Characterisation of the SULT1A1 polymorphism in a
South African Tswana population group
By
Hlengiwe P. Mbongwa
MMedSci: Physiology (UKZN)
BTech: Biotechnology (DUT)
N Dip: Biotechnology (DUT)
Submitted in partial fulfilment of the requirements for the Doctoral
degree of Biochemistry in the Biochemistry Department
School of Physical and Chemical Sciences
North-West University (Potchefstroom Campus)
Promoter:
Professor C.J. Reinecke
Co-promoters: Professor P.J. Pretorius
Doctor G. Koekemoer
ii
ABSTRACT
This dissertation brings to the fore the “Characterization of the SULT1A1 polymorphism in a
South Africa Tswana population group.” The primary experimental group studied came from
South African homogeneous Tswana individuals who participated voluntarily in an ongoing large-scale epidemiological Prospective Urban and Rural Epidemiological (PURE) study the North-West University (Potchefstroom Campus) participates in, as one of the 16 low- middle- and high-income countries across the world.
The primary aspect investigated was the comprehensive profile of the single nucleotide polymorphism (SNP) and copy number variation (CNP) of the SULT1A1 gene. Using the PCR-based RFLP method, SULT1A1 genotypes, and allele frequency distributions in an experimental group of 1 867 individuals were determined. According to the literature this is by far the largest and most homogeneous group from which such information has been acquired to date. The SULT1A1*1, SULT1A1*1/*2 and SULT1A1*2 genotypes were found to be present at a percentage of 43.76, 47.12 and 9.11 respectively. In comparison to similar studies in other population groups, results from this study indicate that there are ethnic differences in the
SULT1A1 genotypes incidence. Asian group differs from Caucasian and Tswana groups
because of its exceptionally high prevalence of individuals with the SULT1A1*1 genotype and a very low incidence of the SULT1A1*2 genotype. The SULT1A1*1 genotype profiles of Caucasian and Tswana groups were comparable, but notable differences were observed for the
SULT1A1*2 genotype.
Using a quantitative multiplex PCR method for the CNV study, the numbers of copies of the SULT1A1 gene in the Tswana population were determined, and the results showed 1 to ~5 copies: only 0.65% of the subjects had a single copy, whereas 59.69% of the subjects had 3 or more copies. This result shows a significant discrepancy between the Caucasian-American samples, which showed that only 26% from that group had more than three copies. However, there is a significant relationship with the African-American population, which presented 63% with 3 or more copies. This finding confirms results from a much smaller African-American study, and suggests a possible genetic link between the African Tswana and the heritage of the African-Americans. These findings were submitted for publication to the South African Journal
of Science, as that journal specializes in publication of new knowledge that has a regional focus
iii
Simultaneous phenotypic consequences of the SNP and CNP of the SULT1A1 gene, as well as the thermo-stable and thermo-labile forms of the sulfotransferases were determined. For this, the formation of [35S]-4-nitrophenyl sulphate from 4-nitrophenol and [35 S]-3’-phosphoadenosine-5’-phosphosulfate ([35S]-PAPS) in platelet homogenates were measured, with the data normalized to a common platelet count. This investigation required fresh blood for enzyme activity. These samples came from 98 Caucasian subjects who voluntarily participated in this part of the study. The experimental data presented a unique challenge to develop a statistical model to accommodate the complexity of the distribution of the data in the phenotype and genotype components, which could be achieved by the development of a mixed model. The model indicated that product formation increased through increasing copy number, but did not differ for SULT1A1*1 and SULT1A1*1/*2. However, the rate of increase in product for the thermo-stable forms of the SULTs was greater than that of thermo-labile forms. In contrast, copy number effect for SULT1A1*2 differed considerably from that of the other two genotypes. Since genotype is also a significant factor, it was concluded from Tukey post-hoc tests that the population group means for product formation differ significantly (for all levels). These results are presently being prepared for publication in an accredited international journal.
Finally, perturbations in 23 biochemical parameters measured in the PURE study were analyzed as a function of the SULT1A1 SNP and CNP were evaluated. No group separation in this regard could be found. It could be shown however, that sulfonation of the iodothyronines, which are endogenous substrates for the SULTs, was influenced by the SULT1A1 genotype. The relative concentrations in plasma of the sulphonated iodothyronines may be expressed as T2S > T3S >> T4S, which coincides with the substrate preference of the SULT1A1 enzymes. This observation may, however, only be qualitatively interpreted as (1) the targeted metabolomics mass spectrometric method used for the quantitative analysis of these substances needs further development, and (2) the influence of deiodonation was not taken into account in these studies. In conclusion, three perspectives are given at the end of the thesis which might be considered for further investigations.
Key words: PURE study, South African Tswana population, copy number polymorphism, single
iv
SAMEVATTING
Die onderwerp van hierdie proefskrif is die “Karakterisering van die SULT1A1 polimorfisme in ‘n
Suid-Afrikaanse Tswana bevolkingsgroep” (goedgekeur as die Engelse weergawe hiervan). Die
hoof groep eksperimentele deelnemers aan hierdie ondersoek was afkomstig van ‘n homogene groep Suid-Afrikaanse Tswana wat vrywillig deelneem aan ‘n omvattende epidemiologiese studie, bekend as die Prospective Urban and Rural Epidemiological (PURE) studie. Die Noordwes Universiteit (Potchefstroom kampus) is een van 16 laag-, middel- en hoë-inkomste lande van oor die wêreld wat hieran deelneem.
Die omvattende profiel van die enkelnukleotied (ENP) en kopiegetal (KGP) polimorfismes van die SULT1A1 geen was die eerste aspek wat nagevors is. Hiervoor is die PCR-RFLP metode gebruik, en daarmee die SULT1A1 genotipe en alleel frekwensie bepaal vir ‘n finale eksperimentele groep van 1 867 individue. In vergelyking met soortgelyke studies wat in die vakliteratuur beskryf is, is dit by verre die grootste en die mees homogene groep waarvoor sulke inligting verkry is. Dit het geblyk dat die SULT1A1*1, SULT1A1*1/*2 en SULT1A1*2 genotipes in die bevolkingsgroep voorkom as 43.76, 47.12 en 9.11 persent respektiewelik. ‘n Vergelyking van die resultate met soortgelyke navorsing op ander bevolkingsgroepe toon aan dat die Asiërgroep baie duidelik verskil van die Kaukasiër- en Tswanagroepe deur die buitengewone hoë voorkoms by hulle van individue met die SULT1A1*1 genotipe in vergelyking met die baie lae voorkoms van die SULT1A1*2 genotipe. Die profiel van die SULT1A1*1 genotipe is meer vergelybaar tussen die Kaukasiër en Tswana groepe, en betekenisvolle verskille het ook voorgekom ten opsigte van die SULT1A1*2 genotipe.
Hierna is die kwantitatiewe meervoudige PCR- metode vir die bepaling van die KGP gebruik, en bevind dat die kopiegetal van die SULT1A1 geen in die Tswanabevolking van 1 tot ~5 kopieë insluit: slegs 0.65% van die individue het net een kopie, terwyl 59.69% van die individue 3 of meer kopië het. Hierdie resultaat verskil betekenisvol van die gegewens van die Kaukasiese Amerikaners waar slegs 26% van daardie groep drie kopieë of meer het. Daarteenoor, is daar is daar ‘n betekenisvolle verwantskap met die Afrika-Amerikaners by wie ook 63% meer as 3 kopieë voorkom. Hierdie waarnemings bevestig dus dié van die veel kleiner vorige Afrika-Amerikaanse studies, en suggereer dat daar moontlik ‘n genetiese band bestaan tussen die Afrika-Amerikaners en die Tswanas bestaan. Hierdie resultate is vir publikasie voorgelê aan die
South African Journal of Science, aangesien hierdie tydskrif spesialiseer in die publikasie van
v
Vervolgens is die gesamentlike fenotipiese gevolge van die ENP en die KGP van die SULT1A1- geen, sowel as van die termostabiele and die termolabiele vorme van die sulfotransferases bestudeer. Hiervoor is die vorming van [35S]-4-nitrofenielsulfaat vanaf 4-nitrofenol and [35 S]-3’-fiosfoadenosien-5’-fosfosulfaat ([35S]-PAPS) in homogenate van bloedplaatjies bepaal, en is die gegewens tot ‘n gemeenskaplike plaatjietelling genormaliseer. Vir hierdie analise word vars bloed vir aktiewe ensieme vereis. Monsters hiervoor is van 98 Kaukasiërs verkry wat vrywillig aan hierdie deel van die navosing deelgeneem het. Hierdie was ‘n unieke uitdaging om ‘n statistiese model te ontwikkel wat die komplekse verspreiding van die genotipe en fenotipe gegewens kon akkomodeer, wat slegs met die ontwikkeling van ‘n gemengde statistiese model bereik kon word. Met hierdie model kon aangetoon word dat die vorming van die produk toeneem met toename in kopiegetal, maar nie t.o.v die SULT1A1*1 en SULT1A1*1/*2 genotipes verskil het nie. Die reksiesnelheid van produkvorming was egter hoër vir die termostabiele as die termolabiele vorme van die SULTs. Hierteenoor het die effek van die kopiegetal grootliks vir die SULT1A1*2 met die ander twee genotipes verskil. Aangesien die genotipe dus ‘n betekenisvolle invloed uitoefen, is Tukey post-hoc toets gebruik wat aangetoon het dat die vorming van die produk op elke vlak betekenisvol van die ander verskil. Hierdie resultate word tans vir publiksie in ‘n geakkrediteerde internasionale vaktydskif voorberei.
Laastens is moontlike versteurings van 23 biochemiese veranderlikes wat in die PURE studie gemeet is as funksie van die SULT1A1 GNP en KGP bepaal. Hier is geen groep skeiding waargeneem nie. Dit kon egter aangetoon word dat sulfonering van die jodotironiene, wat endogene substrate van die SULTs is, wel deur die SULT1A1 genotipe beinvloed word. Die relatiewe plasmakonsentrasie van die gesulfoneerde jodotironiene is in die orde van T2S > T3S > T4S, wat ooreenstem met die substraatvoorkeur vab die SULT1A1’s. Hierdie waarneming moet egter slegs kwalitatief beoordeel word, omdat (1) die geteikende metabolomika massaspektometriese metode wat vir die bepaling an hierdie verbindings gebruik is, nog verder ontwikkel moet word, en (2) die invloed van dejodonering nie in ag geneem is nie. Ten slotte is saaklik drie perspektiewe vir toekomstige navorsing geformuleer.
Sleutelwoorde: PURE studie, Suid Afrikaanse Tswana populasie, SULT1A1 polimorfisme,
kopiegetal polimorfisme, enkelnukleotied polimorfisme, sulfotransferases, geteikende metabolomika
vi
AUTHOR’S DECLARATION
I, Hlengiwe Prosperity Mbongwa declare herewith that the thesis entitled, Characterisation of the SULT1A1 polymorphism in a South African Tswana population group, which I submit to the North-West University as completion/partial completion of the requirements set for the PhD degree, is my own work, has been text edited and has not already been submitted to any other university.
Signature of student: ____________________University number:_________________
Signed at_____________________this _____day of ___________________20...
Declared before me on this ________day of___________________20...
vii
DEDICATION
Ngiyamthanda uJehova ngokuba walizwa izwi lami, wezwa ukukhala kwami ngicela umusa. Ngokuba wangipha indlebe Yakhe, ngizakumbiza Yena yedwa uma ngisaphila.
Amahubo 116:1 – 2.
I love the Lord, for He heard my voice; He heard my cry for mercy. Because He turned His ear to me, I will call on Him as long as I live.
Psalm 116:1 – 2
This manuscript is dedicated to the light of my life, my son Langelihle, whose presence in this world makes me strive to be the best that I can be. Also, to my family:
The late Mr P.A. Mbongwa (April 1939 - August 2010, Dad). Thank you for teaching me and my siblings that nothing in life worth having will be served to you on a silver platter. Also, for the sacrifices you and mom had to make to give us (your kids) a better chance at life. I see bits and pieces of you everyday in myself and my siblings. The day we lost you, we gained a guardian angel. Even though you are gone, you will forever be fondly thought of. We will always love and miss you.
Mrs. H.J. Mbongwa (Mom)
Mthokozisi and Mbongeni Mbongwa (brothers)
Zamambuyisa, Nelisiwe and Nokukhanya Mbongwa (sisters) Nomfundo, Nduduzo and the rest of my nieces and nephews
Kinina boMbuyisa, Mvemnyama, Luthi, Sondisa ukwanda kwaliwa umthakathi! Ngiphelelwa ngamazwi okudlulisa ukubonga ngothando, ukubekezela nokubambisana enikukhombise kimi noLanga kuleminyaka ngisesikoleni! INkosi ize inibusise.
Without your unending love, guidance and support, I would not have persevered this far! Thank you for the sacrifices you had to make on my behalf. You are my rock on which I know I can always lean when the going gets tough. You guys are my guardian angels and I love you all dearly!
viii
ACKNOWLEDGEMENTS
There are many people to whom I am indebted for their contributions to this study and I wish to express my gratitude and sincerest thanks to them.
First and foremost, the biggest thank you goes to the Almighty God through whom all things are possible.
To Professors Carools Reinecke and Piet Pretorius, and Doctor Gerhard Koekemoer the project promoter and co-promoters, thank you for affording me the opportunity to conduct this study with the Biochemistry Department. Thank you for trusting and believing in me, especially since Molecular Biology was not my forte! Your guidance, patience and constructive criticism regarding all aspects of the study are greatly appreciated. Also, your assistance with the preparation of this manuscript is highly appreciated. Your insightful, relevant and critical assessment of the writing was most helpful.
Sincerest thanks to the Tswana people of the North West Province for participating in the PURE study.
I am grateful to Professors Esté Vorster and Annemarie Kruger for letting me use the PURE samples for my study and for providing clinical tests results.
Professor Scott Hebbring (Mayo Clinic College of Medicine, Rochester, Minnesota), thank you for providing the copy number primer sequences, and your guidance on the copy number estimation is greatly appreciated.
To Dr Charlotte Mienie (Agricultural Research Council), thank you for your help with the genetic analyzer.
Professors Richard Weinshilboum and Thomas Wood (Mayo Clinic College of Medicine, Rochester, Minnesota), sincerest thank you goes out to you for your contributions while optimising the enzyme assay method and for providing us with radio-labelled PAPS.
ix To Mr P.J. van Rensburg, thank you for your help with the iodothyronines and their sulfonated counterparts analysis. Your efforts are greatly appreciated.
To Professor T.J. Visser (Department of Internal Medicine, Erasmus Medical Centre, Rotterdam), thank you for providing us with some of the iodothyronine standards; range of normal values for T3, rT3. 3,3’-T2 and T4S; and your guidance through the process of synthesising the sulfonated iodothyronines and analysis of the iodothyronine findings.
Thanks to Ms H.C. Sieberhagen (Translator and Editor) for proof-reading my document.
To my friends, Ms Donella Wright and Ms Krina Reddy, thank you for being there for me whenever I needed to moan. I truly appreciate it!
Sincerest thanks to staff and students of the Biochemistry Department, NWU, for invaluable technical assistance and help with many aspects of life in Potchefstroom, especially during my early days in town!
This study would not have been possible without financial assistance, and sincerest thanks go to the National Research Foundation (NRF) Scarce Skills Scholarship, North-West University Postgraduate Fund and Biotechnology Partnership and Development (BioPAD) in this regard.
x
TABLE OF CONTENTS
ABSTRACT --- ii
AUTHOR’S DECLARATION --- vi
DEDICATION --- vii
ACKNOWLEDGEMENTS --- viii
LIST OF FIGURES --- xvi
LIST OF TABLES --- xix
LIST OF ABBREVIATIONS --- xxi
CHAPTER 1 --- 1
INTRODUCTION --- 1
CHAPTER 2 --- 6
LITERATURE REVIEW --- 6
2.1. GENETIC POLYMORPHISMS --- 6
2.1.1. Single Nucleotide Polymorphisms --- 6
2.1.2. Copy Number Polymorphisms --- 8
2.1.3. The SULT genes --- 9
2.1.3.1. Nomenclature --- 10
2.1.3.2. Location --- 10
2.1.4. SULT1A1 polymorphisms --- 12
2.1.4.1. SULT1A1 SNPs --- 12
2.1.4.2. SULT1A1 CNVs --- 12
2.1.4.3. Population studies on SULT1A1 variability --- 14
2.1.5. Objectives regarding the SULT1A1 variability --- 18
xi
2.2.1. SULT1A1 isoforms --- 19
2.2.2. Enzymatic characteristics of the SULT1A1 enzymes --- 21
2.2.3. Phenotypic consequences of SULT1A1 variability --- 22
2.2.4. Perspective from CNV findings and functional implications --- 24
2.2.5. Objectives regarding the SULT1A1 genotype-phenotype characteristics - 26 2.3. EXPERIMENTAL DESIGN --- 27
2.3.1. Experimental subjects --- 27
2.3.1.1. The PURE study --- 27
2.3.2. Research flow diagram --- 31
2.3.3. Required biochemical research methods --- 32
2.3.3.1. PCR-RFLP --- 32
2.3.3.2. Fluorescent-based quantitative PCR --- 32
2.3.3.3. Radiochemical enzymatic assay --- 33
2.3.3.4. LC-MS-MS --- 33
2.3.4. Biostatistics methods --- 34
2.4. AIMS OF THIS INVESTIGATION --- 35
CHAPTER 3 --- 36
SULT1A1GENOTYPING --- 36
3.1 INTRODUCTION--- 36
OBJECTIVES --- 37
3.2 MATERIALS AND METHODS --- 38
3.2.1. Materials --- 38
3.2.1.1. Samples from the PURE study --- 38
3.2.1.2. PURE eligibility criteria for participants --- 39
3.2.1.3. Overview of data collection --- 39
3.2.1.4. Objectives of the PURE study --- 39
3.2.1.5. Other materials --- 41
3.2.2. Methods --- 42
3.2.2.1. DNA isolation from blood --- 42
3.2.2.2. SULT1A1 genotyping using PCR – based RFLP --- 43
3.2.2.3. Allele frequency calculation --- 43
xii
3.2.2.5. Descriptive and inferential statistics --- 45
3.3. RESULTS AND DISCUSSION --- 46
3.4. CONCLUSIONS --- 54
CHAPTER 4 --- 55
SULT1A1COPYNUMBERESTIMATION --- 55
4.1. INTRODUCTION --- 55
OBJECTIVES --- 55
4.2.MATERIALS AND METHODS --- 57
4.2.1. Materials --- 57
4.2.1.1. Samples from the PURE study --- 57
4.2.1.2. Other materials --- 57
4.2.2. Methods --- 58
4.2.2.1. Estimation of SULT1A1 gene copy number --- 58
4.2.2.2. Statistical methods --- 58
4.3. RESULTS AND DISCUSSION --- 60
4.4. CONCLUSIONS --- 66
CHAPTER 5 --- 67
MODULATION OFSULT1A1 ENZYME ACTIVITY BY SINGLE NUCLEOTIDE AND COPYNUMBERPOLYMORPHISMS --- 67
5.1. INTRODUCTION --- 67
OBJECTIVES --- 68
5.2. MATERIALS AND METHODS --- 69
5.2.1. Experimental subjects --- 69
5.2.2. Materials --- 69
5.2.3. Methods --- 70
5.2.2.1. DNA isolation from blood --- 70
5.2.2.2. SULT1A1 genotyping using PCR – based RFLP --- 71
5.2.2.3. Allele frequency calculation --- 71
5.2.2.4. Estimation of SULT1A1 gene copy number --- 71
xiii 5.2.2.6. Phenol sulfotransferase enzyme activity and thermal stability assay - 71
5.2.2.7. Statistical analysis --- 72
5.3. RESULTS AND DISCUSSION --- 73
5.3.1. SULT1A1 allele frequency distribution --- 73
5.3.2. Copy number estimation --- 73
5.3.3. Characterisation of SULT activity --- 75
5.3.4. Exploration for further developments --- 77
5.3.5. Statistical modelling --- 79
5.4. CONCLUSIONS --- 85
CHAPTER 6 --- 86
AN ANALYSIS OF THE FUNCTIONAL IMPACT OF SULT1A1 VARIABILITY ON SELECTEDBIOLOGICALVARIABLESFROMTHEPUREDATA --- 86
6.1. INTRODUCTION --- 86
OBJECTIVES --- 87
6.2.1 Objectives of the PURE study --- 87
6.2.2 Diagnostic parameters used in the PURE study --- 89
6.2.3 Objective of this investigation related to the PURE study: --- 98
6.3. METHODS --- 99
6.3.1 The biochemical parameters used in the PURE study --- 99
6.3.2 Statistical methods --- 99
6.3.2.1. Principal component analysis --- 99
6.3.2.2. Data pre-treatment --- 100
6.4. RESULTS AND DISCUSSION --- 102
6.4.1. Information from the complete data of selected cases and biochemical variables --- 102
6.4.2 Identification of possible perturbation markers due to the SNPs and CNVs --- 109
xiv
CHAPTER 7 --- 113
AN ANALYSISOFTHEFUNCTIONALIMPACT OFSULT1A1 GENEVARIABILITY ONTHEIODOTHYRONINESUBSTRATESOF THESULT1A1ENZYME --- 113
7.1. INTRODUCTION --- 113
OBJECTIVES --- 114
7.2. MATERIALS AND METHODS --- 115
7.2.1. Materials --- 115
7.2.2. Methods --- 116
7.2.2.1. Synthesis of iodothyronine sulfate --- 116
7.2.2.2. Plasma sample preparation for free and sulfonated iodothyronine quantification --- 116
7.2.2.3. Targeted LC-MS-MS analyses of iodothyronines --- 118
7.2.2.4. Profile of the iodothyronines --- 126
7.2.2.5. Profile of the sulfonated iodothyronines --- 129
7.4. CONCLUSIONS --- 131
CHAPTER 8 --- 132
DISCUSSION AND FUTURE PERSPECTIVES --- 132
8.1. A COMPREHENSIVE PROFILE OF SNP AND CNV FOR THE SULT1A1 GENE OF THE SOUTH AFRICAN TSWANA POPULATION GROUP --- 132
8.1.1 Background --- 132
8.1.2 Noticeable aspects from the investigation of the SULT1A1 SNP’s --- 133
8.1.3 Comparative results of the SULT1A1 CNP --- 137
8.2. A SIMULTANEOUS PHENOTYPIC CONSEQUENCES OF THE SNP AND CNV OF THE SULT1A1 GENE AS DETERMINED BY THE ACTIVITY OF THE RESPECTIVE SULT ALLOZYMES --- 140
8.3. PERTURBATIONS IN BLOOD PARAMETERS UNRELATED AS WELL AS RELATED TO THE FUNCTIONAL ROLE OF THE SULT’S --- 142
8.3.1. Biochemical parameters unrelated to the functional role of the SULT’s - 142 8.3.1.1. Detoxification --- 142
8.3.1.2. Modulation of thyroid hormone functions --- 142 8.3.2. Biochemical parameters related to the physiological role of the SULTS 143 8.3.3. Biochemical parameters related to the physiological role of the SULT’s 144
xv 8.4. FUTURE PROSPECTS --- 146
LITERATURE LIST --- 148
APPENDICES --- 162
Appendix 1 --- 162 Appendix 2 --- 165xvi
LIST OF FIGURES
Figure 2.1: Single-nucleotide genomic changes --- 7
Figure 2.2: Dissection diagram of a representative SULT allele name --- 10
Figure 2.3: A typical Tswana homestead --- 28
Figure 2.4: South African North West Province map --- 30
Figure 2.5: Flow diagram of the proposed study --- 31
Figure 3.1: The sulphonation of 4-nitrophenol --- 36
Figure 3.2: Countries involved in the PURE study --- 38
Figure 3.3: PURE blood samples treatment. --- 40
Figure 3.4: An electrogram showing SULT1A1 genotypes 1A1*1/*1; 1A1*1/*2; and 1A1*2/*2. Electrophoresis conditions were as described in materials and methods. --- 46
Figure 3.5: Illustrations of the estimated genotype probabilities.--- 48
Figure 3.6: SULT1A1 genotypes distribution per four areas sampled. --- 51
Figure 3.7: Genotype distribution per gender --- 52
Figure 3.8: Boxplots representing the age distribution per SULT1A1 genotype --- 53
Figure 4.1: 204, 208 & 212 base pairs fragments --- 60
Figure 4.2: Copy number estimation --- 61
Figure 4.3: A1/A2-ratio (X-axis) and the number of samples per measurement (Y-axis) --- 61
Figure 4.4: Copy number distribution per SULT1A1 genotype --- 62
Figure 4.5: SULT1A1 gene copy number distribution per areas sampled --- 63
Figure 4.6: Copy number distribution per gender --- 64
xvii
Figure 5.1: A1/A2- ratios. The SULT1A1 copy number of 1, 2, 3, 4 and 4+, as indicated --- 74
Figure 5.2: A1/A2-ratio (X-axis) and the number of samples per measurement (Y-axis) --- 74
Figure 5.3: Incubation time, 4-nitrophenol, homogenate and enzyme concentration variations75 Figure 5.4A and B: [35S]-4-nitrophenyl sulphate product formed --- 76
Figure 5.5A and B: A: Product formed under thermostable and thermolabile conditions. B: A1/A2-ratio per product formed --- 77
Figure 5.6: Product formed by the thermostable and thermolabile forms of the enzyme for all genotypes. Solid lines show the pairing of data points for a specific individual. --- 79
Figure 5.7: A1/A2-ratio versus the product formation for different genotype and thermal stability levels with estimated cell equations derived from the mixed model. --- 83
Figure 6.1: Flow diagram depicting the analysis of the remainder of the PURE data --- 102
Figure 6.2 A & B: Data of 23 variables with and without outliers --- 103
Figure 6.3: Two distinct groups of data after Pareto scaling --- 103
Figure 6.4 A and B: Creatinine kernel density (A) and Scatter (B) plots --- 104
Figure 6.5 A and B: Auto scaling PCA representing the 22 variables for wild type (1), heterozygous (2) and homozygous (3) genotypes (all have SULT1A1 gene copy 2). Three components extracted explain 40.4% of the variation in the data. --- 106
Figure 6.6 A, B and C: Auto scaling PCA representing the 22 variables. A represents wild type, B is heterozygous, and C is homozygous genotypes at copy numbers 2, 3 and 4. Three components extracted explain variation in the data by 43.5%, 41.6% and 51.4% for A, B and C, respectively. --- 107
Figure 6.7 A and B: Auto scaling PCA representing the 22 variables for wild type copy number 4 (labelled as 4), and homozygous copy number 2 (labelled as 2). Three components extracted explain 52.6% of the variation in the data. --- 108
Figure 6.8: Effect size (Auto scaling) of wild type versus the grouping of heterozygous and homozygous at copy number 2. --- 109
Figure 6.9: A bar chart representing top 3 variables means after effect size analysis versus 3 genotypes, all with copy number 2. --- 110
Figure 6.10: Bar chart representing top 3 variables after effect size analysis being compared with wild type, copy number 4; and homozygous, copy number 2. --- 111
xviii
Figure 7.1: Structures of T4, T3, rT3, 3,5-T2, 3’,5’-T2 and 3,3’-T2 --- 119
Figure 7.2: Negative ion ESI-MS/MS spectra of the [M-H]– ions formed from T4, T3, & T2 ---- 120
Figure 7.3: MS1 and MS2 spectra of T4 under negative ionization using m/z 775.5 as precursor ion --- 122
Figure 7.4: MS1 and MS2 spectra of T3 under negative ionization using m/z 649.6 as precursor ion --- 122
Figure 7.5: MS1 and MS2 spectra of 3,5-T2 under negative ionization using m/z 523.7 as precursor ion --- 123
Figure 7.6: MS2 spectra of 3’,5’-T2 under negative ionization using m/z 523.7 as precursor ion --- 124
Figure 7.7: MS2 spectra of 3,3’-T2 contaminated with 3,5-T2 under negative ionization using m/z 523.7 as precursor ion --- 124
Figure 7.8: T4 four level calibration curve --- 126
Figure 7.9: T4 reference values when compared to those obtained for PURE and fresh plasm samples --- 128
Figure 7.10: T4 values for the 3 genotypes at copy number 2 --- 128
Figure 7.11: Means of sulfonated T4, T3 and T2 with respect to SULT1A1 genotypes---130
Figure 8.1: Number of cases per group versus CI for SULT1A1*1 --- 135
Figure 8.2: SULT1A1 genotypes for Asian, Tswana and Caucasians --- 136
xix
LIST OF TABLES
Table 2.1: Human SULT gene family --- 11
Table 2.2: SULT1A1 genotype distribution in previously studied populations --- 15
Table 2.3: SULT1A1 copy number differences in the Tswana and three other population groups --- 17
Table 2.4: Human SULT1A1 allozymes --- 20
Table 2.5: Clinical consequences of the SULT1A1 Arg638His polymorphism --- 23
Table 2.6: PURE sampled areas in the North West province of South Africa --- 29
Table 3.1: Manufacturer and catalogue/CAS number of the materials used --- 41
Table 3.2: Genotype probability of the preliminary sample size of 323 --- 47
Table 3.3: Sample size calculation based on a preliminary sample size of 323 --- 49
Table 3.4: Estimated probabilities and simultaneous confidence intervals using a sample size of 1867 --- 50
Table 3.5: Genotype distribution per areas sampled --- 51
Table 3.6: Genotype distribution versus gender --- 52
Table 4.1: Manufacturer and catalogue/CAS number of materials used --- 57
Table 4.2: Distribution of SULT1A1 copy number --- 62
Table 4.3: Copy number distribution per genotype --- 63
Table 4.4: Copy number distribution per area sampled --- 64
Table 4.5: Copy number distribution when compared to gender --- 65
Table 5.1: Materials, manufacturer and catalogue/CAS number used in this part of the study: - 69 Table 5.2: Characteristics stratified by SULT1A1 genotypes of the experimental group --- 73
Table 5.3: SULT1A1 copy number distribution --- 75
Table 5.4: Dummy variables used in the model --- 80
Table 5.5: ANOVA table of the mixed model used for product formation --- 81
Table 6.1: Diabetes mellitus test --- 89
Table 6.2: Cardiovascular disorder tests --- 90
Table 6.3: Liver function tests --- 92
Table 6.4: Iron status tests --- 93
Table 6.5: Nutritional status --- 94
xx
Table 6.7: Kidney function tests --- 97
Table 6.8: Overview of six pre-treatment methods used in this study --- 100
Table 7.1: Manufacturer and catalogue/CAS number of materials used in this part of the study --- 115
Table 7.2: MRM Setting --- 117
Table 7.3: MRM Setting --- 125
Table 7.3: Means of sulfonated T4, T3 and T2 values obtained from fresh plasma samples with respect to the 3 genotypes and grand means. --- 130
Table 8.1: SULT1A1 genotype distribution in previously studied populations --- 134
Table 8.2: SULT1A1 genotypes for Asian, Tswana and Caucasians --- 136
Table 8.3: SULT1A1 copy number differences in the Tswana and three other population groups --- 138
Table 8.4: Thermostable and thermolabile conditions per A1/A2-ratio --- 141
Table 8.5: Preferences of the SULT1A1 allozyme on iodothyronine substrates --- 144
xxi
LIST OF ABBREVIATIONS
% percentage
< less than
[35S]-PAPS radio-labelled 3'-phosphoadenosine-5'-phosphosulfate
> more than
≤ less than or equal to
≥ more than or equal to
µl microlitre
µM micromolar
A adenine
AIDS acquired immunodeficiency syndrome ANOVA analysis of variance
Arg arginine
BMD bone mineral density
BP blood pressure
BSA bovine serum albumin
C cytosine
cDNA cloned deoxyribonucleic acid CHD coronary heart disease
CI confidence interval
CiSO3H chlorosulfonic acid
CNP/s copy number polymorphism/s CNV copy number variation
CPM counts per minute CVD cardio-vascular disease
dL decilitre
DMF dimethylformamide
DNA deoxyribonucleic acid
dNTPS deoxynucleotide triphosphates DPM disintegrations per minute DTT dithiothreitol
xxii EtBr ethidium bromide
g grams
G guanine
His histidine
HIV human immunodeficiency virus
HPLC high performance liquid chromatography
i.e. that is
IU/L international units per litre
km kilometre
LC-MS liquid chromatography mass spectrometry
M molar m/z mass-to-charge ratio mg milligrams MgCl2 magnesium chloride ml millilitre mM millimolar MS mass spectrometry
N/n total sample number
Na-EDTA sodium
ng nanograms
NWU North-West University
o
C degrees Celsius
PAPS 3'-Phosphoadenosine-5'-phosphosulfate PBS phosphate buffer saline
PCA principal component analysis PCR Polymerase Chain Reaction
pmol picomoles
PST phenol sulfotransferase
PUFA omega-6 polyunsaturated fatty acid
PURE Prospective Urban and Rural Epidemiological Study RFLP Restriction Fragment Length Polymorphism
RNA ribonucleic acid rpm rotations per minute rT3 3,5,3’-triiodothyronine
xxiii
SA South Africa
SNP/s single nucleotide polymorphism/s SULT1A1 sulfotransferase 1A1
SULT1A1*1 sulfotransferase 1A1 allozyme 1 SULT1A1*2 sulfotransferase 1A1 allozyme 2 SULT1A1*3 sulfotransferase 1A1 allozyme 3 SULT1A2 sulfotransferase 1A2
SULT1A3 sulfotransferase 1A3 SULT1A4 sulfotransferase 1A4 SULTs sulfotransferases SV structural variant T thymine T2 3,3’-diiodothyronine T3 3,5,3-triiodothyronine T4 thyroxine
TAE Tris-Acetate-EDTA buffer TIBC total iron binding capacity
TL thermolabile
To temperature
TS thermostable
UAE United Arab Emirates
UV ultraviolet
w/v weight over volume
Page | 1
CHAPTER 1
INTRODUCTION
“The complete Khoisan and Bantu genomes from southern Africa” was recently
published in Nature, co-authored by 48 scientists from several laboratories in the US, Australia, Namibia and South Africa. The experimental group included four Namibian hunter-gatherers !Gubi, G/aq’o, D#kgao and !Aî, and one Bantu individual, Archbishop Desmond Tutu, who represents Sotho-Tswana and Nguni speakers (Schuster, et al, 2010). Part of the Abstract to their paper states:
“The genetic structure of the indigenous hunter-gatherer peoples of Southern Africa, the
oldest lineage of modern human, is important for understanding human diversity. Observed genomic differences between hunter-gatherers and others may help to pinpoint genetic adaptations to an agricultural lifestyle. Adding the described variants to current databases will facilitate inclusion of southern Africans in medical research efforts, particularly when family and medical histories can be correlated with genomic-wide data”.
The results presented in this thesis include genomic and phenotypic characteristics of the SULT1A1 gene from a large homogeneous South African Tswana population group, as well as from a more limited number of Caucasian South Africans. Apart from the topicality of the relationship between genotypes and phenotypes, new information on the population characteristics of the Tswana as described here might also provide some complementary detail to the Schuster and associates’ (2010) recently published study which includes one member from the Sotho-Tswana population group.
The super-family of sulfotransferase (SULT) genes code for enzymes that catalyze the sulphoconjugation of different endogenous and exogenous substrates. The SULT1A1 group is one of the largest of the SULT family and the SULT1A1 gene contains probably of the most important genetic information of the human SULT family for detoxification of xenobiotics (Glatt et al., 2000) and biotransformation of neurotransmitters (Eisenhofer et al., 1998), and hormones, like the iodothyronines (Visser et al., 1998),
Page | 2 and therapeutic drugs in humans (Weinshilboum, 1990; reviewed by Blanchard et al., 2004). In addition, genetic polymorphisms are natural variations in the genomic DNA sequence present in more than 1% of the population. Single Nucleotide Polymorphisms (SNPs), and the more recently described Copy Number Polymorphisms (CNPs), are being widely used to better understand disease processes, thereby paving the way for genetic-based diagnostics and therapeutics.
Variable SULT1A1 allele frequencies have been reported in different populations, with
SULT1A1*1 being the most frequent allele in Caucasians, followed by SULT1A1*2 and,
the SULT1A1*3 allele with the lowest frequency (Coughtrie et al., 1999; Carlini et al, 2001). The SULT1A1*1 allele was the most common variant in Chinese subjects, however, with very low frequencies for the other two alleles. In contrast, both the
SULT1A1*2 and SULT1A1*3 alleles were common in African American subjects. Much
less is known on the SULT1A1 copy number variation (CNV) in different ethnic population groups (Carlini et al, 2001). These ethnic population differences specifically in SULT1A1 allele frequencies were interpreted as a contributing factor to the known variability in drug metabolism and disposition among different ethnic groups.
The best characterised polymorphism, SULT1A1*2, results in an amino acid change of Arginine213 to Histidine (Coughtrie et al., 1999; Ozawa et al., 1999). Population studies on SULT1A1 polymorphisms have shown that compromised sulphonation of xenobiotics by the less active SULT1A1*2 enzyme may predispose a patient to cancer, in particular in combination with other risk factors such as smoking and age (Hempel et al., 2007), and is uniformly associated with low activity and low thermal stability of sulfotransferase activity, as determined in platelets, where this enzyme is expressed (Raftogianis et al., 1997).
Normally, variability in gene copy number is a common occurrence throughout the human genome (McCarroll et al., 2006). CNV involves structural changes in the human genome which result in either the deletion; or extra copies of genes (or parts of them) ranging from large, microscopically visible chromosome anomalies to single-nucleotide changes (Redon et al., 2006). As with SNPs, CNVs can be associated with disease as well as with evolution of the genome itself, and have been observed for a number of genes encoding drug metabolising enzymes (Hebbring et al., 2007).
Page | 3 Investigation of these complex SULT1A1 genotype-phenotype characteristics required the participation of a large number of experimental subjects. The difficulty to obtain sufficient sample size was made considerably easier by the availability of the international Prospective Urban and Rural Epidemiological (PURE) samples. The North-West University participates in the PURE study, (Teo et al, 2009) for which a collection of blood samples from more than 2 000 voluntary participants of the homogeneous South African Tswana population group was available for research purposes. This enabled the definition of the following objectives for the current PhD-study:
1. The first objective was to determine the SULT1A1 genotypes and consequently the allele frequency distribution in a Tswana population group of the North West Province of South Africa.
This is described in Chapter 3, and was achieved through following these broad steps: DNA extraction, PCR-RFLP and gel electrophoresis. DNA was isolated from blood cell sediments. The extracted DNA was amplified for the amplicon of interest as per Coughtrie et al., (1999) which is a 333 base-pair fragment in this instance. This fragment was digested with a restriction endonuclease enzyme HaeII. A 3% agarose gel was used to resolve the resulting fragment/s.
2. The second objective was to determine the SULT1A1 gene copy number polymorphism in the same Tswana population group.
This is described in Chapter 4, and was achieved by using a fluorescent-based quantitative PCR method as per Hebbring et al., (2007) using two labelled standards,
SULT1A2 and the coagulation factor V gene. The two sets of primers yield three
fragment sizes i.e. 204 (representing coagulation factor V), 208 (representing
SULT1A2) and 212 (representing SULT1A1) respectively. The SULT1A1/SULT1A2
peak height and area ratios were used to estimate the number of copies. These peaks were obtained by running PCR products in a genetic analyser.
3. The third objective was to identify and quantify the combined effect of the genotype, copy number and thermal influence on SULT activity.
The results obtained for objective 3 are described in Chapter 5. For this purpose a statistical model was developed based on our experimental observations. The platelet homogenates were tested for thermal stability or lack thereof profiles all the while [35
S]-Page | 4 4-nitrophenyl sulphate product formed determined the homogenates enzyme activity. The method used was adapted from that of Van Loon and Weinshilboum, (1984) using 4-nitrophenol and [35S]-PAPS as substrates.
4. A wide range of biochemical, nutritional and life-style physiological parameters is included in the South African component of the PURE study. The fourth objective was to analyse the 23 biochemical parameters used in the PURE (SA) study with relation to the results obtained in Chapters 3 and 4.
Multivariate analysis (Auto, Pareto, Level, Range, and Non-parametric scaling as well as a Log transformation) was used as part of this investigation, and is described in Chapter 6.
5. The fifth objective was to identify and quantify iodothyronines and their sulfonated counterparts from plasma, and to compare the values to the genotypes and CNP of the SULT1A1 gene for the respective individuals.
For this purpose a targeted metabolomics approach was followed which is presented in Chapter 7. The method for the synthesis of sulphonated iodothyronines used was adapted from that by Mol and Visser, (1985).
These objectives emanated from a broad literature survey presented in Chapter 2, and are based on the characteristics of the SULT1A1 gene and physiological role of sulfotransferase (SULT) allozymes and their translational product. This survey was closely linked to the existence of polymorphic forms of the SULT1A1 gene, and of anticipated genotype-phenotype consequences. The outcome of this process was the formulation of three broad aims for this study, each linked to the objectives defined above. Chapter 8 discusses findings of the current investigation, focussing on the defined aims and objectives.
It should be noted that a population study on genotype-phenotype characteristics entails comprehensive statistical analyses, which forms a key aspect of the investigation. Some of these are standardized methods like ANOVA’s, the Pearson Chi-square test, kernel density estimations, sample size determinations, confidence interval construction and a bootstrap analysis. The development of a mixed model to accommodate the results contained in the holistic data set emanating from this investigation on the modulation of
Page | 5 the SULT activity due to the SNP, CNV, and thermal stability of the SULTs became a new and unique quantitative statistical challenge.
From this introduction it should be clear that this investigation could only be completed as a trans-disciplinary study which could not be pursued without the inputs and close collaboration from a variety of experts. Their contributions are specified in the Acknowledgements section of this document.
Page | 6
CHAPTER 2
LITERATURE REVIEW
2.1. GENETIC POLYMORPHISMS
All organisms vary in subtle to profound ways that involve every aspect of biological systems, including morphology, physiology, development, and susceptibility to common diseases (Glazier et al., 2002). Many of phenotype traits are controlled by multiple genes (genetically complex traits), in contrast to monogenic or Mendelian phenotypes that are controlled by single genes. In this review, the focus will be on two types of gene variations: SNPs and CNPs that may impact phenotypic variability.
2.1.1. Single Nucleotide Polymorphisms
A SNP is a DNA sequence variation occurring when a single nucleotide (A, T, C, or G) in the genome (or other limited shared sequence) differs between members of a species or paired chromosomes in an individual. A SNP in which both forms lead to the same polypeptide sequence is termed synonymous or a silent mutation. If a different polypeptide sequence is produced they are termed synonymous. A non-synonymous change may either be missense or nonsense (Figure 2.1). When a missense change occurs, it results in a different amino acid, while a nonsense change results in a premature stop codon (reviewed by Pollex and Hegele, 2007). Amino-acid substitutions, which result from common non-synonymous (NS) polymorphisms, may dramatically alter the function of the encoded protein. Gaining insight into how these substitutions alter function is a step toward acquiring predictability. Back in 2001, a review by Kruglyak and Nickerson reported that the human genome contains an estimated 11 million common single nucleotide changes with allele frequencies greater than 1%. A vast majority of these sequence variations are believed to be functionally neutral, yet a subset alters the structure of a gene product. Such structural changes are often detrimental to function, but some variant proteins have normal, or even improved, function.
Page | 7 When genomic variants are discussed, the terms, mutation and polymorphism are used, sometimes interchangeably. “By convention in human genetic research, any genomic
variant with population frequency <1% is termed a mutation, whereas a variant with population frequency >1% is termed a polymorphism” (Pollex and Hegele, 2007).
Figure 2.1: Single-nucleotide genomic changes.
Figure 2.1 demonstrates how the SNPs occur and the two resulting alleles. The switch of a single nucleotide with another, such as the replacement of the wild-type guanine (allele 1) with adenine (allele 2), is referred to either as a mutation, if present in <1% of the general population, or as SNP, if present at a frequency >1% (as reviewed by Pollex and Hegele, 2007). These changes can occur within coding or non-coding regions of the genome, and both may have functional effects in the respective regions. Most single-nucleotide changes are found outside coding regions (non-coding) and have no impact on the structure or biological function of a protein (silent), though they may affect gene expression or splicing (as reviewed by Pollex and Hegele, 2007). However, variants found within the coding region may code for functional changes in amino acid structure (missense) or predict premature protein truncation (nonsense) and thus may have a possible direct association with disease. The most common form of genetic variation, SNPs, can affect the way an individual responds to the environment and modify disease risk.
Although most of the millions of SNPs have little or no effect on gene regulation and protein activity, there are many circumstances where base changes can have deleterious effects (Chorley et al., 2008). Non-synonymous SNPs that result in amino acid changes in proteins have been studied because of their obvious impact on protein
Page | 8 activity. It is well known that SNPs within regulatory regions of the genome can result in dysregulation of gene transcription. A 2003 review by Loktionov on common gene
polymorphisms and nutrition: emerging links with pathogenesis of multifactorial chronic diseases states that rapid progress in human genome decoding has accelerated search
for the role of gene polymorphisms in the pathogenesis of complex multifactorial diseases There is an apparent progress in the field with hundreds of new gene polymorphisms discovered and characterized, however firm evidence consistently linking them with pathogenesis of complex chronic diseases is still limited.
2.1.2. Copy Number Polymorphisms
The dominating type of variation explored so far in the genome has been SNPs, overshadowing the issue of CNPs (Buckley et al., 2005). Deletions and amplifications of the human genomic sequence (CNPs) represent a greatly underestimated aspect of human genetic variation and are causes of numerous diseases and a potential cause of phenotypic variation in the normal population (Urban et al., 2006).
In the early 1990s, Lupski was one of the pioneers to elucidate a new mechanism in parts of the structure of the DNA itself was grossly duplicated or deleted, which changed number of copies of a gene that occurred in genetic material. The “copy number variation” concept wrote a new chapter in the understanding of genetic variation (reviewed by Lupski, 2009).
Polymorphism of tandem repeats are two or more adjacent and approximate copies of a sequence of nucleotides within protein-coding sequences is known to modulate disease risks and can effect changes in the protein products of genes, leading to diseases such as myotonic dystrophy (O’Dushlaine et al., 2005). While SNPs are currently the subject of extensive research, tandem repeats can exhibit high levels of length polymorphism that will potentially alter protein function.
Copy number variation of DNA sequences is considered to be functionally significant but this phenomenon has yet to be fully ascertained (Sebat et al., 2004). Genetic variation in the human genome takes many forms, ranging from large, microscopically visible chromosome anomalies to single nucleotide changes. Although large-scale copy number variation is an important contributor to genomic diversity of organisms
Page | 9 belonging to the same species, whether these variants frequently contribute to human phenotype differences, remains unknown (Redon et al., 2006. If they have few functional consequences, then CNVs might be expected both to be distributed uniformly throughout the human genome and to encode genes that are characteristic of the genome as a whole (Nguyen et al., 2006). Advances in technology in the 21st century have increased the interest in genome structural variants (SVs), in particular CNVs (large-scale duplications and deletions). Although not immediately evident, CNV surveys make a conceptual connection between the fields of population genetics and protein families, in particular with regard to the stability and expandability of families (Korbel et al., 2008). CNV structural changes associated with disease, and also with evolution of the genome itself (McCarroll et al., 2006). CNV has also been observed for a number of genes encoding drug metabolising enzymes (Hebbring et al., 2007).
A 2008 review by Korbel and associates on the current excitement about CNV and how
it relates to gene duplications and protein families mentions that the results from the
field of genetics argue that gene duplication has occurred frequently during the recent history of the human population and that gene duplicates occur in humans in variable numbers and may have been constantly generated from the beginning of the species. Most CNVs are benevolent variants that will not directly cause disease. However, there are several instances where CNVs that affect critical developmental genes do cause disease (Kuiper et al., 2010). To increase the value of the data, a Database of Genomic Variants has been established by Hospital for Sick Children and it houses CNVs found in the general population. The Wellcome Trust Sanger Institute also developed a database of CNVs (which is called DECIPHER) associated with clinical conditions (Daar
et al., 2006).
2.1.3. The SULT genes
In this investigation results on the polymorphisms of the cytosolic SULT1A1 gene is presented. This research was partially motivated by their role in encoding the detoxification enzyme SULT1A1, which is one of the major fields of research interests in the Division of Biochemistry at the North-West University. The SULT super family encodes enzymes catalyzing the sulfate conjugation for a wide variety of endogenous and exogenous substrates. Sulfotransferase 1A1 (SULT1A1) is one of the major conjugating sulfotransferase enzymes in humans. It has an important role in modulating
Page | 10 the biological activity of numerous potent endogenous substances. It also functions as an important component in detoxification pathways of numerous xenobiotics (Blanchard
et al., 2004).
2.1.3.1. Nomenclature
The nomenclature for the SULT’s was addressed at a workshop dedicated to this issue, and the following guidelines were agreed upon at that meeting:
“Attendees of the Third International Sulfation Workshop held in Drymen, Scotland, in 1996, first agreed that “SULT”1 would be adopted as the abbreviation for cytosolic sulfotransferase enzymes, and this symbol was accepted by the HUGO Gene Nomenclature Committee. It was agreed that members of the SULT super-family would be assigned family and subfamily designations on the basis of their amino acid sequence identity. SULTs sharing at least 45% amino acid sequence identity should be considered members of the same family, and subfamily members should share at least 60% identity (reviewed by Blanchard et al., 2004).
SULT families were designated by an Arabic numerical immediately following the name, and subfamilies were identified by alphabetic categories, with the first-identified subfamily of a new family labelled “A”. Unique isoforms within subfamily were identified using an Arabic numeral following subfamily designation”. Figure 2.2 below illustrates
SULT allele name (adapted from Blanchard et al., 2004)
Figure 2.2: Dissection diagram of a representative SULT allele name
2.1.3.2. Location
The SULT1A1 gene is located on chromosome 16p11.2-12.1, in close proximity to its related isoform SULT1A2 (Hempel et al., 2005). The human SULT1A1 and SULT1A2 genes are tandemly arranged 10 kilobase pairs (kbp) apart in the pericentromeric region
1
In case of the genomic abbreviation, “SULT” will be written in italics, whereas the abbreviation “SULT” will refer to the respective enzymes.
Family Isoform
Subfamily
Suballele
Allele or Allozyme Species Superfamily
Page | 11 of chromosome 16, while the SULT1A3 gene is located ~1.7 million base pairs (Mbp) away. In addition to the three known SULT1A genes, a fourth gene was found by Kent in 2002, SULT1A4, by searching the human genome with the BLAST-like alignment tool (Kent, 2002). SULT1A4 was located midway between the SULT1A1/1A2 gene cluster and the SULT1A3 gene. The SULT1A4 is 99.9% identical to SULT1A3 (Hildebrandt et
al., 2004) and is not listed in Table 2.1.
Table 2.1: Human SULT gene family
SULT1A1 SULT1A2 SULT1A3 SULT1B1 SULT1C1 SULT1C2 SULT1E1
SULT1A1 - 96 93 53 53 51 50 SULT1A2 - 90 53 53 51 49 SULT1A3 - 52 53 50 48 SULT1B1 - 53 52 54 SULT1C1 - 62 48 SULT1C2 - 47 SULT1E1 -
Table 2.1 shows the relationship (in percentage) between different members of human SULT gene family depicted using amino acid sequence comparisons (adapted from Coughtrie and Johnston, 2001).
The gene structure (number and length of exons) is similar among family members. This gene encodes one of two phenol sulfotransferases with thermostable enzyme activity. SULT1A members exhibit probably the widest tissue distribution of any cytosolic SULT subfamily. SULT1A1 is by far the major adult liver SULT1A subfamily member and has also been identified in brain (Richard et al., 2001), breast (Windmill et
al., 1998), intestine (Teubner et al., 1998), endometrium (Falany et al., 1998), adrenal
gland, platelets, and placenta (Heroux et al., 1989), kidney and lung (Vietri et al., 2003), and jejunum (Sundaram et al., 1989).
In human lung cytosol, SULT1A1 and SULT1A3 proteins are detectable, and histological studies have shown that these proteins are present in epithelial cells of the respiratory bronchioles (Windmill et al., 1998; Hempel et al., 2005). Since both the intestine and lungs are major portals of entry of drugs and xenobiotics into the body, the above localization pattern suggests that both SULT1A1 and SULT1A3 may play a significant role in the extrahepatic detoxification and metabolic activation of these
Page | 12 chemicals. From a developmental perspective, both SULT1A1 and SULT1A3 are abundantly expressed in the foetal liver, but SULT1A3 almost disappears in adult liver and kidney (Richard et al., 2001). The findings by previous studies suggest a role for
SULT1A members in protecting the foetus from exogenous toxins and in the
homeostasis of hormones such as dopamine and iodothyronines. A study by Stanley et
al. (2001) on placenta cytosols showed the presence of both SULT1A1 and SULT1A3,
indicating they may have a potential role in the metabolism of xenobiotics entering the foetal circulation from the mother.
2.1.4. SULT1A1 polymorphisms
2.1.4.1. SULT1A1 SNPs2
The major form of the SULT gene in adult human liver SULT1A1 is subject to a common genetic polymorphism. A wild type codon for amino acid 213 in the SULT1A1 gene is CGC which encodes arginine amino acid. A single mutation in the SULT1A1 gene codon that encodes arginine at amino acid 213 results in an Arg213His amino acid substitution, which affects the activity and expression of the protein, apparently through reduced protein stability (Raftogianis et al., 1997). Individuals who are homozygous for the SULT1A1*2 genotype (i.e. His213/His213) have significantly reduced platelet sulfotransferase activity (Raftogianis et al., 1997), and platelet enzyme activity correlates strongly with protein expression (Jones et al., 1993).
A number of other mutations in SULT1A1 have been identified but these are present in the population at allele frequencies of less than 1% and do not appear to have functional consequences for the SULT1A1 enzyme (Raftogianis et al., 1999). Sulfation, as other conjugation reactions, is often associated with the inactivation of drugs and toxic chemicals. However, more than other conjugation reactions, sulfation additionally bears a high risk of the formation of reactive products (Glatt, 1997).
2.1.4.2. SULT1A1 CNVs
The human genome is comprised of 6 billion nucleotides of DNA packaged into two sets of 23 chromosomes, one set inherited from each parent (Sebat et al., 2004). The DNA
2Throughout this document SULT1A1*1, SULT1A1*1/*2, SULT1A1*2 in italics denotes the wild type,
heterozygous and homozygous genotypes and SULT1A1*1 and SULT1A1*2 (normal) denotes the allozymes.
Page | 13 encodes between 20,000 and 30,000 genes. It was generally thought that genes were almost always present in two copies in a genome. However, recent discoveries have revealed that large segments of DNA, ranging in size from thousands to millions of DNA bases, can vary in copy number (Redon et al., 2006). Such CNVs can encompass genes leading to dosage imbalances. For example, genes that were thought to always occur in two copies per genome have now been found to sometimes be present in one, three, or more than three copies. In a few rare instances the genes are missing altogether. The new findings indicate that our DNA is less than 99.9% identical, contrary to what was previously thought (Lupski, 2007).
Differences in the DNA sequence of our genomes contribute to our uniqueness. These changes influence most traits, including susceptibility to disease. Copy number variants have already been associated with some diseases and disease susceptibilities and are likely to prove as significant as sequence polymorphisms in this respect. Changes in copy number of parts of the genome are known to be a feature of many cancers, and their analysis is expected to reveal genes involved in carcinogenesis (Dear, 2009). It was thought that SNPs in DNA were the most prevalent and important form of genetic variation (Feuk et al., 2006). Current studies reveal that CNVs comprise at least three times the total nucleotide content of SNPs. Since CNVs often encompass genes, they may have important roles both in human disease and drug response. Understanding the mechanisms of CNV formation may also help to better understand human genome evolution (Feuk et al., 2006; Urban et al., 2006; Schuster et al., 2010).
Gene polymorphisms are also of importance in pharmacogenetic studies, which focus on the role of inheritance as a possible basis for the variation in relation to drugs by different individuals (Raftogianis et al., 1999). Drug response phenotypes can vary from adverse drug reactions at one end of the spectrum to equally serious lack of the desired effect of drug therapy at the other. Many of the current important examples of pharmacogenetics involve inherited variation in drug metabolism. Sulfate conjugation catalyzed by cytosolic SULT enzymes, particularly SULT1A1, is a major pathway for drug metabolism in humans (Price et al., 1989). An abstract of a study by Hebbring et
al., (2008) states that pharmacogenetic studies of SULT1A1 began over a quarter of a century ago and have advanced from biochemical genetic experiments to include cDNA and gene cloning, gene resequencing, and functional studies of the effects of single
Page | 14
nucleotide polymorphisms (SNPs). SNP genotyping, in turn, led to the discovery of functionally important copy number variations (CNVs) in the SULT1A1 gene. SULT1A1 represents one example of the potential importance of CNV for the evolving disciplines of pharmacogenetics and pharmacogenomics.
2.1.4.3. Population studies on SULT1A1 variability
An important field of interest in gene polymorphic investigations is population studies.
SULT1A1 genotyping studies have been conducted previously on different populations
and some of the findings are presented in Table 2.2. The most common characterised polymorphism, SULT1A1*2, results from a point mutation (CGC→→→→CAC) and produce an amino acid change of arginine 213 to histidine (Coughtrie et al., 1999). The SULT1A1*2 polymorphism has been the focus in many genotyping studies because of its high frequency in the population and its consequence on the sulphonation of drugs, xenobiotics and carcinogens (reviewed by Banoglu, 2000).
Page | 15
Table 2.2: SULT1A1 genotype distribution in previously studied populations
Population
Genotype
N
SULT1A1*1 SULT1A1*1/*2 SULT1A1*2
Population studies on Caucasian groups
Caucasian (Coughtrie et al., 1999) 46.76 42.32 10.92 293 Caucasian (Nowell et al., 2004) 35.16 46.77 18.06 310 Caucasian (Sun et al., 2005) 39.94 45.50 14.56 666 Caucasian (Gjerde et al.., 2007) 47.68 37.09 15.23 151
Population studies on Asian groups
Japanese (Ozawa et al., 1999) 69.23 27.97 2.80 143 Chinese (Han et al., 2004) 83.33 14.32 2.35 426 Japanese (Ohtake et al., 2006) 70.87 25.24 3.88 103
Population studies on African groups
Nigerian
(Coughtrie et al., 1999) 53.85 38.46 7.69 52 African-American
(Nowell et al., 2004) 49.46 44.09 6.45 93 Black South African
(Dandara et al., 2006) 46.82 27.71 25.48 314 Mixed-ancestry South African
(Dandara et al., 2006) 53.19 24.47 22.34 188 Tswana* Unknown Unknown Unknown 1 867
*This study
Table 2.2 gives the SULT1A1 genotypes (sorted according to the year in which the results were published) of some of the Caucasian, Asian and African population groups previously studied. Comparing the Caucasians and Asians, there are big differences in the genotype distribution i.e. the SULT1A1*2 genotype occurrences is between 10 and 16 percent in the Caucasian populations, and between only 2 and 4 percent in the Asian populations. The Nigerian and African-American populations on the other hand compare favourably to the Caucasians. South African black and mixed-ancestry populations have the highest SULT1A1*2 genotype, 22 to about 26%. The mixed-ancestry individuals
Page | 16 (sometimes referred to as ‘coloureds’) are a mix of nationalities resulting from gene flow among black South Africans, Western Europeans, the San, the Khoikhoi, Indonesians and Malaysians who settled in the Cape from the 17th century onwards (Dandara et al., 2006). These results are from non-homogeneous populations, however. Table 2.2 shows clearly that there are genotype discrepancies (in the South African studied populations, especially) when comparing populations of different ancestry. These discrepancies were partially the motivation for this study. The SULT1A1 genotypes were investigated on a largest group studied to date (1867) made up of normal subjects of a homogeneous Tswana population.
After SULT1A1 genotyping and allele frequency distribution determination, more literature survey was performed and an article by Hebbring et al., (2007) in which they report on Human SULT1A1 gene: copy number differences and functional implications was found and one other article reporting on the SULT1A1 gene copy number in Norwegian cancer patients (Gjerde et al., 2007) was found. Table 2.3 represents findings from these two studies. Copy number differences in the Tswana population group were investigated using Hebbring and associates (2007) method as an attempt to shed more light on genetic variations of Tswana homogeneous population group.
