Journal of Biomedicine and BiotechnologyVolume 2011 (2011), Article ID 304612, 11 pagesdoi:10.1155/2011/304612

Research Article

Identification of Missense Mutation (I12T) in the BSND Gene and Bioinformatics Analysis

Hina Iqbal,¹ Tayyba Sarfaraz,² Farida Anjum,³ Zubair Anwar,¹ and Asif Mir¹

¹Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad 44000, Pakistan²Department of Biosciences, COMSATS Institute of Information Technology, Islamabad 44000, Pakistan³Research and Development Section, Higher Education Commission, Islamabad 44000, Pakistan

Received 20 October 2010; Revised 21 December 2010; Accepted 4 February 2011

Academic Editor: Susan Blanton

Copyright © 2011 Hina Iqbal et al. Hina Iqbal et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Nonsyndromic hearing loss is a paradigm of genetic heterogeneity with 85 loci and 39 nuclear disease genes reported so far. Mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness and nonsyndromic nearing loss. We studied a Pakistani consanguineous family. Clinical examinations of affected individuals did not reveal the presence of any associated signs, which are hallmarks of the Bartter syndrome type IV. Linkage analysis identified an area of 18.36 Mb shared by all affected individuals between markers D1S2706 and D1S1596. A maximum two-point LOD score of 2.55 with markers D1S2700 and multipoint LOD score of 3.42 with marker D1S1661 were obtained. BSND mutation, that is, p.I12T, cosegregated in all extant members of our pedigree. BSND mutations can cause nonsyndromic hearing loss, and it is a second report for this mutation. The respected protein, that is, BSND, was first modeled, and then, the identified mutation was further analyzed by using different bioinformatics tools; finally, this protein and its mutant was docked with CLCNKB and REN, interactions of BSND, respectively.

1. Introduction

Deafness or hearing loss can be due to genetic or environmental causes or a combination of both. The genetic hearing loss is classified as syndromic or nonsyndromic. Among the many disorders classified as syndromic hearing loss, the pathology varies widely, but in nonsyndromic hearing loss, the defect is generally sensorineural. Seventy percent of deafness has genetic causes and is classified as nonsyndromic. Autosomal recessive nonsyndromic deafness is genetically heterogeneous and is the most common form of inherited hearing loss. Autosomal recessive genes are responsible for about 77% of the cases of hereditary nonsyndromic deafness, with over 85 loci and 21 different genes identified to date (http://hereditaryhearingloss.org/). The high degree of genetic heterogeneity of deafness reflects the great diversity of specialized proteins that are required to make sense of sound, and continuing discovery of common and rare mutations associated with deafness in humans has provided many serendipitous points of entry into the biology of hearing.

Bartter's syndrome (BS) is characterized by hypokalemic, hypochloremic metabolic alkalosis with normal or low blood pressure despite high plasma renin activity and serum aldosterone. The inheritance pattern is autosomal recessive. Antenatal BS with bilateral sensorineural deafness (BSND) was first described in children born to a consanguineous couple from a Bedouin family of Southern Israel [1]. BS type IV, BSND variant, occurs because of a mutation in the BSND gene on chromosome 1p31 coding for protein “barttin” which forms the β subunit of CICKb and CICKa channels located on the basolateral membrane of TAL and inner ear epithelium [2]. The clinical features of Bartter syndrome type IV include sensorineural deafness and peculiar facies, distinguished by the triangular face, large eyes, and protruding ears (1).

Mutation in BSND gene is analyzed by model prediction using SAM_T08 server. Comparison of the protein sequence of BSND with closely related species takes place and find out conserved regions. Other bioinformatics analysis such as protein modeling and proteins docking are also carried out for more bioinformation.

2. Materials and Methods

Before the onset of the study, approval from the Institutional Review Board, Islamabad, and informed consent was obtained from all individuals. Clinical examination was performed at hospitals of the respective area. Audiometry was performed on selected individuals to detect the level of hearing loss. The families were visited at their places of residence to generate pedigrees [3] and collect other relevant information. Blood samples from available affected and normal individuals of each family were collected for DNA extraction.

2.1. Genotyping and Linkage Studies

High molecular weight DNA was extracted from leukocytes following the standard method as described by Sambrook et al. [4]. Genomic DNA was quantified by spectrophotometer readings at OD₂₆₀ and diluted to 40 ng/uL for amplification by polymerase chain reaction (PCR).

To elucidate the gene defect in the family presented here, an initial search for linkage was carried out by using polymorphic microsatellite markers mapped within autosomal recessive nonsyndromic deafness loci listed on the hereditary hearing loss homepage [5]. Genome-wide screening was conducted with microsatellite markers (Linkage Mapping Set 10, Invitrogen, USA). Two-point linkage analysis was carried out using MLINK of the FASTLINK computer package [6]. Multipoint linkage analysis was performed using ALLEGRO [7]. For the analysis, an autosomal recessive mode of inheritance with complete penetrance and a disease allele frequency of 0.001 were assumed. Equal allele frequencies were used in the analysis. However, since it is well known that using allele frequencies, which are too low, can lead to false positive results, a sensitivity analysis was performed. Haplotypes were constructed using SIMWALK2 [8, 9].

2.2. Mutation Screening

To screen for mutation in the BSND gene, exons and splice junction sites were PCR amplified from genomic DNA using the four primer sets. The purified PCR products were subjected to cycle sequencing using big dye terminator V 3.1 ready reaction mix and sequencing buffer (PE Applied Biosystems, Foster City, Calif, USA). The sequencing products were purified to remove unincorporated nucleotides and primers with Centriflex TM Gel Filtration Cartridge (Edge Biosystems, Gaithersburg, Md, USA). These purified products were resuspended in 10 μL of TSR (Template Suppression Reagent) and were placed in 0.5 mL septa tubes to be directly sequenced in an ABI Prism 310 Automated Sequencer (PE Applied Biosystems, Foster City, Calif, USA). Chromatograms from normal and affected individuals were compared with the corresponding control gene sequences from NCBI (National Center for Biotechnology Information) database to identify the aberrant nucleotide base-pair change. (http://www.ncbi.nlm.nih.gov/).

2.3. Protein Modeling, Docking, and Phylogenetics Analysis

SAM_T08 [10–14] was used to model the BSND protein and its interacting protein with highest confidence score (i.e., 0.990) CLCNKB. For their further evaluation, RAMPAGE server [15] was considered. ClustalW program was subjected to analyze the phylogenetics relationship of different animals having BSND protein. Conserved amino acids were predicted by using UniportKB tool available on Expasy server.

Identified BSND mutation was generated by using Swisspdb server. Protein interactor of BSND was found out by STRING server, an online database of known and predicted protein interactions which includes direct (physical) and indirect (functional) associations [16]. Protein docking of both the normal and the mutant BSND was carried out using HEX tool with its interactive proteins CLCNKB and REN.

3. Results

3.1. Clinical Assessments

Ages of affected individuals varied between 12 and 65 years at the time of study. Clinical examination of the hearing impaired individuals did not reveal the presence of signs or symptoms, which are hallmarks of the Bartter syndrome type IV. Urinary and blood biochemistry was tested at the Islamabad Diagnostic Centre, including testing for liver function, renal function, electrolytes, and hematology. Blood hematology and biochemistry markers were within the normal range, except that renin level was at borderline in one of the affected subject (VI: 5, 2.53 mL/h). Detailed clinical and biochemical features of normal and affected members are mentioned in Table 1. The height and weight were within normal range for the local population. An audiometric evaluation of selected affected members by measuring the threshold of hearing at 250–8000 Hz for pure-tune air conduction and bone conduction showed severe hearing loss across all frequencies.

Table 1: Clinical and biochemical features of normal and affected members of studied family.

Features
Reference ranges
Normal members
Affected members from the family with hearing loss

V : 3
V : 5
V : 1
VI : 3
VI : 5

Age (y)

30
35
45
12
10

Other symptoms of BSIV

no
no
No
No
No

Na (mmol/L)
136–148
141
140
135
137
134

SK (mmol/L)
3.6–5.0
3.9
4.6
3.4
3.5
3.5

SCl (mmol/L)
104–114
105
107
97
102
101

SMg (mg/dl)
1.9–2.5
2.3
2.2
2.0
1.9
2.1

SHCO3 (mmol/L)
17.5–27.5
30.0
27.5
30.0
30.2
31.0

SCa (mg/dl)
8.6–10.5
8.7
9.5
9.2
9.8
10.0

SCreatinine (mg/dL)
0.85–1.35
0.7
0.8
0.6
0.5
0.4

Renin (ng/ml/hr)
0.15–2.33
0.31
0.6
2.13
2.03
2.53

SAldosterone (ng/dL)
1–6; 4–31
>1.7f
13.0
10.3
11.9
6.3

SOsmolality (mosm/kg)
275–295
293
289
281
290
287

Na (mmol/L)
30–150
17.5
154.6
39.1
16.9
13.3

UK (mmol/L)
20–67
3.4
91.31
12.13
8.43
4.12

UCa (mg/dL)

6.0
16.0
15.3
11.3
13.0

UMg (mg/dL)

3.6
14.4
3.1
2.4
1.12

UCl (mmol/L)
46–168
16.3
174.3
46.56
19.2
10.5

Uosmolality (mosm/kg)
50–1400
74
903
179
105
56

NephrocacinosisS

Absent
Absent
Absent
Absent
Absent

Renal sonography ruled out the presence of nephrocalcinosis or metabolic alkalosis in these affected individuals. Nor have the affected individuals displayed problems with polydipsia, polyuria, nocturnal enuresis, and hypocalciuria.

3.2. Genotyping Results

Analysis of the results obtained from genome search performed on the ten individuals of the family (III-1, III-3, IV-1, V-1, V-2, V-4, V-5, VI-1, VI-3, and VI-5) (Figure 1) identified an area of interest on chromosome 1. Two-point analysis generated LOD score of 2.027 at marker D1S193 (73.21 cm) and 2.05 at marker D1S3462 (247.23 cm) on chromosome 1 (Table 2). In order to test linkage to these two regions, additional markers located in the vicinity of D1S3462 and D1S193 were chosen from Marshfield map [17] and genotyped in all the ten family members. The maximum two-point LOD score was increased to 2.55 with marker D1S2700. Multipoint linkage analysis for the family derived a maximum LOD score of 3.42 at marker D1S1661 (Table 3). The three-unit multipoint support interval contained a 10.48 Mb region, which span from markers D1S3721 to D1S2690. Haplotypes analysis delimited the centromeric boundary defined by a recombination between markers D1S1596 and D1S2770 in individual V-1. Recombination event between D1S2706 and D1S3721, defined the telomeric boundary in the same individual (V-1). Therefore, the minimum critical region of 18.36 Mb identified for disease locus and shared by all the affected individuals between the markers D1S2706 and D1S1596. The critical interval (17.27 cm) identified in this Pakistani family overlaps with the critical region to which DFNA2 was mapped on 1p34 in a large Indonesian family with autosomal dominant, progressive and sensorineural hearing loss [18]. The DFNA2 locus maps between markers D1S255 (65.47 cm) and D1S211 (73.81 cm), and thus shares a region of 2.68 cm with DFNB interval identified in our family. Riazuddin et al. [19] identified 1.5 Mb in four kindred segregating nonsyndromic deafness at the chromosome 1.

Table 2: Two-point LOD score results between the locus identified in family and chromosome 1p34.2–p32.1 markers.

Marker
deCode map postion¹
Marshfield map postion²
Physical map postion³
LOD score AT

0.00
0.01
0.05
0.1
0.2
0.3

D1S472
57.95
65.47
37,102,828
−999.99
−0.68
−0.05
0.14
0.20
0.13

D1S186
60.14
67.22
37,900,316
−999.99
−.069
−0.10
0.05
0.10
0.06

D1S432
—
69.86
39,046,164
−1.334
−0.69
−0.18
−0.02
0.03
0.01

D1S1598
63.51
70.41
40,011,183
−1.544
−0.61
−0.09
0.03
0.04
0.00

D1S2706
65.31
71.13
40,741,361
−999.99
−1.34
−0.18
015
0.24
0.14

D1S3721
65.87
72.59
41,395,560
2.027
1.97
1.75
1.49
1.49
0.56

D1S193
66.64
73.21
42,688,369
2.027
1.97
1.75
1.49
0.99
0.56

D1S2713
67.87
73.81
44,182,569
1.726
1.67
1.47
1.24
0.80
0.43

D1S2134
71.29
75.66
47,993,241
1.690
1.66
1.52
1.32
0.91
0.52

D1S1661
72.42
78.25
51,142,551
1.228
1.19
1.06
0.90
0.60
0.33

D1S2652
77.15
80.77
55,178,852
1.319
1.28
1.13
0.96
0.62
0.34

D1S2690
79.63
83.07
56,867,560
2.027
1.97
1.75
1.49
0.99
0.56

D1S2700
83.07
87.31
58,828,955
2.556
2.48
2.21
1.88
1.23
0.66

D1S2770
85.02
88.40
59,442,255
−999.99
−0.26
0.26
0.35
0.29
0.17

D1S1596
—
89.49
59,670,589
−1.55
−0.52
−0.00
0.10
0.08
0.01

D1S2737
85.47
90.58
61,038,037
−999.99
−1.10
0.07
0.40
0.46
0.29

D1S2788
87.25
93.86
61,568,857
−999.99
−2.16
−0.90
−0.46
−0.14
−0.03

D1S230
88.12
95.31
62,314,306
−999.99
−2.16
−0.90
−0.46
−0.14
−0.03

D1S1648
99.62
101.48
73,125,563
−999.99
−2.27
−1.00
−0.55
−0.20
−0.06

D1S1665
99.62
102.02
73,941,402
−999.99
−2.34
−1.04
−0.57
−0.20
−0.06

¹Sex-average Kosambi cM map distances from the deCode genetic map [7].²Sex-average Kosambi cM map distances from the Marshfield genetic map [17].³Sequence-based physical map distance in bases according to the Human Genome Project-Santa Cruz (http://genome.ucsc.edu/, Assembly: May 2004).

Table 3: Multipoint LOD score results between linked locus identified in family and chromosome 1p34.2–p32.1 markers.

Marker
Physical position
Location
LOD

ATA79C10
—
0.0000
−2.0630

D1S472
37,102,828
3.0940
nan

D1S186
37,900,316
5.1350
nan

D1S432
39,046,164
8.2290
−2.4708

D1S1598
40,011,183
9.2390
−0.0755

D1S2706
40,741,361
10.2490
−8.2103

D1S3721
41,395,560
11.2590
3.0063

D1S193
42,688,369
12.2690
3.2113

D1S2713
44,182,569
14.3100
3.3672

D1S2134
47,993,241
16.3510
3.4190

D1S1661
51,142,551
18.3920
3.4201

D1S2652
55,178,852
21.4860
3.3868

D1S2690
56,867,560
24.5800
3.2867

D1S2700
58,828,955
28.7490
2.8398

D1S2770
59,442,255
29.7590
1.3404

D1S1596
59,670,589
30.7690
nan

D1S2737
61,038,037
31.7800
−inf

D1S2788
61,568,857
34.8730
nan

D1S230
62,314,306
36.9140
−inf

D1S1648
73,125,563
43.3060
−inf

D1S1665
73,941,402
44.3160
nan

GATA152F05L
85,238,770
45.3260
nan

Figure 1: Pedigree of family with nonsyndromic autosomal recessive hearing loss associated haplotypes. Haplotypes for the most closely linked STRPs are shown below each symbol. The genetic map distances according to the Marshfield genetic map in centimorgans (cM) are shown in parenthesis next to the marker name. Arrows adjacent to the haplotypes indicate key recombination events. The alleles are denoted 1–3 according to their allele size.

3.3. Mutation Analysis of Candidate Genes

Through a database search, we identified several genes mapping between the linkage interval in the family (Human Genome Project-Santa Cruz; http://genome.ucsc.edu/, May 2004). Among these, KCNQ4 (MIM 603537), CLDN19, FOXE3 (MIM 601094), FOXD2 (MIM 602211), TSPAN1, and BSND (MIM 606412) were plausible candidate candidates in the interval of our family. We started sequencing of these and completely sequenced two genes (KCNQ4 and CLDIN19), but we found no mutation in both. Recent studies of Riazuddin et al. [19] showed that mutation in BSND can cause nonsyndromic deafness and is the molecular basis of DFNB73 locus in four Pakistani families segregating nonsyndromic deafness [19]. As the critical interval identified in the family contains potential candidate gene barttin (BSND, MIM 606412) as well; therefore, it was sequenced to search for the mutation. Sequence analysis of exon 1 of the BSND gene in our family also revealed missense mutation involving T to C transition at nucleotide number 35 (35T > C), which resulted in substitution of isoleucine to threonine at amino acid position 12 (I12T) (Figure 2). BSND mutation; that is, p.I12T cosegregated in all extant members of the pedigree. This mutation was present in the heterozygous state in obligate carriers within the family.

(a)

(b)

(c)

Figure 2: Representative chromatogram generated by Big Dye Terminator, sequencing of translated exon1 of BSND gene indicating a T to C transition at nucleotide 35 (35T > C) from an affected female individual (VI-1) (a), a heterozygous carrier (VI-1) (b) and a normal male individual (V-2) (c) of the family. The T to C transition at nucleotide position 35 results in isoleucine to threonine change at amino acid 12 (I12T). Arrows indicate the nucleotide change in the sequence.

3.4. Bioinformatics Analysis

Models of BSND CLCNKB and REN proteins were generated by SAM_T08 server and visualized by Rasmol (Figure 3(a)–3(c)). Rampage values for BSND were number of residues in favored region 95.6%, the number of residues in allowed region 3.1%, and the number of residues in outlier region 1.4% (Figure 4(a)); however, the CLCNKB and REN values were little varying. Its values were the number of residues in favored region: 94.8%, 97%, the number of residues in allowed region: 4.2%, 3%, and the number of residues in outlier region is 0.9% for CLCNKB and 0% for REN (Figure 4(c)).

(a)

(b)

(c)

Figure 3: Protein models of BSND (a) CLCNKB (b), and REN (c) generated by SAM_T08 and visualized by Rasmol (display = ribbon; colors = group).

(a)

(b)

(c)

Figure 4: Rampage results of residues for protein model of human BSND protein (a) CLCNKB (b) and REN (c).

Comparison of conserved amino acid of human BSND with closely related species like mouse, rat, and rabbit shows that isoleucine at position 12 is highly conserved, so mutation at this point can be significant (Figure 5(a)). The evolutionary relationship between human, mouse, rat, and rabbit were evaluated by phylogenetic analysis. The analysis represents that the BSND of mouse and rat is closely related to each other and shows homology with human and rabbit (Figure 5(b)).

(a)

(b)

Figure 5: (a) Comparison of conserved regions of BSND protein between human, mouse, rat, and rabbit by using UniportKB ClustalW. Dark region shows conserved amino acid sequence between species. (b) Phylogenetic analysis of BSND protein. It shows evolutionary relationship between human, mouse, rat, and rabbit species.

Our mutation (I12T) of BSND was generated by Swisspdb server and was analyzed by Viewerlite. This substitution of isoleucine with threonine altered the bonding capacity with other side chains as well as other interactors (Figure 6(a)-(b)).

Figure 6: (a) Normal and mutated model (Il12T) of BSND are superimposed and visulized in Viewerlite. (b) Focused normal model is yellow highlighted and displayed in stick format, and mutated is displayed in ball and stick format.

According to STRING database, the physical and functional interaction of BSND was very much significant with CLCNKB protein. Clinical assessment showed the reduced chlorine level in all the affected individuals. According to further clinical assessments, renin level was at border range in one affected individual, so REN protein was also selected to check the effect of the respected mutation on this protein. However, other interactors are COL7A1, ARL2, CLCN5, KCNJ1, CASR, SLC12A1, SLC12A3, and CLCNKA (Figure 7). Both the proteins were docked with HEX v 6.1, taking BSND as receptor and CLCNKB and REN as a ligand. The total energy value calculated for BSND-CLCNKB molecules was −229094.6 and BSND-REN was −1145.7. However, the total energy value for the mutant (I12T) on docking with CLCNKB was maximized to −218782.3 and with REN was −1149.8. This values fluctuation ultimately results in reduced binding affinity with CLCNKB and increased binding affinity with REN and so may be the case with other interacting proteins.

Figure 7: The interactions of BSND with other functionally similar proteins retrieved by STRING online database. CLCNKB showed highest confidence score, that is, 0.990 with BSND.

4. Discussion

Pathophysiological pathway leading from a specific mutation to a specific phenotype has remained elusive in syndromic as well as nonsyndromic hearing loss families. Individuals with the same mutation can fall along a clinical spectrum ranging from asymptomatic to severely affected and can even have completely different diseases with different mutation in the same gene. Our results also support the findings of Riazuddin et al. [19] that pathogenic mutation of BSND gene can cause the nonsyndromic hearing loss. But there are no subclinical renal metabolic changes in our family as reported by Riazuddin et al. [19]. Renin level is at boarder range only in one affected individual, while renal sonography ruled out the presence of nephrocalcinosis in these affected individuals. Nor have the affected individuals displayed problems with polydipsia, polyuria, nocturnal enuresis, metabolic alkalosis, and hypocalciuria. It might be possible that partial loss of function of barttin induced by T12I allele cause only selectively hearing loss and appear to have less pronounced effects. However, the genotype does not always predict the clinical phenotype, which varies both within and between families carrying the mutation in the same gene, implying the existence of other genetic and/or environmental factors that influence phenotype. Several disease-causing BSND mutations have been identified and functionally analyzed. In all cases, there is a genotype-phenotype relationship in that the level of function of mutant barttin predicts the renal phenotype. Our results support the relationship between missense BSND mutation (I12T) and nonsyndromic hearing loss. It is a secondary report that the BSND as causal gene in nonsyndromic deafness.

The protein sequence of BSND is highly conserved in closely related species of human, mouse, rat, and rabbit, and it contains 2 putative transmembrane domain starting from 7 to 26 amino acid and 31 to 53 amino acid [20].

Bioinformatics analysis of mutation in BSND protein suggest that isoleucine, a hydrophobic amino acid, is being converted into threonine, a polar amino acid, which is more susceptible to posttranslation modifications, affecting the quaternary structure of protein resulting in mutant protein.

Barttin (BSND) is an accessory subunit that modifies protein stability, subcellular distribution, and voltage-dependent gating of ClC-K chloride channels expressed in renal and inner ear epithelia. ClC-K channels are double-barreled channels with two identical protopores that may be opened by individual or common gating processes [21].

Potassium (K⁺) secretion by strial marginal cells and vestibular dark cells require Cl⁻ to recycle in the basolateral membrane via a major Cl⁻ conductance. This Cl⁻ conductance is composed of the Cl⁻ channels CLCNKA/BSND and CLCNKB/BSND. Cl⁻ channels CLCNKA/BSND and CLCNKB/BSND consist of the pore-forming CLCNKA α-subunits and CLCNKB and the BSND β-subunit. Mutations of BSND reduce channel conductivity and surface expression [22].

Estévez et al. [2] proved that BSND protein is an essential β subunit for CLCNKA and CLCNKB chloride channels, with which it colocalizes in basolateral membranes of renal tubules and of potassium-secreting epithelia of the inner ear. Disease-causing mutations in either CLCNKB or BSND compromise currents through heteromeric channels [2].

The CLCNKA and CLCNKB channels are members of the ClC family, which comprises at least 9 mammalian chloride channels. Each is believed to have 12 transmembrane domains and intracellular N and C termini. The prototype of the family in torpedo is gated by both voltage and chloride [23].

Docking results of both normal and mutant interaction with CLCNKB suggested that there is a reduced binding affinity on mutation which may results the reduced down regulating or up regulating interactions; however, the mutant does not effect the interaction with renin (REN). So, we can conclude that the boarder range of renin level in one affected individual is not dependent upon this mutation.

Identification of mutation in BSND gene in more families will give valuable insight into the genetic mechanisms underlying nonsyndromic hearing disorder. This work is a fundamental step in the bioinformatics analysis of BSND and provides basics for further analysis.

Electronic-Database Information

(i)Marshfield Medical Research Foundation database website: http://research.marshfieldclinic.org/genetics/.(ii)National Center for Biotechnology Information: http://www.ncbi.nlm.nih.gov/.(iii) UCSC Genome Browser: http://genome.ucsc.edu/.

Abbreviations

BSND: | Bilateral sensorineural deafness
CLCNKB: | Chloride Channel, Kidney, B
REN: | Renin
ARF: | ADP-ribosylation factor
I: | Isoleucine
T: | Threonine.

Acknowledgment

This work was supported by Higher Education Commission (HEC), Islamabad, Pakistan.

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