Mutational Analysis of Exon15 of PKD1 Encoding PKD Domains
The Polycystic Kidney Disorder (PKD) is a hereditary disorder associated with the formation of multiple cysts in kidneys. Autosomal dominant polycystic kidney disorders (ADPKD) are caused due to mutation in genes PKD1 and PKD2. Mutation in PKD1 is the most prevalent of the three, affecting 1 in every 600 to 1000 live births with ADPKD. Nearly 200 different mutations are observed in PKD1 gene and most of them lead to abnormal polycystin1 protein. ADPKD is a middle age onset disorder with formation of multiple cysts in the kidneys. The formation of cyst leads to failure in normal functioning of kidney and other associated complications. The most serious complication is end stage renal disorder. The extra-renal complications involve Cysts in liver, pancreas and other organs, hypertension, cerebral aneurysms, and mitral valve prolapse. The PKD1 is a nearly 54-kb long gene with a transcript size of 14.5-kb encoding Polycystin1. The PKD1 transcript contains 46 Exons. The Exon15 encodes for PKD1 Domains. The PKD Domains are necessary for the normal functioning of PC1 and mutations in PKD domains are associated with the pathogenesis related to ADPKD. The aim of the study was to identify the most prevalent gene sequence variation in Exon 15 of the PKD1 gene in Indian population. The analysis revealed two prominent mutations in the Exon15. The first mutation observed is a transition from C to T at 4754 position of the mRNA. The second mutation was a single nucleotide deletion at 5223 position of the mRNA.
Keywords: Polycystic Kidney Disorder, Polycystin-1, PKD1 Gene, PKD Domain
Polycystic kidney disorder is a genetic disorder associated with main clinical manifestation of fluid filled cysts in kidney leading to end stage failure . The autosomal dominant form of the disease is associated with PKD1 and PKD2 genes, while PKHD1 is associated with the autosomal recessive form of the disorder.
The PKD1 and PKD2 genes are located on chromosome 16p13.3 and 4q12-23 and encode membrane protein polycystin1 and polycystin2 respectively  . The PKHD1 is located on chromosome 6p21.1-p12 and encodes another membrane protein named fibrocystin .
Gene sequence analysis of Exon 15 of PKD1 encoding the PKD Domain
2.1 To isolate Genomic DNA from the blood samples of PKD patients
2.2 To amplify the Exon 15 of PKD1 using PCR
2.3 To purify the PCR product by ExoSAP
2.4 To sequence the PCR Product
2.5 To analyse the Gene Sequence variation in Exon15 of PKD1 in patient samples
MATERIALS AND METHODS
GENOMIC DNA ISOLATION
|Peripheral Blood is one of the most common sources for isolation of DNA by non-enzymatic method. The RBCs are separated by lysing it using solution A, and then the WBCs are lysed using solution B to release the DNA present inside the cell. Further the DNA is purified by precipitating the proteins using solution C and chloroform. The DNA is finally is precipitated in ethanol and dissolved in TE buffer for further experimental purposes.|
Genomic DNA was isolated from the blood samples of patients with Polycystic Kidney Disorders by non-enzymatic method.
1) Solution A
· 1M MgCl2- 5 ml
· 100X Triton X- 10 ml
· Sucrose- 109 gm.
· Make up to 1 litre using distilled water
2) Solution B
· 1M Tris-Cl (pH-8)- 40 ml
· 0.5M EDTA(pH-8)- 12 ml
· 1M NaCl- 15 ml
· Make up to 100 ml using distilled water
3) Solution C
· 5M sodium perchlorate
4) 0.9% NaCl (Saline)
5) 20% SDS
6) Chilled chloroform
7) Absolute Ethanol, 70% ethanol
8) TE Buffer (10X)
· 100mM Tris-Cl(pH-8)
· 100mM EDTA(pH-8)
1) Whole blood was collected in a syringe containing heparin (anticoagulant).
2) 3-4 ml of blood was transferred into a 15 ml centrifuge tube and 3 volumes of Saline (0.9% NaCl) was added; and was mixed properly.
3) Samples were centrifuged at 5000 rpm for 5 mins and the supernatant was discarded.
4) 3 volumes of Solution A was added to the cell pellet and mixed properly.
5) The suspension was centrifuged at 5000 rpm for 5 mins and the supernatant was discarded.
6) The cell pellet was dissolved in 2 ml of Solution B by vigorous mixing.
7) Then 100 µl of 20% SDS, 0.5 ml of solution C and 2 ml of chilled chloroform was added to it and mixed gently.
8) The mixture was then centrifuged at 5000 rpm for 5 mins.
9) Two distinct layers were observed; the transparent aqueous layer was transferred into a fresh sterile centrifuge tube and chilled absolute ethanol was added up to 5ml into it to get the DNA precipitated.
10) The precipitated DNA was then washed with 70% of alcohol twice.
11) It was kept for drying at 37⁰ C overnight.
12) After drying, 150µl of 1X TE buffer was added to the precipitated DNA and was kept at 37⁰ C for dissolution.
13) After complete dissolution, the DNA was stored at 4⁰C.
Amplification of Exon-15 of PKD1 gene using PCR
| PCR stands for Polymerase Chain Reaction developed by Kary Mullis in 1983  . PCR is a molecular technique used to make multiple copies of a specific region of a polynucleotide chain (DNA or RNA). It is based on the fact that DNA polymerase can synthesize a new strand of DNA complementary to the template strand provided to it under suitable conditions. The enzyme Taq DNA polymerase is used for amplification during PCR as it can withstand the high temperature condition necessary fordenaturation of DNA. The following are the three stages during a PCR reaction. ·|
Denaturation- The temperature of the reagent mixture containing the template DNA is raised to 94⁰C to break the hydrogen bonds present between the complementary strands (formation of ssDNA from dsDNA). ·
Annealing- At temperature ranging between 55⁰C to 68⁰C the primers bind to the specific region of the DNA strand complementary to it (binding of primer to template DNA). ·
Extension- at nearly 72⁰C the Taq DNA polymerase efficiently add nucleotides to the 3’ end of the primer, complementary to the sequence present in the template (synthesis of complementary daughter strand).
PCR was performed to amplify the Exon 15 of PKD1 gene. The Exon 15 was amplified into 13 smaller fragments using specific primer set.
25μl PCR reaction mixture with specific primers and was prepared to amplify each fragment of Exon 15 of PKD1 gene.
|10X PCR Buffer||2.5μl|
|Taq DNA Polymerase Enzyme||0.3μl|
Thermo-Cycler Temperature Setup
|95⁰ C||5 mins|
|Step 2: cycles: 30|
|95⁰ C||1 min|
|65/68⁰ C||30 sec|
|72⁰ C||1 min|
|72⁰ C||5 mins|
The PCR products were stored at 4⁰ C.
The PCR product was run on 2% Agarose Gel for confirmation.
PURIFICATION of PCR PRODUCT
|The ExoSAP protocol is generally followed to clean-up PCR products before sequencing it. The Exonuclease I removes leftover primers, while the Shrimp Alkaline Phosphatase removes any remaining dNTPs.|
The PCR products were cleaned up during ExoSAP before sending it for sequencing.
|Exonuclease I Enzyme||0.18μl|
|Alkaline phosphatase Enzyme||0.3μl|
Thermo-Cycler Temperature Setup
|37⁰ C||60 mins|
|85⁰ C||15 mins|
SEQUENCING of Exon 15
|Sanger Sequencing technique was devised by Fred Sanger in 1960s and is based on the chain termination using dideoxy-nucleotide. Replication of the DNA template strand proceeds with a reaction mixture of four standard dNTPs and all four ddNTP, each labelled with a different fluorescent dye (ddATP, ddCTP, ddGTP, and ddATP). Random incorporation of the labelled ddNTPs produces a series of DNA fragments in which chain growth has been terminated at each successive position, each one nucleotide longer than the previous. Separation of the fragments by size produces a sequencing ladder as a series of coloured bands. In an Automated DNA Sequencer, the fluorescent dye of each band is activated by a scanning laser as it passes a set point at the bottom of the electrophoretic gel. The color of each succesive band is read by a fluorometer, and a computer assembles these as a gel image, which can be read from bottom to top, like a conventional radioactively-labelled sequencing ladder. Multiple sequencing reactions on separate templates are run in parallel: the bands in each ladder are read as a separate electropherogram or chromatogram.|
The Exon 15 of PKD1 gene was sequenced through Automated Sanger Sequencing Method.
ANALYSIS OF SEQUENCE DATA
|FinchTV is a free bioinformatics tool developed by Geospiza for working with DNA sequence data. It can read chromatogram in almost all formats. It is used to read and edit the sequence data, find its reverse complement and perform BLAST searches.|
The sequence chromatogram was analysed using FinchTV and the gene sequence variation in the sample sequence was found by comparing with sequence data of NC 000016.10 and NM 001009944.2 through MegaBLAST.
GENOMIC DNA ISOLATION
=DNA SEQUENCE CHROMATOGRAM AND SEQUENCE ALIGNMENT
The PCR products showed a prominent single band when run on 2% Agarose Gel, the size of the band was confirmed by comparing with the size of the sample 86.1of respective sizes, which was used as the positive control for the PCR reaction. The analysis of chromatogram showed the presence of two DNA sequence variants in the Exon15 of the PKD gene. The first mutation observed was a transition from C to T at 4754 position of mRNA. The second mutation was a single nucleotide deletion at 5223 position of mRNA
The analysis of sequence data of Exon 15 of PKD1 Gene showed two prominent mutations. The first mutation observed is a transition from C to T at 4754 position of mRNA. The second mutation was a single nucleotide deletion at 5223 position of mRNA. Both the mutations were found in the PKD1 gene that codes for Polycystin1 Protein. Polycystin1 is an integral membrane protein and regulates the cellular proliferation, differentiation and maturation. Exon 15 of PKD1 codes for the Ig like PKD domains. The PKD domain is found in the extracellular region of the PC1. The earlier functional and structural analyses on cell lines show that these domains play an important role in cellular interaction by homophilic interactions and they are necessary for the normal functioning of PC1. There are predictions that mutations in the PKD domains are associated with cystogenesis. Hence in my opinion it will not be wrong to say that these two new mutations directly or indirectly are associated with ADPKD due to the PKD1 gene. Further validation on the effects on these mutations can only be done by structural and functional analysis of the mutated PC1 protein, which is beyond the scope of this project.
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