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UNIVERSITI PUTRA MALAYSIA

REAL-TIME HIGH RESOLUTION MELTING ANALYSIS-PCR FOR ASSESSMENT OF GENE MUTATIONS IN HUMAN AUTOSOMAL

DOMINANT POLYCYSTIC KIDNEY DISEASE

RUSNI BINTI MOHD JAS

FPSK(m) 2011 31

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REAL-TIME HIGH RESOLUTION MELTING ANALYSIS-PCR FOR

ASSESSMENT OF GENE MUTATIONS IN HUMAN AUTOSOMAL


By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in

Fulfilment of the Requirement for the Degree of Master of Science

September 2011

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of

the requirement for the degree of Master of Science

REAL-TIME HIGH RESOLUTION MELTING ANALYSIS-PCR FOR

ASSESSMENT OF GENE MUTATIONS IN HUMAN AUTOSOMAL


By


September 2011

Chair: Patimah Ismail, PhD

Faculty: Medicine and Health Sciences

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common

hereditary renal diseases that affect 1 in 400 to 1 in 1000 life birth and cause by

mutations in two known genes; PKD1 and PKD2. Although the manifestation of Type1

ADPKD is similar to Type2, it is more severe and patients suffer total renal failure by

the fifth decade of life. In finding the mutations occurring in both genes, PCR-based

methodology have been used and all require technical expertise, otherwise are time

consuming and costly. Therefore, this study aims to develop a much rapid screening

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procedure that is cost effective but have the same sensitivity to widely used

methodology to differentiate ADPKD samples from control. Fourteen primers were

selected and used to amplify the target genes by using Real-time High Resolution

Melting Analysis PCR (Real-time HRMA PCR) and was compared to PCR-SSCP in

terms of rapidity, sensitivity and reliability. PCR-SSCP revealed same number of bands

in control and case sample, with different only in the bands’ size. All primers

successfully amplified respective genes in Real-time HRMA PCR. Ten primers had two

apparent melting curves which enabled control and case samples to be differentiated.

The mean melting temperature for control samples of the ten primers was 81.38±1.70oC

whereas in case samples the mean was 81.00±1.68oC. The p value was 0.001. The

melting curves of control were all shifted to the right having higher melting temperature

while case melting curves were shifted to the left due to lower melting temperature.

Possible mutations were found to be present in five of these primers due to lone case

samples where the melting curve were shifted differently from control and case melting

curves and cannot be classified either in control or case group (p<0.001). Three primers

were found to be less useful to be used in Real-time HRMA PCR as control and case

samples melted in the same manner with same exact temperature, resulting in

inconclusive results. While other primers conferred single melting domain, a primer

amplifying the PKD1 gene was melted twice and evidence in both samples. The PCR-

SSCP needs more time to be completed (mean; 13.93±0.94 hours) before analysis can

be done while the Real-time HRMA PCR can be completed in a very short time (mean;

2.40±0.54 hours) including the analysis. Both methodologies are able to discriminate

case from control but Real-time HRMA PCR is more rapid (p<0.001). Since Real-time

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HRMA PCR is more rapid and less procedure is needed, it is proven to be better for

gene mutation screening. In conclusion, the Real-time HRMA PCR is not only sensitive,

but it can screen for control and ADPKD samples more rapidly and more reliable with

the application of commonly used primers in conventional PCR.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Master Sains

REAL-TIME HIGH RESOLUTION PCR UNTUK PENYARINGAN MUTASI

GEN PESAKIT AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE

Oleh


September 2011

Pengerusi: Patimah Ismail, PhD

Fakulti: Perubatan dan Sains Kesihatan

Autosomal dominant polycystic kidney disease (ADPKD) merupakan salah satu

penyakit genetik buah pinggang yang paling biasa dialami dengan nisbah 1 dalam 400

atau 1 dalam 1000 kelahiran hidup dan penyakit ini disebabkan mutasi yang berlaku

kepada dua gen yang telah dikenalpasti; gen PKD1 dan gen PKD2. Walaupun ciri-ciri

Jenis1 dan Jenis2 adalah serupa, Jenis1 lebih serius dan pesakit akan mengalami

kegagalan buah pinggang yang memerlukan rawatan dialisis sepanjang hayat semasa

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mereka berumur lewat 50-an. Untuk mengenalpasti mutasi yang berlaku di dalam

kedua-dua gen tersebut, teknologi yang berasaskan PCR telah digunakan dan kesemua

teknologi ini memerlukan kemahiran teknikal, paling tidak pun memerlukan banyak

masa dengan kos yang mahal. Oleh itu, tujuan utama kajian ini adalah untuk membina-

uji satu kaedah yang lebih pantas dan efektif bukan sahaja daripada segi kos, tetapi juga

mempunyai sensitiviti yang setaraf dengan kaedah yang biasa digunakan untuk

membezakan sampel pesakit ADPKD daripada sampel kawalan. Sebanyak 14 primer

dipilih dan digunakan untuk kaedah Real-time HRMA PCR dan dibandingkan dengan

kaedah PCR-SSCP daripada segi kepantasan, sensitiviti dan ‘realibility’. Hasil PCR-

SSCP menunjukkan kehadiran jalur-jalur DNA dalam jumlah yang sama diantara

pesakit dan kawalan, cuma berbeza daripada segi saiz jalur. Kesemua primer berjaya

digunakan di dalam kaedah Real-time HRMA PCR. Sepuluh primer mempunyai dua

lengkung pencairan yang berbeza, setiap satu mewakili sampel pesakit dan kawalan

yang membolehkan perbezaan sampel dilakukan. Purata suhu pencairan bagi kawalan

adalah 81.37±1.70oC manakala bagi sampel pesakit adalah 81.01±1.68

oC. Lengkung

pencairan untuk kawalan beranjak lebih ke kanan kerana suhu pencairan yang lebih

tinggi manakala lengkung pencairan pesakit beranjak ke kiri disebabkan suhu pencairan

yang lebih rendah (p<0.001). Tiga primer didapati tidak boleh digunakan dalam kaedah

Real-time HRMA PCR kerana sampel pesakit dan kawalan mempunyai suhu dan

lengkung pencairan yang sama menyebabkan tiada kesimpulan dapat dibuat. Hanya satu

primer didapati mempunyai dua domain pencairan; kedua-dua sampel pesakit dan

kawalan dicairkan dua kali, sementara yang lain hanya mempunyai satu domain

pencairan. PCR-SSCP memerlukan lebih masa untuk disiapkan (purata masa;

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13.93±0.94 jam) sementara Real-time HRMA PCR memerlukan masa yang sangat

singkat (purata masa; 2.40±0.54 jam). Kedua-dua cara berupaya untuk membezakan

sampel pesakit daripada sampel kawalan tetapi Real-time HRMA PCR adalah lebih

pantas (p<0.001). Disebabkan kepantasan dan kurangnya prosedur eksperimen, Real-

time HRMA PCR dibuktikan lebih baik untuk penyaringan mutasi gen. Sebagai

kesimpulan, Real-time HRMA PCR bukan sahaja sensitif, tetapi ia boleh menyaring

sampel kawalan dan pesakit ADPKD dengan lebih cepat dan ‘reliable’ menggunakan

primer sedia ada yang digunakan di dalam PCR biasa.

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ACKNOWLEDGEMENTS

In the name of Allah swt, the most Gracious and most Merciful, and selawat to the

prophet, Muhammad saw, Alhamdulillah.

I have completed my research.

Thank you to my supervisor, Prof. Dr. Patimah Ismail for her priceless guidance, help

and patience throughout the research and to my co-supervisor, Assoc. Prof. Dr. Saidi

Moin and Assoc. Prof. Dr. Abdul Halim Abdul Gafor.

Thank you to Dr. R.Vasudevan for the advice in my laboratory work and all the staffs in

Molecular Biology Lab, FMHS, UPM.

To Mimi Soraya M and Aisyah A, all my labmates and friends, thank you for all the

sweat, blood and tears we all went through. It is indeed, a journey of adding values.

With love to my family members, for their understandings and support.

May Allah bless us all.

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I certify that a Thesis Examination Committee has met on 30 September 2011 to

conduct a final examination of Rusni binti Mohd Jas on her thesis entitled “Real-time

High Resolution Melting Analysis-PCR for Assessment of Gene Mutations in

Human Autosomal Dominant Polycystic Kidney Disease” in accordance with the

Universities and University Colleges Act 1971 and the Constitution of the Universiti

Putra Malaysia [P.U.A(A) 106] 15 March 1998. The Committee recommends that the

student be awarded the Master of Science.

Members of the Thesis Examination Committee were as follows:

Fauziah Othman, PhD

Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Chong Pei Pei, PhD

Associate Professor



(Internal Examiner)

Abdul Aziz Dollah, PhD

Associate Professor



(Internal Examiner)

Srikumar Chakravarthy, PhD

Lecturer

Department of Pathology

International Medical University (IMU)

(External Examiner)

________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date: 20 December 2011

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Patimah Ismail, PhD

Professor



(Chairman)

Saidi Moin, PhD

Associate Professor



(Member)

Abdul Halim Abdul Gafor

Associate Professor

Faculty of Medicine

Universiti Kebangsaan Malaysia

(Member)

______________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citation which

have been dully acknowledged. I also declare that it has not been previously, and is not

concurrently, submitted for any other degree at Universiti Putra Malaysia or at any other

institution.

_______________________


Date: 30 September 2011

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION x1

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVIATIONS xviii

CHAPTER

1 INTRODUCTION 1

1.1 Introduction 1

1.2 Problem Statement 3

1.3 Objectives 4

1.4 Hypothesis 5

2 LITERATURE REVIEW 6

2.1 Autosomal Dominant Polycystic Kidney Disease 6

2.1.1 Polycystic Kidney Disease 6

2.1.2 Epidemiology 6

2.1.3 Molecular Genetics

2.1.3.1 Polycystic Kidney Disease (PKD) Genes and

Polycystins

2.1.3.2 Polycystins Roles in ADPKD

7

8

11

2.1.4 Pathogenesis of Cyst

2.1.4.1 The Two-hit Model

2.1.4.1.1 Loss of Heterozygosity (LOH)

2.1.4.2 The trans-heterozygous Mutations

2.1.4.3 Pathological Features of ADPKD

13

14

16

16

17

2.1.5 Complications, Renal and Extra Renal Manifestation 18

2.1.6 Disease Outcome 20

2.1.7 Clinical Diagnosis 21

2.1.8 Factors Affecting Disease Progression 24

2.1.9 Treatment 26

2.2 Mutation Screening 26

2.2.1 Introduction 26

2.2.2 Polymerase Chain Reaction (PCR)

2.2.2.1 Background

2.2.2.2 Principle

28

28

28

2.2.3 Agarose Gel Electrophoresis 29

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2.2.3.1 Background

2.2.3.2 Viewing the Gel

29

30

2.2.4 Single Stranded Conformation Polymorphism (SSCP)

2.2.4.1 Background

2.2.4.2 The Principle of SSCP

2.2.4.3 Applications in Genetics

31

31

31

33

2.2.5 Real-Time PCR 33

2.2.6 High Resolution Melting Analysis (HRMA)

2.2.6.1 Background

2.2.6.2 The Principle of HRMA

2.2.6.3 Primer Design

2.2.6.4 Saturating Dyes

2.2.6.5 Applications of HRMA

2.2.6.6 Advantages and Drawbacks of HRMA

34

34

35

36

37

39

42

3 METHODOLOGY 44

3.1 Approval of Study 44

3.2 Location 44

3.3 Study Design 45

3.4 Sampling

3.4.1 Study Population

3.4.2 Sample Unit

45

45

45

3.5 Instrument of Study

3.5.1 Sample Collection and Preparation

3.5.1.1 DNA Extraction

3.5.1.2 DNA Quantification

3.5.2 Polymerase Chain Reaction (PCR)

3.5.2.1 Optimization of PCR

3.5.2.2 Agarose Gel Electrophoresis

3.5.3 PCR-SSCP

3.5.4 Real-time HRMA with Pre-amplification

46

46

47

48

48

50

51

52

52

3.6 Data Analysis 53

4 RESULTS AND DISCUSSION 54

4.1 Subjects 54

4.2 DNA Extraction from Buccal Samples 58

4.3 PCR Optimizations 59

4.4 PCR-SSCP Analysis 59

4.5 DNA Amplification in Real-time PCR 62

4.6 HRMA

4.6.1 Primers with Distinguishable Melting Point for

Control and Case

4.6.2 Primers with a Single Melting Point for Control and

Case

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69

79

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4.6.3 Primers with Two Melting Domains 83

4.7 Comparison of PCR-SSCP and Real-time HRMA PCR 86

5 SUMARRY, CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH

89

5.1 Summary 89

5.1 Conclusion 90

5.2 Recommendations for Future Research 90

REFERENCES 92

APPENDICES 100

BIODATA OF STUDENT 114

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