PATHWAY NETWORK ANALYSIS OF GENES INVOLVED IN …€¦ · Salma Hafeez 1, Tahira Aslam2, Mirza Jawad Ul Hasnain3 1, 2Student, Department of Biology, Virtual University of Pakistan,

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PATHWAY NETWORK ANALYSIS OF GENES INVOLVED IN

DOMINANT SYNDROMES

Salma Hafeez *1, Tahira Aslam *2, Mirza Jawad Ul Hasnain*3

*1, 2Student, Department of Biology, Virtual University of Pakistan, Lahore.

*3Lecturer, Department of Bioinformatics & Computational Biology, Virtual University of

Pakistan, Lahore.

ABSTRACT

The autosomal dominant hearing loss and deafness is one of the unique phenotypical effects that is

observed in multiple dominant syndromes. The hereditary sensorineural hearing loss & Diegenic non-

syndromic hearing impairment are the most familiar and frequent auditory diseases. These auditory

disorders are the main causes of autosomal dominant human deafness and hearing loss. There are a set of

genes that are associated with this sensorineural hearing loss and played a vital role in these disorders.

This research study exploring the set of genes that are closely associated with hearing impairment. The

FGFR and FGF protein family of cell signalling have a significant role in this respect. These proteins are

involved in several anomalies of ears at the embryonic level. The SALL1 protein, Connexin,

Microphthalmia-associated transcription factors, protein tyrosine phosphate and Homeobox protein SIX1

are closely related to syndromic and non -syndromic hearing loss. In this research study, we performed

the biological pathway analysis using online tools and databases such as STRING database, DAVID

(Database for Annotation, Visualization and Integrated Discovery), and IPA (Inguenity Pathway Analysis

software), to find out the Cellular Enrichment Components and Biological Enrichment Process followed

by gene network analysis to determine the interaction of the associated genes. This research study

explains the set of genes that are associated with human autosomal dominant deafness.

KEYWORDS: Biological pathway, gene network, gene annotation, hearing impairment.

I. INTRODUCTION Hearing is a remarkable process that is involved in many types of cells and various cellular and molecular

mechanisms. The inner ear is the sensory organ whose function and development are due to a productive

interaction among various cells [1]. In humans, Congenital hearing loss is mainly caused by the external

and inner ear anomalies [2]. There are multiple dominant syndromes in which hearing impairment is

observed. There is a large no of genes which are involved in hereditary sensorineural hearing loss in

these dominant disorders. These genes are involved in cellular signalling mechanism of the development

of the ear [3]. The genes are involved in 50% of Congenital hearing loss, 15-25 % of autosomal dominant

hearing loss and 70% of non-syndromic hearing loss among them 30% genetic hearing loss is syndromic.

The diagnosis of genetic hearing impairment plays a significant role in the therapies of affected families

[4]. The NGS provides more specific information of the genomic cellular activity as compare to other past

technologies. With the advancement of genomics, more than 150 genes and more than 6000 mutations

are identified as a cause of hearing loss [5].With microarray set of genes must be specified highlighted the

variant copy no in hereditary hearing loss[6]. The next-generation sequencing efficiently identified the

mutation associated with autosomal dominant hearing loss [7]. Through next-generation sequencing, a

large no of genes can be examined in one diagnosis test [8]. The WES successfully identified the type of

mutation related to human dominant hearing loss as Congenital hearing loss affects 1-2 in 1000 birth and

about 50% of the population of developed countries and more than 1000 mutation are causes of hearing

impairment [9-10]. In human inner ear have sensory organs as organ of corti in cochlea and cristae and

maculae in vestibule, each one comprised with sensory epithelia that are responsible for hearing and

balance. In human the mutation of specific miRNA genes are cause of hearing loss. To sequenced short

RNA molecule from cochlear and auditory vestibular epithelial used high throughput RNA-seq analysis.

By RNA-seq analysis it is possible to identify the miRNA that are related to inner ear and demonstrates

the expression profiles of known mi RNA as well as it discovered the novel miRNA in the inner ear. RNA-



[242]

seq analysis also highlighted these mi RNA and their regulatory role in various molecular pathways in the

development of inner ear at embryonic level [11].The RNA-seq analysis revealed the key microRNA that

are caused of sensorineural hearing loss[12]. The syndromic and non-syndromic hearing loss is

associated with various dominant syndromes gives rise dysfunction of ears, vestibular system,

deformities of bones, facial features, craniocytosis and skeletal anomalies [13]. The large no of genes are

involved in these multiple syndromes. These genes are-

GJB2,LORICRINACTG1,COL11A1,COL2A1,COL11A2,CHD7,SEMA3E,MITF,PAX3,GJB6,KCQN4,TECTA,GJB3,D

IAPH1,MYH14,CEACAM16,GSDME,WFS1,COCH,EYA4,IFNLR1,LMX1A,

MYO7A,POU4F3,MYH9,MYO6,SIX1,SLC17A8,REST,GRHL2,NLRP3,TMC1,P2RX2,CRYM,

CCDC50,MIR96,TJP2,TNC,DIABLO,TBC1D24,CD164,OSBPL2,HOMER2,KITLG,MCM2,PTPRQ,DMXL2,MYO3

A,PDE1C,TRRAP,PLS1,MYO1A,COL4A3,FGFR2,EYA1,SIX5,SALL1,PMP22,MPZ,MFN2,NIBPL,RAD21,SMC3,F

GFR1,TWIST1,EDN3,EDNRB,SOX10,TCOF1,POLR1D,FGFR3,FGF10,NF2,TBX1,COMT,FGF18,COL1A1,COL1

A2,IFITM5,NSD1,PAX2,,SF3B4,GATA2,KMT2D,PTPN11,RAF1,BRAF,MAP2K1,SALL4,GATA3,FAM136A,DN

MT1,DSPP,EFTUD2,NOP56,GJA1,POLG,TWNK,RRM2B,SLC254A,SQSTM1,TNFRSF11A,TNFRSF11B,SPTLC1

,NOTCH2,SOX9,KRIT1,CCM2,PDCD10,AFF4,TGFB1,GNAI3,PLCB4,MYCN,MIR17HG,CYLD and OPA3 [14-

83].Therefore we aim to study the biological pathway to analyze the mode of genes associated to

dominant sensorineural hearing loss in multiple autosomal dominant syndromes by analyzing the

behavior of candidate genes with respect to disease and effects of the drug on them by constructing their

associated network. We used different tools and databases of bioinformatics to explore the candidate

genes in human dominant hearing loss and deafness across multiple biological aspects including disease

process, molecular interaction and cellular process. We also investigate the biological behavior of most

targeted protein families and highlighted the set of genes that are involved in sensorineural hearing loss

for diagnosis and remedy purposes.

II. METHODOLOGY

To find out biological pathway and gene network that are associated with human autosomal dominant

deafness following methods are used.

a) Gene Mining associated with Hearing Impairment

Gene mining is a supervised learning process for the identification of contributed genes in a specific

biological pathway. Gene mining can also be employed to figure out the gene associated with any

syndrome. For the purpose of acquiring the data of genes allies associated with all autosomal syndromes

associated with Hearing Impairment (HI) we performed an exhaustive literature survey as well as from

databases in order to find out the genes exclusively associated with all the syndromes associated with

hearing loss and hearing impairment using the analytical method based on the Pearson`s correlation

matrix by ensemble decision analysis of all genes.

b) Functional Protein Association Network

We will upload our candidate genes in STRING database and will filter out the genes having interaction

among them in the larger cluster/network. STRING is a search tool/database or web resource

https://string-db.org/ [84], used for retrieval of interacting genes. It is used to show the interaction of

genes with each other, involved in a related function [85-86]. Filtered genes will be the input of next

tools/software. Filtered genes will be uploaded in DAVID tool.

c) Cellular Enrichment Components and Biological Enrichment Process

The behavior of the candidate genes in biological pathways and the labeling of the associated genes were

predicted by analyzing the gene ontology, cellular enrichment components and biological enrichment

process of hearing impairment gene using DAVID, a Database for Annotation, Visualization and Integrated

Discovery database (https://david.ncifcrf.gov/ ) [87-88].

d) Gene Network

The output of gene ontology predicting the biological pathways is the broad range of information which is

necessary to be narrowed down in order to find the genes of large family responsible for the hearing

impairment especially for hearing loss. For this purpose, the confident score



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cell signaling and metabolic pathways including molecular and biochemical studies, we used network

analysis using Ingenuity Pathway Analysis software (IPA; Ingenuity Systems, Redwood City, CA, USA), a

software connected to world`s largest knowledge based biological network system[89-90]. We referred to

the most interconnected molecules in a network as central node. The network of each group was studied

to estimate the likelihood that all genes of a group fit in to the same network. Statistical analysis of

network and pathways was performed in IPA using the right-tailed Fisher`s exact test to filter pathways

by using the cut off value (p



[244]

COL11A1

1

Myopia, Hearing loss, Joints

problems

Non-syndromic hearing

loss

Sensorineural hearing loss [27-28]

GNAI3

Auriculo-Condylar

syndrome

Abnormalities of ears,

Micrognathia,Hearing

loss,Microstomia,Facial asymmetry

[79]

SF3B4

Nager syndrome

Micrognathia,Hearing loss,

Syndactyly, Clinodactyly,

Deformities

[50]

MFN2 Charcot-Marie Tooth

disease

Muscle atropy,Itching,Loss of

vision, Hearing loss(deafness)

[33]

GJB3 Non-syndromic hearing

loss


COL4A3

2

Alport syndrome Kidney disease, Hearing loss, Eye

anomalies, Hematuria, proteinuria

[30]

PAX3

Waardenburg

Syndrome

Hearing loss, Pigmentation [38-39]

Craniofacial deafness

hand syndrome

Hypoplastic maxilla, Sensorineural

deafness, Ocular Hypotelorism,

Ulnar deviation, Camptodactyly

[26]

RAF1

3

Noonan syndrome with

lentigines(LEOPARD)

syndrome

Hypertelorism, Heart defects,

Pigmentation, Hearing loss,

Decrease fertility, Short stature,

Hypospadias, Cryptorchidism

[54]

PDCD10 Cereberal cavernous

malformations

Headaches, seizure, paralysis,

hearing or vision loss, cerebral

haemorrhage

[74]

MCM2 Non-syndromic hearing

loss


GATA2 Emberger syndrome Leukemia, anemia, sensorineural

hearing loss, Hypertelorism

[51]

MITF Tietz syndrome

Hearing loss, pigmentation of eyes

& skin

[25]

Waardenburg

syndrome

REST

4


loss


WFS1

FGFR3 Lacrimo-Auriculo-

Dental-Digital (LAAD)

syndrome

Keratoconjunctivitis,

Dacryocystitis, Sensorineural

deafness, Xerostomia, dental

anomalies, Hydronephrosis,

deformities of fingers

[41]

DIAPH1

5


loss


SQSTM1 Paget disease Arthritis, Hearing loss,

Osteosarcoma, Abnormalities of

bones, Dizziness

[69]

FGF10 Lacrimo-Auriculo- Keratoconjunctivitis, [41]



[245]


syndrome





FGF18 Di George syndrome Cardiovascular defects,

Hypocalcemia, cleft palate, hearing

loss, Arthritis, Grave disease

[43]

COL11A2

6


loss


Stickler syndrome Scoliosis, cataract,

Glucoma,Myopia, Hearing loss,

Joints problems

[18]

EYA4 Non-syndromic hearing

loss


MYO6

GJA1 Oculodentodigital

Dysplasia

Syndactyly, Microcephaly, Hearing

loss, Ataxia, anomalies of eyes &

teeth, Dysarthria, Palmoplantar

keratoderma

[64]

COL1A2

7

Dentinogenesis

Imperfecta

Hearing loss , dental anomalies [60]

[44] Osteogenesis

Imperfecta

Blue or purple sclera , joints or

muscular disorders, hearing loss,

deformities of bones, Kyphosis,

Scoliosis, fracture by minor trauma

BRAF Noonan syndrome with

lentigines(LEOPARD)

syndrome

Hypertelorism,Heart defects,

Pigmentation,Hearing loss,



[54]

KRIT1 Cereberal cavernous

malformations

Headaches, seizure, paralysis,

hearing or vision loss, cerebral

hemorrhage

[74]

CCM2

EYA1

8

Branchiootorenal(BOR)

syndrome

Deformities of tissues,

malformation of ear & neck

[32]

FGFR1 Pfeiffer syndrome Hearing loss, Ankylosis, dental

problems

Craniosynostosis,Syndactyly,

Brachydactyly,

[36]

RAD21 Cornelia de Lang

syndrome

Synophrys ,Microcephaly,

Hypertrichosis, hearing loss, vision

problem, heart defects, digestive

problems, abnormalities of bones

[34]

TNC 9 Non-syndromic hearing

loss


Pfeiffer syndrome Craniosynostosis, Brachydactyly,

Syndactyly, Ankylosis, Hearing loss,

dental problems

[36]

Lacrimo-Auriculo-


syndrome

Keratoconjunctivitis,



[41]



[246]

FGFR2

10



Crouzon syndrome Craniosynostosis, hearing loss,

Strabismus, dental problems, vision

problems

[35]

[31] Apert syndrome Craniosynostosis, Syndactyly,

exophthalmos, Hypertelorism,

Strabismus, Ocular proptosis,

hearing loss, malformation of ears,

hyperhidrosis, Polydactyly,

SMC3 Cornelia de Lang

syndrome

Synophrys , Microcephaly,

Hypertrichosis, hearing loss, vision

problem, heart defects, digestive

problems, abnormalities of bones

[34]

GATA3 Barakat syndrome Hypoparathyroidism,

Sensorineural hearing loss, renal

dysplasia, Cardiomyopathy,

Muscular pain

[56]

MYO3A Non-syndromic hearing

loss


COL2A1

12

Stickler syndrome Scoliosis, cataract, Glaucoma,

Myopia, Hearing loss, Joints

problems

[18]

Czech Dysplasia Progressive hearing loss,

osteoarthritis, Kyphoscoliosis,

platyspondyly

[19]

Kniest Dysplasia Myopia, hearing loss,

Kyphoscoliosis, dwarfism,

platyspondyly

[23]

[20-22] Spondyloperipheral

Dysplasia

Brachydactyly, platyspondyly,

lordosis, Myopia, hearing loss

Spondyloperipheral

Dysplasia congenita

Hearing loss, Myopia, dwarfism,

skeletal anomalies, lordosis

KITLG Non-syndromic hearing

loss


PTPN11 Noonan syndrome with

lentigines(LEOPARD)

syndrome

Hypertelorism, Heart defects,

Pigmentation, Hearing loss,



[53]

MYO1A Non-syndromic hearing

loss


GJB2

13


loss


Vohwinkel syndrome Erythroderma, Ichthyosis, hearing

loss, Palmoplantar keratosis

[14]

[15]

Bart-Pumphrey

syndrome

Leukonychia, hearing loss, knuckle

pads, Palmoplantar keratoderma,

Pseudoainhum

Hystrix –like ichthyosis Hearing loss, ichthyosis, Squamous [15]



[247]

with deafness cell carcinoma

KID syndrome Photophobia, blindness, hearing

loss, ichthyosis, palmoplantar

keratoderma, Squamous cell

carcinoma, erythrokeratoderma

[16]

Palmoplantar

Keratoderma with

deafness

Palmoplantar keratoderma,

hearing loss, skin anomalies

GJB6 Non-syndromic hearing

loss


EDN3 Waardenburg

syndrome


SIX1 14 Non-syndromic hearing

loss


BOR syndrome Deformities of tissues,

malformation of ears, kidneys &

neck, sensorineural hearing loss

[32]

MAP2K1 15 Noonan syndrome with

lentigines(LEOPARD)

syndrome

Hypertelorism,Heart defects,

Pigmentation,Hearing loss,



[54]

SALL1

16

Towne-Brocks

syndrome

Anomalies of ears, Sensorineural

hearing loss, Anorectal

malformation, renal abnormalities,

heart defects deformities

[61]

BOR syndrome Deformities of tissues,

malformation of ears, kidneys &

neck, sensorineural hearing loss

[32]

CYLD Multiple familial

trichoepithelioma

Multiple skin tumors,

trichoepitheliomas, spiradenomas,

cylindromas, vision disorders,

hearing disorders

[82]

COL1A1

17

Dentinogenesis

Imperfecta

Hearing loss , dental anomalies [60]

[44] Osteogenesis

Imperfecta

Blue or purple sclera , joints or

muscular disorders, hearing loss,

deformities of bones, Kyphosis,

Scoliosis, fracture by minor trauma

ACTG1 Non-syndromic hearing

loss


Baraitser- winter

syndrome

Unusual facial appearances,

hearing loss, dystonia, heart defects

[17]

EFTUD2 MFDM syndrome Progressive microcephaly,

Micrognathia, hearing loss, ears

infections, choanal atresia, heart

problems ,esophageal atresia

[62]

SMAD4 18 Myhre syndrome Fibrosis, dwarfism, Hearing loss

Hypertension, Myopia, Distinct

facial features

[48-49]

TGFB1 19 Camurauti Engelmann Hyperostosis, Contractures, [77]



[248]

disease Erythema of limbs, Macrocephaly,

Ocular proptosis, headache,

Hearing loss, vision problems,

ringing of ears(tinnitus),

dizziness(vertigo), facial paralysis,

dentition, anemia, hyperhidrotic,

hepatosplenomegaly

MYH14 Non-syndromic hearing

loss


SALL4 20 Duane-radial ray

syndrome

Distinctive facial features, hearing

loss, bones & eyes anomalies, heart

& kidney defects

[55]

PLCB4 Auriculo-Condylar

syndrome

Abnormalities of ears,

Micrognathia, Hearing loss,

Microstomia, Facial asymmetry

[78]

EDNRB Waardenburg

syndrome


MYH9 22 Non-syndromic hearing

loss


SOX10 Waardenburg

syndrome


Functional Protein Association Network

STRING database showed experimentally proved interaction in 57 genes displaying by different clusters

of significant interaction. The COL11A1, COL11A2, COL1A1, COL1A2, COL4A3, COL2A1, TGFB1, SALL1,

SALL4, REST, SMAD4 GATA2 and GATA3 genes shows a specific pattern of interaction in STRING

database. Next GJA1, GJB2, GJB3, GJB6, MYH14, MYH9, MYO1A MYO3A, MYO6, ACTG1 and DIAPH1 genes

shows another unique pattern of interaction in protein associated network database. FGF10, FGF18,

FGFR1, FGFR2, FGFR3, EDN3, EDNRB, KITLG, MAP2K1, MCM2, PTPN11, BRAF, RAF1, GNAI3 and PLCB4

genes shows another large interaction among themselves. These three network of interaction are

assumed as large association of protein-protein interaction in STRING database among these 57 genes.

However there are small pattern of interaction in some genes as EYA1, EYA4, SIX1, PAX3, MITF, SOX10,

CYLD, EFTU2D, SF3B4, WFS1, TNC, SQSTM1, MFN2, RAD21, SMC3, KRIT1, CCM2 and PDCD10. Figure 1.1

Clusters of significant interaction in 57 genes associated with one phenotypical effect of hearing loss in

multiple dominant syndromes (Fig 1.1 STRING database).

Cellular enrichment components and biological enrichment process

In order to estimate the signal transmission in cell these filtered 115 genes were added to the DAVID

database, among these 115 genes 57 genes are experimentally proved interaction protein-protein

interaction associated genes which are under specific consideration in this research and cellular

enrichment components, as well as the pathway description of each cluster of genes, was procured. Here

we found that our 57 experimentally proved associated genes are present in the largest group of 112

genes with Pathway ID GO: 0044464 involving in cell part pathway to show protein-protein interaction in

the signal transduction in the cell. Here on DAVID database our 57 genes of interest are present in

another large group of 104 genes with Pathway ID GO: 0005622 to show the intracellular pathway .It is

to be noted that among these 57 gene, 56 genes are also present in another large group of 103 genes with

Pathway ID GO:0044424 involving in intracellular part pathway. All of these pathway describes the

intracellular signal transduction in cell. Table 1.2 the whole large group genomic pathway statistic

background is concluded.

The DAVID database also gives biological process descripting that in which biological pathway the group

or sub group of genes are involved. Here it is shown that large no of 108 genes with Pathway ID GO:



[249]

0009987 involved in cellular process with FDR value 2.04×106.The 57 genes ACTG1, BRAF, CCM2,

COL11A1, COL11A2,COL1A1, COL1A2, COL4A3, COL2A1, CYLD, DIAPH1, EDN3, EDNRB, EFTU2D, EYA1,

EYA4, FGF10, FGF18, FGFR1, FGFR2, FGFR3, GATA2, GATA3, GJA1, GJB2, GJB3, GJB6, GNAI3, KITLG,

KRIT1, MAP2K1 ,MCM2, MFN2, MITF, MYH14, MYH9, MYO1A MYO3A, MYO6, PAX3, PDCD10, PLCB4,

PTPN11, RAD21, RAF1, REST, SALL1, SALL4, SF3B4, SIX1, SOX10, SMAD4, SMC3, SQSTM1, TGFB1, TNC,

WFS1 that are associated with unique phenotypical effect hearing loss in all multiple dominant

syndromes most of them are involved in overall biological enrichment process in Gene Ontology (Table

1.3)

Table 1. 2: Gene Ontology behavior of genes: cellular enrichment analysis keeping whole genomic

statistics background (DAVID database)

Cellular Enrichment Components(GO)

Pathway ID Pathway

description

Observed

gene

count

False

discovery rate

Matching protein clusters(labels)

GO: 0044464

Cell part

112

5.19e-05

ACTG1, BRAF, CCM2, COL11A1,

COL11A2,COL1A1, COL1A2, COL4A3,

COL2A1, CYLD, DIAPH1, EDN3, EDNRB,

EFTU2D, EYA1, EYA4, FGF10, FGF18,

FGFR1, FGFR2, FGFR3, GATA2, GATA3,

GJA1, GJB2, GJB3, GJB6, GNAI3, KITLG,

KRIT1, MAP2K1 ,MCM2, MFN2, MITF,

MYH14, MYH9, MYO1A MYO3A, MYO6,

PAX3, PDCD10, PLCB4, PTPN11, RAD21,

RAF1, REST, SALL1, SALL4, SF3B4, SIX1,

SOX10, SMAD4, SMC3, SQSTM1, TGFB1,

TNC, WFS1

GO: 0005622

Intracellular

104

7.86e-05



COL2A1, CYLD, DIAPH1, EDN3, EDNRB,









TNC, WFS1

GO: 0044424

Intracellular

part

103

7.40e-05



COL2A1, CYLD, DIAPH1, EDNRB,









TNC, WFS1



[250]

Table 1.3: Gene Ontology (GO): biological enrichment analysis keeping the whole genome as statistical

background for identification of multiple dominant syndromes with common phenotypical effect of

Hearing loss (DAVID databse)

Biological Enrichment Process(GO)

Pathway

ID

Pathway

description

Observed

gene

count

False

discovery

rate

Matching protein (labels)

GO:

0009987

Cellular

process

108

2.04e-06

ACTG1,BRAF,CCM2, COL11A1, COL11A2,

COL1A1, COL1A2, COL4A3, COL2A1, CYLD,

IAPH1, EDN3, EDNRB, EFTU2D, EYA1, EYA4,

FGF10, FGF18, FGFR1, FGFR2, FGFR3, GATA2,

GATA3, GJA1, JB2, GJB3, GJB6, GNAI3,

KITLG,KRIT1, MAP2K1 ,MCM2, MFN2, MITF,

MYH14, MYH9, MYO1A MYO3A, MYO6, PAX3,

PDCD10, PLCB4, PTPN11, RAD21, RAF1, REST,

SALL1, SALL4, SF3B4,SIX1,SOX10, SMAD4, SMC3,

SQSTM1, TGFB1, TNC, WFS1

GO:

0065007

Biological

regulation

98

1.16e-07

BRAF,CCM2,COL11A1,COL11A2,COL1A1,COL1A2,

COL4A3, COL2A1, CYLD,

DIAPH1,EDN3,EDNRB,EYA1, EYA4, FGF10,

FGF18, FGFR1, FGFR2, FGFR3, GATA2, GATA3,

GJA1, GJB2, GJB3, GJB6,GNAI3,KITLG, KRIT1,

MAP2K1 ,MCM2, MFN2, MITF, MYH14, MYH9,

MYO1A MYO3A, MYO6, PAX3, PDCD10, PLCB4,

PTPN11, RAF1, REST, SALL1, SALL4, SIX1, SOX10,

SMAD4, SMC3, TGFB1, TNC, WFS1

GO:

0032501

Multicellular

organismal

process

95

2.66e-24

ACTG1,BRAF,CCM2,COL1A1,COL1A2,

COL4A3,COL2A1,CYLD,DIAPH1,EDN3,

EDNRB,EYA1,EYA4,FGF10, FGF18, FGFR1,

FGFR2,FGFR3,GATA2,GATA3, GJA1, GJB6, GNAI3,

KITLG, KRIT1, MAP2K1 ,MCM2,

MFN2,MITF,MYH14, MYH9, MYO6, PAX3,

PDCD10, PLCB4, PTPN11, RAD21, RAF1, REST,

SALL1, SALL4, SF3B4, SIX1, SOX10,

SMAD4,SMC3,SQSTM1,TGFB1,TNC, WFS1

Gene Network

The network analysis of genes including auditory and vestibular development and system, embryonic

development, organ development, cellular development, connective tissue disorders, auditory diseases,

hereditary disorders, cell morphology and neurological disease but important features is auditory

diseases such as hearing and speech disorder malformation of external and inner ears, pigmentation of

skin, eyes and hairs and others anomalies of ears and eyes are studied which explained three network

but the network with 35 molecules with 13 focus genes is selected with score 27 and p=10-21 in (Fig 1.4 &

Fig 1.5). We rejected rest of two networks as all these networks consists of 35 molecules but the focus

genes no is different among these network, rest of two network have focus genes no 10 and 6 respectively

(Fig 1.3). All of these network are involved in auditory disorder on top, neurological disorders on second

no and hereditary disorder on third no (Fig 1.2). As our focus is to determine genes which are involved in

sensorineural hearing loss or hearing impairment with respect to multiple dominant syndrome, so we

found a very significant interaction in network with the middle nodule Erk1/2 dimer with 20 edges while

10 and 7 edges are connected to Integrin and PLC gamma respectively. Detailed information explains that

FGF10 and FGF18 are located on extracellular space, FGFR1, FGFR2, FGFR3, GJB2, GJB6, GJB3 are located



[251]

on plasma membrane, PTPN11 and MAP2K1 are located on cytoplasm, SALL1 and SIX1 are located in

nucleus. Table 1.4 shows that most of our candidate genes are on plasma membrane. We found that most

of our candidate genes are forming clusters in network (Fig 1.4 & Fig 1.5) which proves that these genes

are directly & indirectly interacting with each other in hearing loss as well as associated syndromes. This

network indicates many others genes are playing some direct and indirect role in hearing loss and

associated syndromes and also pointed out our candidate genes in the treatment of auditory diseases. The

GJB2, GJB6, GJB3 are transporters on plasma membrane and have specific role disease progression.

Similarly FGFR1, FGFR2, FGFR3 are kinase and located on plasma membrane & have a great role in

prognosis, diagnosis and response to therapy. FGF10 and FGF18 are growth factors & located on

extracellular space. IPA network analysis also provides information about upstream analysis, upstream

regulators involved in biological pathways interacting with our candidate genes. Table 1.4 shows that

most of our candidate genes or proteins are present in upstream analysis dataset follow different type of

molecularity pattern. These 13 genes as FGFR1, FGFR2, FGFR3, GJB2, GJB6, GJB3, FGF10, FGF18, PTPN11,

MAP2K1, GJB3, SIX1 and SALL1 are more closely biologically pathway in the auditory disorders with

specific phenotypical effects of hearing impairment, development & morphogenesis of ears (Fig1.7 & 1.8).

Among these 13 genes 11 genes are more predicted inhibited to auditory diseases and human autosomal

dominant deafness (Fig 1. 6, Fig 1.10 & Fig 1.11).The Fig 1.9 shows the MAP (molecule activity predicted )

depicts the downstream and upstream effect of activation & inhibition of molecules in network, most of

molecules in network shows activation as well as inhibition effect.



[252]

Fig-1.1: Clusters of significant interaction of 57 genes associated with one phenotypical effect of hearing loss in

multiple dominant syndromes (STRING database).



[253]

Table-1.3: Most common Upstreaming analysis biological pathway interacted with target genes in

hearing loss.

Upstreaming

Regulators

Molecules Type p-value

overlap

Target molecules in dataset

CTNNB1 Transcription

regulators

1.17E-10 SIX1,SALL1,MITF,GJB2,FGF18

TGFB1 Growth factor 2.26E-10 SIX1,MITF,GJB2,MAP2K1,FGFR1,FGFR2,FGF10

TNF Cytokines 2.27E-09 PTPN11,FGFR1,FGFR2,FGF18,FGF10,MITF

EPHB4 Kinase 3.53E-09 FGFR2

FGF2 Growth factor 7.70E-09 MITF,FGFR1,FGFR2,FGFR3

WNT3A Cytokines 2.12E-08 FGF18,MITF

Trichostatin A Chemical drug 3.65E-08 FGFR1.FGFR3

IFNG Cytokines 3.76E-07 SIX1,PTPN11,GJB2,MITF,MAP2K1

HRAS Enzymes 5.50E-06 GJB2,GJB3

ERS2 Ligand-

dependent

nuclear

receptors

6.07E-06 GJB3,FGFR1

TWIST1 Transcription

regulators

4.00E-06 FGFR2,FGFR3,FGF10

SPDEF Transcription

regulators

2.07E--08 FGFR1

Estrogen

receptor

Group 1.74E-07 GJB3,FGFR2,FGFR3,FGFR1

TGFBR2 Kinase 7.65E-07 GJB2,GJB6

TP53 Transcription

regulators

7.66E-07 GJB3,MAP2K1,PTPN11,FGFR1

KRAS Enzyme 3.29E-06 MAP2K1,FGFR1,FGF10

ESR1 Ligand-

dependent

nuclear

receptors

5.11E-06 SIX1,FGFR2,FGF10



[254]

Fig-1.2: Diseases associated with biological expression analysis keeping view on Hearing loss

Fig-1.3: Biological pathway of three network of candidate genes produced by IPA



[255]

Fig-1.4: significant network of genes produced by IPA (Hearing Loss)

Fig-1.5: significant network of highlightened genes produced by IPA associated with hearing loss



[256]

Fig-1.6: Highlighted the genes associated with auditory diseases Network produced by IPA

Fig-1.7: network of genes involved in development of ears

Fig-1.8: Biological pathway showing morphogenesis of ear produced by IPA



[257]

Fig-1.9: MAP (molecule activity predictor)

Fig-1.10: Auditory diseases & their associated genes biological pathway produced by IPA



[258]

Fig-1.11: Auditory diseases & their associated functional genes biological pathway produced by IPA

(prediction inhibited in all syndromes

IV. DISCUSSION In this study of expression analysis of different autosomal dominant syndromes with one common

phenotypical effect of hearing loss different tissues and cells are examined and biological pathway are

analyzed. These networks and pathways are help us to understanding the role of these candidate genes in

human sensorineural hearing loss and effects of drugs on them. Gene mining from NCBI databases[91] as

well as review of research literature, these genes are filtered by STRING database 115 genes are

selected out of 119 among which we select 57 genes which shows most significant interaction among

themselves and are experimentally proved one, and gene enrichment analysis are performed by DAVID.

At last IPA software is used to predict interaction between most associated genes. Most of these 57 gene

are present biological enrichment process to follow different intracellular pathway to covey the signal

transduction in cell at cellular level. These genes as ACTG1, BRAF, CCM2, COL11A1, COL11A2,COL1A1,

COL1A2, COL4A3, COL2A1, CYLD, DIAPH1, EDN3, EDNRB, EFTU2D, EYA1, EYA4, FGF10, FGF18, FGFR1,

FGFR2, FGFR3, GATA2, GATA3, GJA1, GJB2, GJB3, GJB6, GNAI3, KITLG, KRIT1, MAP2K1 ,MCM2, MFN2,

MITF, MYH14, MYH9, MYO1A MYO3A, MYO6, PAX3, PDCD10, PLCB4, PTPN11, RAD21, RAF1, REST,

SALL1, SALL4, SF3B4, SIX1, SOX10, SMAD4, SMC3, SQSTM1, TGFB1, TNC and WFS1 are involved in all

autosomal dominant syndromes having one common phenotypical effects of hearing loss with other one

medical defects. In DAVID these genes are present in all pathways both in cellular enrichment & biological

process to gives a strong interaction of these genes with one another with respect to hearing loss by



[259]

following the pathway of cellular process, biological regulation, cell part, intracellular process and

multicellular organismal process (Table 1.2 & 1.3). In IPA we upload our 57 genes, where 56 genes are

identified except ACTG1 .By core analysis in IPA (Expression analysis) 3 networks are predicted in which

most significant network is selected (fig 1.3). All of these network are related to auditory disease on its

top functional diseases (Fig 1.2). In IPA software one out of three networks, one network with 35

molecules and 13 focus genes is selected (Fig 1.4 & Fig 1.5). Among these 13 target genes 11 genes are the

genes (Fig 1.6) that showed most significant & functional interaction in IPA with respect to development

of ears (fig1.7) in certain auditory diseases as it is top most predicted disease (Fig 1.2) in all these 3

network. These genes FGFR1, FGFR2, FGFR3, GJB2, GJB6, GJB3, FGF10, PTPN11, GJB3, SIX1 and SALL1 (fig

1.10 & fig 1.11) are located on cytoplasm, nucleus, plasma membrane and extracellular space. These

genes are associated with progressive disease of hearing loss & malfunctioning of ears. In IPA a precise

depiction of upstreaming analysis of target genes showing p-value overlap from7.66×107 to 1.17×1010

having different molecules type as cytokines TNF,INFG and transcription regulators as

TP53,SPDEF,TWIST1 ,Trichostatin A as drug. There are ligand –dependent nuclear receptor as ERS1 and

ERS2.KRAS are HRAS are enzymes. In the IPA selected network among these 13 genes 11 genes are

interacting showing clusters with respect to hearing loss (Fig 1.6 & Fig 1.10). So by expression analysis

we found 11 target genes having functional role in the auditory and vestibular system development as

well as in function of particular system with respect to human deafness. As these genes are involved in

auditory disorders so by this predicted scheme of network we identify their location and can be cured

with possible ways with respect to these target genes. Fig 1.10 & 1.11 shows the precise network of target

genes & their associated auditory syndromes, most of these genes are located on plasma membrane. All of

these auditory syndromes such as sensorineural hearing loss, autosomal dominant deafness,

development of inner ear and external ears, diegenic non-syndromic hearing loss & autosomal non-

syndromic hearing impairment, morphogenesis of ears are all showed more and less confident prediction

inhibition with target 11 genes. (Fig1.10 & Fig 1.11)

IV. CONCLUSION

It is concluded that 57 genes that have significantly interacted in STRING database only 11 genes among

them as FGFR1, FGFR2, FGFR3, GJB2, GJB6, GJB3, FGF10, , PTPN11, GJB3, SIX1 and SALL1(fig 1.10) are

showing significant clusters in the IPA selected network. These genes are involved in hereditary hearing

loss and in malformation of ears at embryonic level. These genes are not only involved in hearing loss but

as well as in certain neurological & connective tissue disorders that affect muscular activity in human.

Through this study we found the core location and relevant genes and their relationship with one other

and with neighboring genes that are involved in common phenotypical effect of hearing loss in multiple

dominant syndromes. To compete this query of health we can use different strategy of gene therapy by

gene editing and a specific technique of CRISPR cas-9 [92].There are several therapies of genome editing

and replacing to treat sensorineural hearing loss, also stem cell therapy is a new strategy and hope in the

treatment of hearing loss to regenerate the cells associated with auditory diseases [93-94].

ACKNOWLEDGEMENTS

Especially thanks to Great supervisor Mirza Jawad Ul Hasnain who helped and guides at every step. All

credits goes to Mirza Jawad Ul Hasnain, without his co-operation it was impossible.

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PATHWAY NETWORK ANALYSIS OF GENES INVOLVED IN …€¦ · Salma Hafeez *1, Tahira Aslam*2, Mirza Jawad Ul Hasnain*3 *1, 2Student, Department of Biology, Virtual University of Pakistan,

PATHWAY NETWORK ANALYSIS OF GENES INVOLVED IN …€¦ · Salma Hafeez 1, Tahira Aslam2, Mirza Jawad Ul Hasnain3 1, 2Student, Department of Biology, Virtual University of Pakistan,