeping the whole genome as a statistical background (DAVID Database)

Cellular Enrichment Components (GO) Cellular Enrichment Components (GO) Pathway ID Pathway Observed False discovery Matching proteins clusters (labels) Pathway ID descriptionPathway geneObserved count False ratediscovery Matching proteins clusters (labels) GO.0032420 stereodescription cilium gene 5count 9.95Erate-08 GRXCR1, GRXCR2, LOXHD1, GO.0032420 stereo cilium 5 9.95E-08 GRXCR1,RDX, GRXCR2, STRC LOXHD1, GO.0032421 stereo cilium 5 2.74E-07 GRXCR1,RDX, GRXCR2, STRC LOXHD1, GO.0032421 stereobundle cilium 5 2.74E-07 GRXCR1,RDX, GRXCR2, STRC LOXHD1, bundle RDX, STRC

GO.0005902 microvillus 5 6.93E-06 GRXCR1, GRXCR2, LOXHD1, GO.0005902 microvillus 5 6.93E-06 GRXCR1,RDX, GRXCR2, STRC LOXHD1, GO.0042995 cell 9 0.00238 DIAPH1,RDX, GRXCR1, STRC GRXCR2, cell 9 0.00238 DIAPH1, GRXCR1, GRXCR2, GO.0042995 projectionPathways Network Analysis of Pendred SyndromicLOXHD1, Genes P2RX2, RDX, projectionPathways Network Analysis of Pendred SyndromicSLC26A4,LOXHD1, Genes STRC, P2RX2, TBC1D24 RDX, GO.0060091 Kino cilium 2 0.00445 SLC26A4,GRXCR1, STRC, STRC TBC1D24 GO.0060091 Kino cilium 2 0.00445 GRXCR1, STRC

Table 3. Gene Ontology behavior: biological enrichment analysis keeping the whole genome as a statistical background forTable the 3.identification Gene Ontology of Pendred behavior: syndrome: biological Hearing enrichment Loss analysis(DAVID keeping Database) the whole genome as a statistical background for the identification of Pendred syndrome: Hearing Loss (DAVID Database)

Biological Enrichment Process (GO) Biological Enrichment Process (GO) Pathway ID Pathway Observed False discovery Matching proteins in your network

Pathway ID descriptionPathway geneObserved count False ratediscovery Matching proteins(labels) in your network GO.0007605 descriptionsensory gene15 count 2.59Erate-25 CRYM, DFNA5,(labels) DFNB59, DIAPH1, GO.0007605 perceptionsensory of 15 2.59E-25 CRYM,EYA4, GJB3,DFNA5, GRXCR1, DFNB59, LOXHD1, DIAPH1, perceptionsound of 7 EYA4,LRTOMT GJB3,, MARVELD2, GRXCR1, LOXHD1, P2RX2, 7 sound SLC26A4,LRTOMT SLC26A5,, MARVELD2, STRC, P2RX2, TECTA GO.0007600 sensory 14 6.62E-12 CRYM,SLC26A4, DFNA5, SLC26A5, DFNB59, STRC, DIAPH1, TECTA GJB3, GO.0007600 perceptionsensory 14 6.62E-12 CRYM, DFNA5,GRXCR1, DFNB59, LOXHD1, DIAPH1, GJB3, perception LRTOMT,GRXCR1, MARVELD2, LOXHD1, P2RX2, SLC26A4,LRTOMT, SLC26A5, MARVELD2, STRC, P2RX2, TECTA GO.0050877 neurological 13 6.62E-09 CRYM,SLC26A4, DFNA5, SLC26A5, DFNB59, STRC, DIAPH1, TECTA GJB3, GO.0050877 systemneurological process 13 6.62E-09 CRYM, DFNA5,GRXCR1, DFNB59, LOXHD1, DIAPH1, GJB3, system process LRTOMT,GRXCR1, MARVELD2, LOXHD1, SLC26A4, LRTOMT,SLC26A5, MARVELD2, STRC, TECTA SLC26A4, GO.0048839 inner ear 5 0.00135 DFNA5,SLC26A5, FOXI1, LRTOMT,STRC, TECTA SLC26A5, GO.0048839 developmentinner ear 5 0.00135 DFNA5, FOXI1,STRC LRTOMT, SLC26A5, GO.0043583 developmentear 5 0.00203 DFNA5, FOXI1,STRC LRTOMT, SLC26A5, GO.0043583 developmentear 5 0.00203 DFNA5, FOXI1,STRC LRTOMT, SLC26A5, GO.0042491 developmentauditory 3 0.00608 GRXCR1, LRTOMT,STRC STRC GO.0042491 receptorauditory cell 3 0.00608 GRXCR1, LRTOMT, STRC differentiationreceptor cell GO.0060119 differentiationinner ear 3 0.00856 GRXCR1, LRTOMT, STRC GO.0060119 receptorinner ear cell 3 0.00856 GRXCR1, LRTOMT, STRC developmentreceptor cell GO.0060113 developmentinner ear 3 0.0236 DFNA5, LRTOMT, STRC GO.0060113 receptorinner ear cell 3 0.0236 DFNA5, LRTOMT, STRC differentiationreceptor cell GO.0002064 differentiationepithelial cell 4 0.031 LRTOMT, MARVELD2, RDX, STRC GO.0002064 epitdevelopmenthelial cell 4 0.031 LRTOMT, MARVELD2, RDX, STRC development 3.2. Gene Network mole cules (one was with 3 molecules and one 3.2. Gene Network wasmole havingcules (one 2 molecules) was with with 3 molecules one focused and gene one Network analysis of genes including inwas each having network. 2 molecules) All threewith onenetworks focused weregene audiologicalNetwork analysis features ofsuch genesas hearing including and involvedin each innetwork. auditory All disease three on networks top, hereditary were speechaudiological disorder, features malformation such as of hearingthe external and disorderinvolved inon auditory the diseasesecond on numbertop, hereditary, and earspeech, and disorder, pigmentation malformation anomalies of inthe eye, external skin, neurologicaldisorder on disease the onsecond number number three. ,As andour andear, andhai rpigmentation was studied anomalies which inrevealed eye, skin 3, fneurologicalocus was diseaseto determine on number only three. Pendred As our networksand hair. Twashe networkstudied withwhich the revealed maximum 3 syndromicfocus was genes to networkdetermine so theonly study Pendredfound a networksnumber of. 35The molecules network with with 10 thefocused maximum genes verysyndromic small networkgenes network with the so middle the study nodule found Ca 2+a 2+ wasnumber selected of 35 withmolecules a high with significance 10 focused score genes of verywith small16 edges network while with 14 the and middle 12 edgesnodule wereCa was28 (p selected = 10-27) (withFig 2a) .high The restsignificance of the 2 networks score of connectedwith 16 edges to whileELAVL1 14 andand 12cyclic edges AMPwere -27 were28 (p rejected= 10 ) (becauseFig 2). T ofhe their rest offewer the 2 networks respectively.connected to ELAVL1 and cyclic AMP were rejected because of their fewer respectively.

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Pathways Network Analysis of Pendred Syndromic Genes 21 genes was selected with a high significance score any cluster in the network which proved that these of 28 (p = 10-27) (Fig 2). The rest of the 2 networks genes are indirectly interacting with each other in were rejected because of their fewer molecules Pendred Syndrome. This network indicates many (one was with 3 molecules and one was having 2 other genes playing direct and indirect roles in molecules) with one focused gene in each network. Pendred syndrome which may also be considered All three networks were involved in auditory as candidate genes for auditory disease treatments. disease on top, hereditary disorder on the second Aiming to determine the role of ion transporters in number, and neurological disease on number underlying causes of Pendrin protein found that the three. As our focus was to determine only Pendred SLC26A4 and SLC26A5 protein playing a vital syndromic genes network so the study found a very role in different transport mechanisms such as the small network with the middle nodule Ca2+ with iodide transport mechanism where the thyrocyte 16 edges while 14 and 12 edges were connected to uptakes NA+/I- cotransporter on the basolateral ELAVL1 and cyclic AMP respectively. Detailed membrane where Pendrin (SLC26A4) permits information revealed that DIAPH1, EJB3, iodide ions within the thyrocyte to pass through LRTOMT, MARVELD2, P2RX2, SLC26A4, the apical membrane and into the colloid space SLC2645 were located on the plasma membrane where it quickly binds to Tg by a catalyzed reaction while EYA4, DFNA5 were located on cytoplasm regulated by thyroid peroxidases (TPO) [12]. and GRXCR1, LOXHD1, STRC, AND TECTA were located in extracellular space as shown in Fig. IPA network analysis tool also provided 3. IPA databases also revealed that among the genes information about upstream regulators involved in located on the plasma membrane, GJB3, SLC26A4, biological pathways interacting with our candidate SLC26A5, and RX2 were ion transporters. Also, genes. Table 4 shows Pendrin protein (SLC26A4) found that none of our candidatePathways Networkgenes Analysisformed of Pendredas a vitalSyndromic protein Genes having interactions with most

FigFig 2. 2. The mostmost significant significant gene gene network network produced produced by IPA networkby IPA analysis:network Three analysis: types ofThree nodes types are indicated of nodes are here: central nodes with maroon color, candidate genes with dark blue color, and SLC17A9 gene is highlighted indicatedwith green here: color central as it belongs nodes to with the SLCmaroon family color, and itcandidate is not from genes the candidate with dark gene blue list. color, and SLC17A9 gene is highlighted with green color as it belongs to the SLC family and it is not from the candidate gene list. Detailed information revealed that DIAPH1, transporters. Also, found that none of our EJB3, LRTOMT, MARVELD2, P2RX2, candidate genes formed any cluster in the SLC26A4, SLC2645 were located on the network which proved that these genes are plasma membrane while EYA4, DFNA5 were indirectly interacting with each other in located on cytoplasm and GRXCR1, LOXHD1, Pendred Syndrome. This network indicates STRC, AND TECTA were located in many other genes playing direct and indirect extracellular space as shown in Fig. 3. IPA roles in Pendred syndrome which may also be databases also revealed that among the genes considered as candidate genes for auditory located on the plasma membrane, GJB3, disease treatments. SLC26A4, SLC26A5, and RX2 were ion

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22 Pathways Network AnalysisMirza ofJ. PendredHasnain Syndromicet al Genes

Pathways Network Analysis of Pendred Syndromic Genes

4.15*10-3) found that only Pendred protein was acetazolamide. GUCA2B and FOXI1 are the interacting with GUCA2B, chloride, genes, mineralocorticoid and acetazolamide are

mineralocorticoid, FOXII, iodide, and chemicals and chlorides are iodides are ions. The candidate genes are categorized based upon their working in cellular pathways including ion transportation, Fig Fig.3. The3. candidate genes are categorized based upon their working in cellular pathways including ion transportation, catalytic activities, and their locations in extracellular space, plasma membrane, cytoplasm, and nucleus.catalytic activities, and their locations in extracellular space, plasma membrane, cytoplasm, and nucleus.

Table 4: The most common Pendred syndromic upstream regulator interacting with candidate genes. AimingTable to determine 4. The most th commone role of Pendredion transporters syndromic upstreamquickly regulator binds interacting to Tg withby candidatea catalyzed genes. reaction in underlying causes of Pendrin protein found regulated by thyroid peroxidases (TPO) [12]. that the SLC26A4 and SLC26A5 protein p-value playing a vital role in different transport IPA network analysis toolTarget also moleculesprovided in mechanismsUpstream such Regulator as the iodide transportMolecule Typeinformation about upstreamof regulators involved + the dataset mechanism where the thyrocyte uptakes NA /I- in biological pathwaysoverlap interacting with our cotransporter on the basolateral membrane candidate genes. Table 4 shows Pendrin protein where PendrinGUCA2B (SLC26A4) permits iodideOther ions (SLC26A4) as a 0.000692vital proteinSLC26A4 having within the thyrocyte to pass through the apical interactions with most of the chemical drugs, membraneChloride and into the colloid space wherechemical it - endogenoustranscription mammalian regulators0.00207, andSLC26A4 endogenous mammalians. By having a cut-off value (p = Mineralocorticoid chemical drug 0.00207 SLC26A4 10 FOXI1 transcription regulator 0.00346 SLC26A4 Iodide chemical - endogenous mammalian 0.00415 SLC26A4 Acetazolamide chemical drug 0.00415 SLC26A4 LASP1 Transporter 0.00552 MARVELD2 Hormone chemical drug 0.00896 SLC26A4 miR-9-5p (and other mature microRNA 0.0124 DIAPH1 miRNAs w/seed CUUUGGU) NOX1 Enzyme 0.0124 DIAPH1 ADCY Group 0.0151 CRYM Sucrose chemical - endogenous mammalian 0.0158 CRYM deoxycorticosterone acetate chemical drug 0.0165 SLC26A4 BTG2 transcription regulator 0.0172 DIAPH1 TG Other 0.0212 SLC26A4 miR-200b-3p (and other mature microRNA 0.0287 GJB3 miRNAs w/seed AAUACUG) N-formyl-Met-Leu-Phe chemical reagent 0.03 DIAPH1 Methimazole chemical drug 0.0347 SLC26A5 ESR2 ligand-dependent nuclear receptor 0.0349 EYA4, GJB3 miR-141-3p (and other mature microRNA 0.048 GJB3 miRNAs w/seed AACACUG) PAX7 transcription regulator 0.0494 EYA4

4. DISCUSSION than 1 gene associated with PDS. Genes namely 5. DIAPH1, LRTOMT, RDX, P2RX2, STRC, and In this study, expression of Pendred syndrome CRYM not only cause loss of hearing but also and subcellular localization of Pendrin in non-functioning of audiological features [24]. different tissues and their biological pathways were analyzed. The networks and pathways Gene enrichment and pathway analysis by helped us in understanding the role of candidate DAVID showed a somewhat bigger picture of genes in human deafness and the effect of drugs genes interacting with each other. In cellular upon them [23]. After Gene mining and enrichment analysis, a total of five pathways Ensemble decision analysis, 21 out of 80 genes were identified. The largest pathway was ‘cell were selected and gene enrichment analysis projection’ which consisted of 9 genes was performed through DAVID [20]. IPA including the SLC26A4 gene with 0.00238 software was used to predict interactions FDR value. Other pathways were stereo cilium, among the associated genes. stereo cilium bundle, microvillus, and Kino cilium. Biological enrichment analysis showed Among the shortlisted 21 genes, the SLC26 15 candidate genes involved in sensory gene family was most significant having more perception of the sound pathway with an FDR

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Pathways Network Analysis of Pendred Syndromic Genes 23 of the chemical drugs, transcription regulators, 14, and 16 nodes respectively. Plasma membrane and endogenous mammalians. By having a and cytoplasm genes were identified, and it was cut-off value (p = 4.15*10-3) found that only reported that some genes present on the plasma Pendred protein was interacting with GUCA2B, membrane were ion transporters. These genes are chloride, mineralocorticoid, FOXII, iodide, and GJB3, SLC26A4, SLC26A5, and RX2. SLC26A4 acetazolamide. GUCA2B and FOXI1 are the genes, is the most vital gene in PDS involved in several mineralocorticoid and acetazolamide are chemicals important ion transport mechanisms[26] like the and chlorides are iodides are ions. iodide transport mechanism producing thyroid peroxidases [27]. SLC26A4 also interacts with 4. DISCUSSION several molecules and genes including GUCA2B, chloride, mineralocorticoid, FOXII, iodide, and In this study, expression of Pendred syndrome and acetazolamide that directly initiate PDS [28]. subcellular localization of Pendrin in different tissues FOXI1 gene is a transcriptional activator that and their biological pathways were analyzed. The is responsible for the sense of balance in the networks and pathways helped us in understanding development of normal hearing [29]. It plays a vital the role of candidate genes in human deafness and role in the expression of SLC26A4, JAG1, and the effect of drugs upon them [23]. After Gene COCH genes which contribute to the development mining and Ensemble decision analysis, 21 out of the endolymphatic system in the inner ear and of 80 genes were selected and gene enrichment also help in the differentiation of intercalated cells analysis was performed through DAVID [20]. IPA [30] in the distal renal tubule`s by software was used to predict interactions among the supporting SLC4A1, SLC4A9, and ATP6V1B1 associated genes. Among the shortlisted 21 genes, genes [31]. GUCA2B gene encodes proteolytic the SLC26 gene family was most significant having preprotein [32] for producing multiple proteins such more than 1 gene associated with PDS. Genes as uroguanylin and guanylate cyclase-C receptor`s namely DIAPH1, LRTOMT, RDX, P2RX2, STRC, [33] endogenous ligands. This gene being the and CRYM not only cause loss of hearing but also transcriptional regulator stimulates SLC26A4 non-functioning of audiological features [24]. (PDS) indirectly.

Gene enrichment and pathway analysis by The proposed study has highlighted the DAVID showed a somewhat bigger picture of genes central role of the SLC26 protein family in interacting with each other. In cellular enrichment hearing impairment and hearing loss. The future analysis, a total of five pathways were identified. benefit associate with the proposed system is the The largest pathway was ‘cell projection’ which identification of genes, pathways, or networks that consisted of 9 genes including the SLC26A4 gene will be responsible for hearing impairment and with 0.00238 FDR value. Other pathways were hearing loss. It allows the pharmaceutical scientist stereo cilium, stereo cilium bundle, microvillus, and drug designers to target the respective genes and Kino cilium. Biological enrichment analysis or pathways to develop a candidate drug-using showed 15 candidate genes involved in sensory docking mechanism. perception of the sound pathway with an FDR value of 2.59x10-25. Other pathways identified during 5. CONCLUSIONS biological enrichment analysis were neurological system process, inner ear development, ear This study focused on the cellular enrichment, development, auditory receptor cell differentiation, network and pathway analysis of human Pandered inner ear receptor cell development, inner ear syndromic genes. By studying the ontology receptor cell differentiation, and epithelial cell behavior of genes, we constructed a network development [25]. to understand the role of these genes and their interactions with each other. Our analyses provided In IPA analysis 1 out of 3 gene networks with a platform to understand the role of the Pandered 35 molecules and 10 focused genes were selected. syndrome in different cellular pathways and a ELAVL1, cycAMP, and CA2+ being central nodes cutting-edge manifesto to treat and design new of the selected network were connected to 12, drugs not only for deafness but also for the inner 24 Mirza J. 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