World Journal of Nephrology and Urology, ISSN 1927-1239 print, 1927-1247 online, Open Access
Article copyright, the authors; Journal compilation copyright, World J Nephrol Urol and Elmer Press Inc
Journal website http://www.wjnu.org

Original Article

Volume 9, Number 1, March 2020, pages 15-24

Characterization of Novel Truncated Variants Identified From Indian Autosomal Dominant Polycystic Kidney Disease Cases

Figures

Figure 1.
Figure 1. Chromatogram of PKD1: c.445_445delC deletion in patient 16.1.
Figure 2.
Figure 2. Multiple sequence alignment of wild PC1 and predicted mutant c.445_445delC-PC1 protein. PC1: polycystin 1.
Figure 3.
Figure 3. Hydrophobicity and helix forming capacity calculation of wild and mutant proteins using ProtScale. (a, b) Hydrophobicity of PC1-wild and PC1: p.Q149Sfs*141; (c, d) helix forming capacity around PC1-wild and PC1:p.Q149Sfs*141. PC1: polycystin 1.
Figure 4.
Figure 4. Schematic diagram of (a) excision of GFP-tagged wild PKD1 2kb region using BamHI and XhoI and sub-cloned into pcDNA3.1 mammalian expression vector; (b) confirmation of sub-cloned plasmid using restriction enzyme; (c) colony PCR to confirm SDM after transformation using PKD1 specific primer; (d) Sanger sequencing of plasmids, mutant pcDNA 3.1+2kb ex4delC PKD1, and wild pcDNA 3.1+2kb wild PKD1; (e) expression of wild and mutant proteins in transfected COS7 cells, lane 1 displaying 54kDa mutant and lane 2 displaying 72kDa mutant protein using anti-PC1 antibody, and lane 3 displaying empty vector; (f) lane 1 displayed 54kDa mutant, lane 2 displayed 72kDa mutant protein and lane 3 displayed empty vector using anti-GFP antibody. GFP: green fluorescence protein; PCR: polymerase chain reaction; SDM: site-directed mutagenesis.
Figure 5.
Figure 5. Multiple sequence alignment of wild PC2 and predicted mutants c.854_854delG-PC2 and c.1050_1050delC-PC2 proteins.
Figure 6.
Figure 6. Hydrophobicity and helix forming capacity calculation of wild and mutant proteins using ProtScale. (a-c) Hydrophobicity of PC2-wild, PC2: p.G285Afs*32 and PC2: p.G285Afs*32, and (d-f) helix forming capacity of PC2-wild, PC2: p.G285Afs*32 and PC2: p.G285Afs*32.
Figure 7.
Figure 7. (a) PCR product of c.854_854delG SDM, (b) PCR product of c.1050_1050delC SDM, (a, d) colony PCR to confirm the transformation using PKD2 specific primer, (e, f) Sanger sequencing of plasmids mutants c.854_854delG, c.1050_1050delC, and (g) identification of wild and mutant PC2 proteins using Myc-tag antibody. PCR: polymerase chain reaction; SDM: site-directed mutagenesis; PC2: polycystin 2.

Tables

Table 1. List of Variants Identified in Three Patients
 
Sample IDPKD1/PKD2rs IDProteinDomainIn silicoRemark
Mutation tasterAlign-GVGDPolyPhen2PROVEANSIFTPKDB
ND: not defined; NA: not possible to analyze; Align-GVGD: Align-Grantham Variation Grantham Deviation; PolyPhen2: Polymorphism Phenotyping v2; PROVEAN: Protein Variation Effect Analyzer; SIFT: Sorting Intolerant From Tolerant; PKDB: Polycystic Kidney Disease Mutation Database.
PKD16.1c.445_445delCNAp.Q149Sfs*141LRRCTPathogenicNANANANANADisease causing
c.7165T>Crs2457533p.L2389LREJPolymorphismNANANANALikely neutralLikely neutral
c.7441C>Trs2003782p.L2481LREJPolymorphismNANANANALikely neutralLikely neutral
c.12133A>Grs10906p.I4045VTM10PolymorphismClass C25BenignNeutralToleratedLikely neutralLikely neutral
c.12176C>Trs3209986p.A4059VNDPolymorphismClass C55BenignNeutralToleratedLikely neutralLikely neutral
c.12276A>Grs3087632p.A4092ATM11PolymorphismNANANANALikely neutralLikely neutral
c.12409C>Trs79899502p.L4137LNDPolymorphismNANANANALikely neutralLikely neutral
c.12630T>Crs7203729p.P4210PNDPolymorphismNANANANALikely neutralLikely neutral
IVS3-22G>Ars2725221PolymorphismNANANANALikely neutralLikely neutral
PKD39.1PKD2: c.854_854delGReferencep.G285Afs*32First extracellular loopPathogenicNANANANANADisease causing
c.1119C>Trs199685642p.L373LNDPolymorphismNANANANALikely neutralLikely neutral
PKD64.1PKD2: c.1050_1050delCReferencep.S351Vfs*24First extracellular loopPathogenicNANANANANADisease causing
c.1119C>Trs199685642p.L373LNDPolymorphismNANANANALikely neutralLikely neutral
c.10529C>Trs45478794p.T3510MNDPolymorphismClass C65Possibly damagingNeutralDamagingLikely neutralDamaging

 

Table 2. List of Primers Used in Sub-Cloning and SDM
 
Primer nameSequence 5′-3′
aPrimers used in sub-cloning; bSDM primers; cSite of nucleotide deletion.
PKD1_F_BamHlaTAATATGGATCCGGCATGGTGAGCAAGGG
PKD1_R_XholaTAATATCTCGAGGTAGCAGTGCCCGTTGCC
PKD1_EX4_C.445delC_PFbcAGCCCGAGGCAGCCACGT
PKD1_EX4_C.445delC_PRbCACCACCCGCACCTGCTG
PKD2_EX4_C.854delC_PFbcCTCCTTATTGGATGGGCTG
PKD2_EX4_C.854delC_PRbCTTCTGTGAACTTCCAGAAG
PKD2_EX4_C.1050delC_PFbcAGTAGTGAAGATAGGGCTC
PKD2_EX4_C.1050delC_PRbACAGAGTAGACATCATAGC