ALI BABA ISSAH

127 Pages·2017·5.37 MB·English

by  Ali Babs

KWAME NKRUMAH UNIVERSITY OF SCIENCE AND TECHNOLOGY,
KUMASI, GHANA.

COLLEGE OF SCIENCE
DEPARTMENT OF BIOCHEMISTRY AND BIOTECHNOLOGY

INVESTIGATING THE PHARMACOGENETIC BASIS OF NEVIRAPINE-
INDUCED CUTANEOUS REACTIONS AMONG SELECTED HIV
PATIENTS IN GHANA.

By

ALI BABA ISSAH

JULY, 2017
INVESTIGATING THE PHARMACOGENETIC BASIS OF NEVIRAPINE-
INDUCED CUTANEOUS REACTIONS AMONG SELECTED HIV
PATIENTS IN GHANA.

By

ALI BABA ISSAH

A Thesis submitted to the Department of Biochemistry and Biotechnology,
College of Science,
in partial fulfilment of the requirements for the award of the degree of
MASTER OF SCIENCE
(BIOTECHNOLOGY).

JULY, 2017
DECLARATION
I, ALI BABA ISSAH, hereby declare that the contents of this work are the results of my own
original research and that, to the best of my knowledge, it contains no materials previously
published by another person, nor materials accepted by another university for the award of any
degree; except where due acknowledgement has been made in the text.

…...……………………. ……………………..
(Signature) (Date)
ALI BABA ISSAH (PG2480814)
Student Name & ID

…...………………. ………………… …...………………. …………………
(Signature) (Date) (Signature) (Date)
Dr. KWABENA OWUSU DANQUAH Prof. FAREED K.N. ARTHUR
Supervisor Supervisor

…...……………………. ……………………..
(Signature) (Date)
Dr. PETER TWUMASI
Head of Department
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ACKNOWLEDGEMENT
I thank the Almighty God for his protection and provision for my life, without which I would not
be alive to undertake this project. Lord, you have been good to me and I am eternally grateful.
I also sincerely wish to express my gratitude toward Lady Reverend Dr. Joy Bruce, for making my
stay here in Kumasi worthwhile. I was lost and away from home, but your constant voice in my
life served as a wonderful guide that always kept me at peace and in check. I honestly do not know
how my stay here in Kumasi would have been if I did not have the privilege of knowing you as
my Pastor. God richly bless you, Mummy.
I do wish to acknowledge the immense contributions of my supervisors, Dr. Kwabena O. Danquah,
and Prof. F.K.N. Arthur, whose guidance and supervision ensured the success of this work. I am
very grateful for your support.
Finally, to all my friends and loved ones who have supported me in diverse ways, I say a very big
thank you. To First Love Church, Ayeduase City Campus, words cannot express the love we share.
You guys are the very blessing of a family that I needed at this time of my life.

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DEDICATION
To my lovely mother, Mary Duah. I do not know any other mother who would do what you have
already done for me. I love you, Mom and I am forever grateful.
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TABLE OF CONTENTS
DECLARATION ............................................................................................................................. i
ACKNOWLEDGEMENT .............................................................................................................. ii
DEDICATION ............................................................................................................................... iii
TABLE OF CONTENTS ............................................................................................................... iv
LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ....................................................................................................................... ix
LIST OF ABBREVIATIONS ......................................................................................................... x
ABSTRACT .................................................................................................................................. xii
CHAPTER 1 ................................................................................................................................... 1
1.0 INTRODUCTION .................................................................................................................... 1
1.1 Background ........................................................................................................................... 1
1.2 Problem Statement ................................................................................................................ 3
1.3 Hypothesis and Conceptual Framework ............................................................................... 4
1.4 Aims and Objectives ............................................................................................................. 5
1.4.1 Specific Objectives ......................................................................................................... 5
1.5 Justification ........................................................................................................................... 6
CHAPTER 2 ................................................................................................................................... 8
2.0 LITERATURE REVIEW ......................................................................................................... 8
2.1 Human Immunodeficiency Virus (HIV) ............................................................................... 8
2.1.1 History and Origin of HIV.............................................................................................. 8
2.1.2 Clinical Importance of HIV ............................................................................................ 9
2.1.3 Classification and Structure of HIV ............................................................................. 11
2.1.4 HIV Treatment .............................................................................................................. 13
2.2 Nevirapine ........................................................................................................................... 16
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2.2.1 Nevirapine Structure and Mechanism of Action .......................................................... 16
2.2.2 Nevirapine Pharmacokinetics ....................................................................................... 17
2.2.3 Nevirapine Hypersensitivity ......................................................................................... 19
2.3 Human Leucocyte Antigen (HLA) ...................................................................................... 22
2.3.1 The Major Histocompatibility Complex....................................................................... 22
2.3.2 Classification and Structure of HLA ............................................................................ 22
2.3.3 HLA Nomenclature System.......................................................................................... 24
2.3.4 HLA Typing ................................................................................................................. 28
2.4 Pharmacogenetics and The Role of DNA Sequencing........................................................ 31
CHAPTER 3 ................................................................................................................................. 33
3.0 MATERIALS AND METHODS ............................................................................................ 33
3.1 Patients Recruitment and Ethics Statement ......................................................................... 33
3.2 Study Design ....................................................................................................................... 33
3.3 Study Population ................................................................................................................. 34
3.3.1 Sample Size .................................................................................................................. 34
3.3.2 Inclusion Criteria .......................................................................................................... 34
3.3.3 Exclusion Criteria ......................................................................................................... 34
3.4 Data Collection and Patient Information Management ....................................................... 35
3.5 Blood Sample Collection and Preparation .......................................................................... 36
3.6 Laboratory Analysis ............................................................................................................ 36
3.6.1 DNA Isolation and Purification .................................................................................... 36
3.6.2 Polymerase Chain Reaction (PCR) ............................................................................... 37
3.6.3 Agarose Gel Electrophoresis and Visualization ........................................................... 39
3.6.4 DNA Sequencing .......................................................................................................... 39
3.7 Bioinformatics and DNA Data Mining ............................................................................... 40
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3.8 Statistical Analysis .............................................................................................................. 41
CHAPTER 4 ................................................................................................................................. 42
4.0 RESULTS ............................................................................................................................... 42
4.1 Baseline Characteristics of Study Population ..................................................................... 42
4.2 DNA Extracts ...................................................................................................................... 44
4.3 DNA Amplicons .................................................................................................................. 46
4.4 DNA Sequencing Analysis.................................................................................................. 51
4.4.1 Chromatogram Data ..................................................................................................... 51
4.4.2 DNA Sequences ............................................................................................................ 56
4.5 HLA Typing Analysis ......................................................................................................... 57
4.5.1 BLAST Analysis and Genotype Assignment ............................................................... 57
4.5.2 Typing Resolution and Genotypic Frequencies ............................................................ 59
4.5.3 Allele Frequencies and Associations ............................................................................ 64
CHAPTER 5 ................................................................................................................................. 65
5.0 DISCUSSION ......................................................................................................................... 65
CHAPTER 6 ................................................................................................................................. 69
6.0 CONCLUSIONS, LIMITATIONS AND RECOMMENDATIONS ..................................... 69
6.1 Conclusions ......................................................................................................................... 69
6.2 Limitations .......................................................................................................................... 70
6.3 Recommendations ............................................................................................................... 71
REFERENCES ............................................................................................................................. 72
APPENDICES .............................................................................................................................. 83
Appendix 1 ................................................................................................................................ 83
Appendix 2 ................................................................................................................................ 87
Appendix 3 ................................................................................................................................ 91
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Appendix 4 ................................................................................................................................ 93
Appendix 5 ................................................................................................................................ 99
Appendix 6 .............................................................................................................................. 106
Appendix 7 .............................................................................................................................. 110

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LIST OF TABLES
Title Page
Table 2.1: Pharmacogenetic associations between various SNPs/HLA alleles and
Nevirapine-induced hypersensitivity reactions………………………………………........... 21
Table 3.1: Primer Sequences used for PCR………………………………………………... 38
Table 4.1: Baseline characteristics of study participants and their association to NVP-
rash………………………………………………………………………………………….. 43
Table 4.2: DNA yield and purity check by NanoDrop™………………………………….. 45
Table 4.3: HLA typing results from BLAST analysis in IPD-IMGT/HLA database............ 58
Table 4.4: Allele distribution and their association to rash among typing population.......... 64

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