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Title Evidence for a genetic mechanism of the chromogranin A and phenylethanolamine-N- methyltransferase genes in the pathogenesis of hypertension in the spontaneously hypertensive rat

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Author Friese, Ryan Scott

Publication Date 2007

Peer reviewed|Thesis/dissertation

eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO

Evidence for a Genetic Mechanism of the Chromogranin A and

Phenylethanolamine-N-methyltransferase Genes in the Pathogenesis of

Hypertension in the Spontaneously Hypertensive Rat

A dissertation submitted in partial satisfaction of the

requirements for the degree Doctor of Philosophy

Bioengineering

Ryan Scott Friese

Committee in charge:

Professor Daniel T. OConnor, Chair Professor Geert W. Schmid-Schönbein, Co-Chair Professor Shu Chien Professor Nicholas J. Schork Professor Shankar Subramaniam

2007

Ryan Scott Friese, 2007

acceptable in quality and form for publication on microfilm:

______

______Co-Chair

______Chair

University of California, San Diego

2007

iii

To Mom and Sis:

Together we can achieve great things

Namasté

TABLE OF CONTENTS

Signature Page………………………………………………………………….. iii

Dedication……………..……………………………………………………….... iv

Table of Contents……..………..………………………………………………. v

List of Abbreviations…………………..……………………………………...... vii

List of Figures…………………..………………………………………...... viii

List of Tables……………………………..………………………………...... x

Acknowledgements………………………………………………………...... xi

Vita and Publications………………………………………………………...... xiii

Abstract………………………………………….…………………………...... xiv

Chapter 1: Introduction……………………….…………………………...... 1

Text………………………………………………………………...... 2 References……………………………………………………………...... 11

Chapter 2: Common Genetic Mechanisms of Blood Pressure Elevation in Two Independent Rodent Models of Human Essential Hypertension……………………...... …... 17

Abstract………………………………………………………….……...... 18 Introduction…………………………………………………………...... 18 Methods………………………………………………………………...... 19 Results…………………………………………………………………...... 20 Discussion………………………………………………………………...... 21 Conclusion…………………………………………………………………... 34 References………………………………………………………………….. 35

Chapter 3: Identification of and Polymorphism Discovery in Candidate Genes for Hypertension in the Spontaneously Hypertensive Rat……………………………………………….. 38

Abstract………………………………………………………….……...... 39 Introduction…………………………………………………………...... 40 Methods………………………………………………………………...... 43 Results…………………………………………………………………...... 47 Discussion…………………………………………………………………... 53 Conclusion…………………………………………………………………... 63 References………………………………………………………………….. 80

Chapter 4: Promoter Polymorphisms Contribute to Adrenal mRNA Differential Expression of the Chromogranin A and Phenylethanolamine-N-methyltransferase Genes in the Spontaneously Hypertensive Rat……………… 84

Abstract………………………………………………………….…………... 85 Introduction………………………………………………………………….. 86 Methods……………………………………………………………………… 88 Results………………………………………………………………………. 95 Discussion………………………………………………………………...... 99 Conclusion…………………………………………………………………... 111 References………………………………………………………………….. 149

Chapter 5: Conclusion…………………….………………………………… 155

Appendix A: PCR and Sequencing Primers……………………………...... 159

Appendix B. Candidate Gene Nucleotide Sequence………………...... 166

Appendix C: Mutagenesis Primers…………………….…………………….. 217

LIST OF ABBREVIATIONS

3-UTR: 3-untranslated region

5-UTR: 5-untranslated region

ANOVA: analysis of variance

BN: Brown Norway rat (normotensive control strain for the SHR)

Chga: chromogranin A

Comt: catechol-O-methyltransferase

Dbh: dopamine beta-hydroxylase

Ednrb: endothelin receptor, type B

Etfdh: electron-transferring-flavoprotein dehydrogenase

GRE: glucocorticoid response element

Npy: neuropeptide Y

PACAP: pituitary adenylate cyclase-activating peptide

PCR: polymerase chain reaction

Pnmt: phenylethanolamine-N-methyltransferase

QTL: quantitative trait locus

SHR: Spontaneously Hypertensive Rat (genetically/hereditary hypertensive rat)

SNP: single nucleotide polymorphism

WKY: Wistar-Kyoto rat (normotensive control strain for the SHR)

vii

LIST OF FIGURES

Figure 2.1: Distribution of significantly differentially expressed orthologs in SHR and BPH……………………………………….. 21

Figure 2.2: Functional classification of shared orthologous genes in SHR and BPH…………………………………..………. 26

Figure 2.3: Catecholamines and sympathetic function in SHR and BPH… 27

Figure 2.4: Steroid hormone biosynthesis/degradation and receptors……. 27

Figure 2.5: Differences in metabolic gene expression patterns between SHR and BPH…………………………………………… 32

Figure 3.1: Candidate gene identification strategy………………………….. 66

Figure 3.2: Rationale for candidate gene selection…………………………. 67

Figure 3.3: The Chga gene is a positional candidate for a RI strain adrenal Chga protein QTL……………………….. 69

Figure 3.4: Polymorphism discovery in the Comt gene…………………….. 70

Figure 3.5: Polymorphism discovery in the Ednrb gene……………………. 71

Figure 3.6: Polymorphism discovery in the Etfdh gene…………………….. 72

Figure 3.7: Polymorphism discovery in the Npy gene………………………. 73

Figure 3.8: Polymorphism discovery in the Chga gene…………………….. 74

Figure 3.9: Polymorphism discovery in the Pnmt gene…………………….. 75

Figure 4.1: Polymorphism within the Chga promoter..……………………… 112

Figure 4.2: Chga promoter constructs..………………………………………. 114

viii

Figure 4.3: Transcriptional response of SHR/WKY Chga promoters to dexamethasone, nicotine, and PACAP………..... 116

Figure 4.4: Polymorphism within the Chga 3-UTR.……………………...…. 117

Figure 4.5: Chga 3-UTR constructs………………………………………...... 119

Figure 4.6: Chga 3-UTR luciferase assays………………………………..... 120

Figure 4.7: Sequence of SHR and WKY Chga protein…………………...... 122

Figure 4.8: Inter-species conservation of Chga protein sequence……...... 124

Figure 4.9: Predicted coiled-coil conformation of SHR and WKY Chga...... 126

Figure 4.10: Chga cDNA/EAP reporter constructs……………...... 128

Figure 4.11: Chga cDNA/EAP reporter assays…..…………...... 130

Figure 4.12: EAP assay relative secretion and sorting index...... 132

Figure 4.13: Polymorphism within the Pnmt promoter...... 133

Figure 4.14: Pnmt promoter constructs...... 135

Figure 4.15: The SHR and BN Pnmt promoters lack differential response to PACAP and nicotine...... 137

Figure 4.16: The SHR Pnmt promoter shows a blunted dose-dependent response to dexamethasone...... 139

Figure 4.17: The T-529C Pnmt promoter SNP is adjacent to a GRE...... 140

Figure 4.18: Pnmt promoter SNP variant luciferase assays…...... 142

LIST OF TABLES

Table 2.1: SHR and BPH ortholog comparison….……………………...…. 21

Table 2.2: Orthologous probe sets overexpressed in both SHR and BPH……………………………………………. 22-23

Table 2.3: Orthologous probe sets underexpressed in both SHR and BPH…………………………………………..... 24-25

Table 2.4: Oxidative stress functional cluster……….…………………….. 28-31

Table 3.1: Candidate genes for hypertension in the SHR……………...... 76

Table 3.2: Positional candidates for SHR blood pressure QTLs...... …. 77

Table 3.3: Polymorphism discovery in candidate genes………………….. 78

Table 3.4: Lack of specificity of the Etfdh probe set...... ………. 79

Table 4.1: SHR and WKY Chga promoters respond differentially to transcriptional stimuli…………………………… 143

Table 4.2: SHR and BN Pnmt promoters lack differential response to PACAP and nicotine……………………………….. 144

Table 4.3: The SHR Pnmt promoter shows a blunted dose-dependent response to dexamethasone………………... 145

Table 4.4: Pnmt promoter SNP constructs………………………………… 146

Table 4.5: Luciferase assay results from the Pnmt SNP variant constructs created from the BN promoter..…………… 147

Table 4.6: Luciferase assay results from the Pnmt SNP variant constructs created from the SHR promoter..…………. 148

ACKNOWLEDGEMENTS

First and foremost, I need to thank my mentor Dr. Daniel OConnor for his tutelage and guidance throughout my graduate career and my co-advisor

Dr. Geert Schmid-Schönbein for his thoughtful and visionary counsel. I also need to thank Dr. Shu Chien, Dr. Nicholas Schork, and Dr. Shankar

Subramaniam for their constructive feedback and suggestions. Each of my committee members challenged me to expand the breadth and depth of my knowledge and fostered my growth as a scientist and, more importantly, as an individual. Thank you all!

Special thanks to Dr. Nitish Mahapatra, my trusted mentor and devoted teacher in the lab. Vafa Mahboubi and Kenton Murthy provided vital instruction and advice on genomic DNA resequencing. Martin Jirout generously contributed Pnmt sequencing data and Chga and Pnmt SHR RI strain QTLs.

Ted Kurtz kindly provided genomic DNA from the SHR and WKY rat strains.

Thank you to Dr. Morton Printz for providing the SHR and WKY adrenal tissue.

The rat chromogranin A cDNA clone was generously provided by Lee Eiden.

Plasmid DNA sequencing was performed by the University of California, San

Diego, Cancer Center, DNA Sequencing Shared Resource. The University of

California, San Diego, Veterans Medical Research Foundation and Center for

AIDS Research Genomics Core performed the Real-Time PCR experiments.

The University of California, San Diego, Veterans Medical Research

Foundation Microarray Center performed the GeneChip® experiments.

Chapter 2, in full, is a reprint of the material as it appears in the

American Journal of Hypertension (Friese RS, Mahboubi P, Mahapatra NR,

Mahata SK, Schork NJ, Schmid-Schönbein GW, O'Connor DT. Common genetic mechanisms of blood pressure elevation in two independent rodent models of human essential hypertension. Am J Hypertens. 2005 May;18(5 Pt

1):633-52). The dissertation author was the primary investigator and author of this paper and the following co-authors directed and supervised the research:

Daniel T. OConnor, Geert W. Schmid-Schönbein, Nicholas J. Schork, Sushil

K. Mahata, Nitish R. Mahapatra, and Payam Mahboubi.

The text of Chapter 3 and Chapter 4, in part or in full, will be submitted for publication. The dissertation author was the primary researcher and author, and the following co-authors directed and supervised the research: Daniel T.

OConnor, Geert W. Schmid-Schönbein, Nitish R. Mahapatra, and Martin

Jirout.

xii

VITA

2001 B.S., Biomedical/Biochemical Engineering, University of Southern California

2003 M.S., Bioengineering, University of California, San Diego

2002-2007 Research Assistant, Departments of Bioengineering and Medicine, University of California, San Diego

2007 Ph.D., Bioengineering, University of California, San Diego

PUBLICATIONS

Friese RS, Mahboubi P, Mahapatra NR, Mahata SK, Schork NJ, Schmid- Schoenbein GW, O'Connor DT. Neuroendocrine transcriptome in genetic hypertension: multiple changes in diverse adrenal physiological systems. Hypertension. 2004 June;43(6):1301-1311.

Friese RS, Mahboubi P, Mahapatra NR, Mahata SK, Schork NJ, Schmid- Schönbein GW, OConnor DT. Common genetic mechanisms of blood pressure elevation in two independent rodent models of human essential hypertension. American Journal of Hypertension. 2005 May;18(5 Pt 1):633- 652.

Zhang K, Rao F, Wen G, Salem R, Vaingankar S, Mahata M, Mahapatra NR, Lillie EO, Cadman PE, Friese RS, Hamilton BA, Hook VY, Mahata SK, Taupenot L, OConnor DT. Catecholamine storage vesicles and the metabolic syndrome: The role of the chromogranin A fragment pancreastatin. Diabetes, Obesity, and Metabolism. 2006 Nov;8(6):621-633.

Friese RS, Rao F, OConnor DT. The Adrenal Medulla in Hypertension. In: Molecular Mechanisms in Hypertension. Editor: Sowers JR. Taylor and Francis Medical Books, London, 2006.

Rao F, Friese RS, Wen G, Zhang L, Taupenot L, Mahata SK, Ziegler MG, OConnor DT. Catecholamines, pheochromocytoma, and hypertension: genomic insights. In: Comprehensive Hypertension. Editor: Schiffrin E. Elsevier, 2007.

xiii

ABSTRACT OF THE DISSERTATION

Evidence for a Genetic Mechanism of the Chromogranin A and

Phenylethanolamine-N-methyltransferase Genes in the Pathogenesis of

Hypertension in the Spontaneously Hypertensive Rat

Ryan Scott Friese

Doctor of Philosophy in Bioengineering

University of California, San Diego, 2007

Professor Daniel T. OConnor, Chair

Professor Geert W. Schmid-Schönbein, Co-Chair

Dissection of the genetic basis of human essential hypertension is greatly hindered by the inherent complexity of the disorder. Indeed, susceptibility genes are estimated to contribute ~20-30% to development of essential hypertension, and gene actions are confounded by environmental variables, such as diet and exercise, and the diverse heterozygosity of the human population. In contrast, identification of susceptibility genes in the

xiv

Spontaneously Hypertensive Rat (SHR) model of human essential hypertension is greatly enhanced by the purely genetic basis of hypertension in the SHR and the ability to strictly control the environment. The large gap in physiology between the immediate action of susceptibility genes and the ultimate development of hypertension, however, looms large, even in the SHR.

The objective of the dissertation, therefore, is to identify polymorphisms within candidate genes for hypertension in the SHR that manifest as intermediate phenotypes for the ultimate hypertensive disease state. Differential expression of adrenal mRNA is proposed as a novel intermediate phenotype for hypertension in the SHR. Initially, a comparison of adrenal gene expression between the SHR rat and BPH mouse, two independent models of human essential hypertension, was performed to uncover common genetic mechanisms of hypertension across mammalian species. A diverse set of differentially expressed genes and biochemical systems within and between the SHR and BPH strains was identified, reinforcing the complexity of the disease. Next, candidate genes for hypertension in the SHR were identified using a method to integrate adrenal gene expression data with blood pressure

QTL data. Seven candidate genes were identified and resequenced.

Polymorphisms discovered in the promoter and/or 3-untranslated region of the chromogranin A (Chga) and phenylethanolamine-N-methyltransferase (Pnmt) genes were hypothesized to contribute to differential expression of Chga

(1.73-fold overexpressed) and Pnmt (0.67-fold underexpressed) adrenal mRNA in the SHR. Finally, luciferase assays were used to demonstrate that promoter polymorphisms are likely to contribute to the adrenal differential mRNA expression of Chga and Pnmt in vivo. The crucial role of Chga and

Pnmt in the biosynthesis and exocytosis of catecholamines makes them strong candidate genes for hypertension in the SHR. More importantly, the association of Chga and Pnmt with human essential hypertension makes them potential therapeutic targets as well.

xvi Chapter 1: Introduction

1 2

What is hypertension?

The American Heart Association estimates that almost one in three

(33%) adults in the U.S has elevated blood pressure, a condition also known as hypertension—this equates to about 72 million people in the U.S., age 20 and older, with the disorder. Untreated, chronic hypertension can lead to stroke, heart attack, heart failure, and kidney failure. The age-adjusted death rate from hypertension increased 25.2% over the ten-year period from 1994 to 2004.

Hypertension is clinically defined and diagnosed in three stages: (1) prehypertension—defined as blood pressure between (120-139)/(80-89) mm Hg; (2) Stage 1 hypertension—defined as blood pressure between

(140-159)/(90-99) mm Hg or higher; and (3) Stage 2 hypertension—defined as blood pressure 160/100 mm Hg or higher. The cause of hypertension in

90-95% of all cases is unknown, and this idiopathic type of hypertension is referred to as essential or primary hypertension. Essential hypertension is often observed with the presence of other risk factors for cardiovascular disease, including abdominal obesity, atherogenic dyslipidemia (high triglycerides, low HDL cholesterol, high LDL cholesterol), insulin resistance/glucose intolerance, a prothrombotic state (e.g. elevated fibrinogen or plasminogen activator inhibitor–1 in the blood), and a proinflammatory state (e.g. elevated C-reactive protein in the blood)—this

3 cluster of cardiovascular risk factors is often referred to as the “metabolic syndrome1.”

Genetic dissection of hypertension

Essential hypertension is commonly observed in families but many people with a positive family history never develop the disorder. Indeed, the contribution of genes to development of essential hypertension is estimated to be ~20-30%2. Traditional approaches to elucidate the genetic underpinnings of human disease, such as linkage analysis and QTL mapping, have conclusively pinpointed susceptibility loci responsible for monogenic,

Mendelian disorders such as Huntington disease3,4 and cystic fibrosis5-7. In contrast, human essential hypertension is a complex trait that involves multiple genes either acting independently or acting interactively with themselves and the environment, and linkage analysis and QTL mapping have thus far yielded weakly tractable results for human essential hypertension. The polygenic, intricate, and complex nature inherent to essential hypertension proves problematic for traditional linkage analysis and QTL mapping8,9.

The SHR/WKY model of hypertension

Model organisms are a potentially powerful system in which to study the genetic basis of essential hypertension. In particular, pairs of hypertensive and

4 normotensive inbred rodent strains, which have been studied for over 40 years10-12, constitute ideal models for genetic studies because the disease progression is completely dictated by genetics and the environment can be strictly controlled. Many rat strains have been developed that exhibit spontaneous hypertension, such as the Spontaneously Hypertensive Rat

(SHR)10 and the Stroke-Prone SHR (SHRSP)13 strains, as well as salt-sensitive hypertension, such as the Dahl11,12 and Sabra strains14.

The Spontaneously Hypertensive Rat (SHR) and its normotensive control, the Wistar-Kyoto rat (WKY), form the current paradigm for essential hypertension research. Kozo Okamoto and Kyuzo Aoki developed the SHR and WKY in 1963 by selective breeding of an outbred stock of Wistar rat10.

Initially, a male Wistar rat showing spontaneous hypertension (systolic blood pressure ranging from 150 – 175 mm Hg for at least 1 month) was mated with a female Wistar rat with a blood pressure slightly above the average (systolic blood pressure ranging from 130 – 140 mm Hg for at least 1 month) to obtain

an F1 population. Then, hypertensive (systolic blood pressure exceeding 150

mm Hg for at least one month) male and females within the F1 population were

mated in brother-sister combinations to produce a F2 generation. Selection on

the basis of hypertension and brother-sister mating was continued in F2 and

through F6. One-hundred percent incidence of spontaneous hypertension was

observed in all generations after and including F3. Starting with F6, the

5 hypertensive rats were inbred to homozygosity to produce the SHR.

Inbreeding of the SHR has now surpassed F70. The WKY was developed concurrently with the SHR in a selective breeding scheme based on normal blood pressure, which Okamoto and Aoki defined as systolic pressure less than 149 mm Hg (the generally accepted threshold for normotension/hypertension at the time of the study). Inbreeding of the

homozygous WKY strain has now surpassed F70.

In addition to elevated blood pressure, the SHR exhibits many of the co- morbidities observed in human essential hypertension, such as insulin resistance15,16, dyslipidemia17,18, and vascular inflammation19. The vascular inflammation observed in the SHR is of particular importance because evidence is increasing for its role in the pathogenesis of vascular lesions, end- organ damage, and even hypertension19. The SHR exhibits elevated levels and activation of leukocytes20,21, increased reactive oxygen species (ROS) and oxidative stress22-30, and enhanced endothelial cell apopotisis31-33 and capillary rarefaction31,34. Evidence suggests that glucocorticoid hormones secreted from the adrenal cortex contribute to vascular inflammation and hypertension in the

SHR35-38.

The adrenal gland in hypertension

In addition to its ability to secrete glucocorticoids, the adrenal gland is a compelling target for the study of essential hypertension because its other secretory products, both medullary and cortical, can directly modulate endocrine, cardiovascular, and sympathetic functions. The adrenal medulla is highly innervated with preganglionic sympathetic fibers from the splanchnic nerve and is capable of biosynthesis and secretion of catecholamines (i.e. dopamine, norepinephrine, epinephrine) directly into the circulation through the adrenal vein. The chromaffin cell is the principal cell type in the adrenal medulla and is specialized for biosynthesis, vesicular storage, and regulated secretion of catecholamines. The adrenal cortex envelops the medulla and is the site of biosynthesis and secretion of several classes of steroid hormones, including mineralocorticoids, glucocorticoids, and sex steroids. The zona glomerulosa, zona fasiculata, and zona reticularis constitute the adrenal cortex, and each zone contains histologically and functionally distinct cells that synthesize and secrete mineralocorticoids, glucocorticoids, and sex steroids, respectively. The entire adrenal gland is richly vascularized with a generally centripetal circulation (cortex to medulla), and all medullary and cortical secretory products exit the gland through the single, large adrenal vein.

Catecholamines function as both neurotransmitters and circulating, endocrine hormones, but in the adrenal medulla both norepinephrine and

7 epinephrine are synthesized and released into the circulation as endocrine hormones. Catecholamines are stored for regulated exocytotic secretion in secretory vesicles, which, in chromaffin cells, are known as chromaffin granules. Biosynthesis of catecholamines begins in the cytosol with the conversion of phenylalanine, an essential dietary amino acid, to the amino acid tyrosine by the enzyme phenylalanine hydroxylase. Tyrosine hydroxylase then catalyzes the rate-limiting step in catecholamine biosynthesis: conversion of tyrosine to dihydroxyphenylalanine (DOPA). The enzyme DOPA decarboxylase converts DOPA to dopamine, which is shuttled into catecholamine storage vesicles via vesicular monoamine transporters. Inside the storage vesicles, dopamine -hydroxylase catalyzes the conversion of dopamine to norepinephrine. Norepinephrine is the final catecholamine product in ~15–20% of adrenal chromaffin cells. In the remaining 80–85% of adrenal chromaffin cells, phenylethanolamine-N-methyltransferase, a cytosolic enzyme, coverts norepinephrine to epinephrine39.

In addition to catecholamines, chromaffin granules contain a variety of soluble proteins and bioactive peptides that are co-released with the catecholamines, including a group of acidic and soluble secretory proteins known as the chromogranins/secretogranins, or granins. The granin family currently consists of seven proteins: chromogranin A, chromogranin B, secretogranin II (or chromogranin C), secretogranin III (or 1B1075),

8 secretogranin IV (or HISL-19), secretogranin V (or 7B2), and secretogranin VI

(or NESP55). The granins have roles in vesiculogenesis39 but also serve as pro-hormones39 that can be proteolytically processed to yield bioactive peptides with a wide variety of effects, including regulation of catecholamine secretion. Granins are ubiquitously distributed in neuroendocrine and nervous tissue and are co-secreted with catecholamines – these properties make the granins useful indicators of sympathoadrenal activity and secretion.

Chromogranin A measurements in particular have proved clinically useful for diagnosis and treatment of pheochromocytoma40,41 and have even provided clues to the pathogenesis of essential hypertension42,43. Chromogranin A was the first granin discovered and has been studied the most extensively.

Bioactive peptide products of chromogranin A are capable of modulating catecholamine secretion (the catestatin fragment)44-46, vasodilation (the vasostatin fragment)39,47, glucose homeostasis (the pancreastatin fragment)48-

53, immune response (the chromacin and prochromacin fragments)39, and vascular inflammation (the vasostatin fragment)54-57.

Synopsis of the dissertation

The genetic basis of hypertension in the SHR remains largely unknown, and although linkage analysis has produced ~80 blood pressure Quantitative

Trait Loci (QTLs) in the SHR, a detailed investigation of candidate genes that

9 includes resequencing and functional testing of discovered polymorphisms is lacking. The large gap in physiological mechanism between candidate gene mutations and development of hypertension looms large. Therefore, the objective of the dissertation is to begin to bridge the mechanistic gap between polymorphism and disease by identifying strong candidate genes for hypertension in the SHR and establishing an initial mechanism whereby polymorphisms in the candidate genes can eventuate in hypertension. A series of experiments described in Chapter 2 through Chapter 4 present a novel approach to accomplish this objective.

First, in Chapter 2, a comparison of gene expression in the adrenal gland of two independent rodent models of human essential hypertension (the

SHR rat and the BPH mouse) is performed with the goal of uncovering common genetic mechanisms of hypertension across mammalian species.

Then, in Chapter 3, candidate genes for hypertension in the SHR are identified using a novel, integrative method that combines adrenal gland microarray data from Chapter 2 with preexisting QTL data. The crucial hypothesis underlying the method is that the nucleotide mutations in the genes responsible for hypertension in the SHR manifest as changes in mRNA transcript abundance in the adrenal gland, even before onset of classical hypertensive disease phenotypes. Polymorphisms in candidate genes are identified and hypothesized to contribute to differential expression of the genes

10 in the SHR adrenal gland.

Finally, in Chapter 4, luciferase reporter experiments are used to show that polymorphisms discovered in the promoter regions of the chromogranin A and phenylethanolamine-N-methyltransferase candidate genes are likely to contribute to mRNA differential expression of these genes in the SHR adrenal gland. The crucial role of chromogranin A and phenylethanolamine-N- methyltransferase in the biosynthesis and exocytosis of catecholamines makes them strong and worthwhile candidate genes for the study of the genetic basis of hypertension in the SHR.

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50. Sanchez-Margalet, V, Gonzalez-Yanes, C, Santos-Alvarez, J & Najib, S. Pancreastatin. Biological effects and mechanisms of action. Adv Exp Med Biol 482, 247-262 (2000).

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Chapter 2: Common Genetic Mechanisms of Blood Pressure Elevation in Two Independent Rodent Models of Human Essential Hypertension

17 18

Common Genetic Mechanisms of Blood Pressure Elevation in Two Independent Rodent Models of Human Essential Hypertension

Ryan S. Friese, Payam Mahboubi, Nitish R. Mahapatra, Sushil K. Mahata, Nicholas J. Schork, Geert W. Schmid-Schönbein, and Daniel T. O Connor

Genetic studies of essential hypertension, a complex, poly- used a systems biology approach to analyze expression genic, and age-dependent disorder, have not been able to patterns in biochemical pathways and networks to isolate completely elucidate the genes responsible for develop- systems involved in hypertension pathology in both SHR ment of the trait. We used a novel strategy to compare and BPH. We found transcript pattern evidence for in- gene expression in the adrenal gland of two independent volvement of several systems in the pathology of hyper- rodent models of human essential hypertension (the spon- tension in SHR and BPH: adrenal catecholamines and taneously hypertensive rat, SHR, and the blood pressure sympathetic function; steroid hormone synthesis, catabo- high mouse, BPH), with the goal of uncovering shared, lism, and its contribution to enhanced glucocorticoid sen- common genetic mechanisms of hypertension across sitivity in SHR; oxidative stress and its role as a common mammalian species that might, therefore, be pertinent to mechanism of vascular and end-organ injury; and inter- human hypertension. We deliberately studied young, 4- to mediary metabolism with global but mechanistically dif- 5-week-old, “prehypertensive” SHR and BPH that had not ferent perturbations in SHR and BPH. Approximately 10% yet developed complete elevations in blood pressure (BP), of the differentially expressed orthologous genes we stud- so that we could minimize the impact of chronic, sustained ied shared a common direction of expression in the two BP elevation, age, and other confounding factors on gene hypertensive rodent strains, suggesting fundamental tran- expression, therefore increasing the likelihood that differ- scriptional mechanisms in common whereby mammals ential expression reﬂects relatively early pathogenic mech- can elevate BP or respond to such elevation; even these anisms in hypertension, rather than later responses to, or shared orthologs spanned a diverse set of biological pro- compensations for BP elevation. We compared transcript cesses, reinforcing the multifactorial and complex nature expression patterns of genes orthologous between the rat of hypertension. Am J Hypertens 2005;18:633–652 and the mouse, and presented candidate genes for hyper- © 2005 American Journal of Hypertension, Ltd. tension that are differentially expressed in the same direction in SHR and BPH (ie, overexpressed in both SHR and Key Words: Hypertension, genetic, hereditary, adrenal BPH, or underexpressed in both SHR and BPH). Then we gland, gene expression, genome, transcriptome.

he complex, polygenic, and age-dependent na- and other facets of the “cardiovascular dysmetabolic ture of human essential hypertension has made it syndrome”2 that often accompany essential hyperten- T difﬁcult to isolate the primary genetic causes of sion. the disease. The exact mechanisms of hypertension are Microarrays are a potentially powerful tool for studying further confounded by environmental factors (eg, diet the genetics of hypertension as they allow measurement of and exercise), which are estimated to contribute up to the expression of thousands of genes simultaneously. In- 70% to development of the disease trait,1 and the pres- bred, homozygous rodent models of human essential hy- ence of dyslipidemia, dysglycemia, insulin resistance, pertension are ideal for microarray research but only a

Received September 16, 2004. First decision October 26, 2004. Accepted This study was supported by the Department of Veterans Affairs and November 24, 2004. National Institutes of Health. From the Departments of Bioengineering (RSF, GWS-S), Medicine Address correspondence and reprint requests to Dr. Daniel T. (PM, NRM, SKM, DTO), Pharmacology (DTO), and Psychiatry (NJS), O’Connor, Department of Medicine and Center for Molecular Genetics, Center for Molecular Genetics (DTO), Polymorphism Research Labora- University of California at San Diego and VASDHS (0838), 9500 Gil- tory, University of California (NJS, DTO), and VA San Diego Healthcare man Drive, La Jolla, CA 92093-0838; e-mail: [email protected] System (SKM, DTO), San Diego, California.

© 2005 by the American Journal of Hypertension, Ltd. 0895-7061/05/$30.00 Published by Elsevier Inc. doi:10.1016/j.amjhyper.2004.11.037 19 limited amount of research using microarrays and animal do not yet have maximal elevation of BP. The SHR and models of essential hypertension has been presented thus BPH models appear to experience the same degree of far.3–9 We present a comparison of adrenal gland gene hypertension: with maturity of the SHR and BPH, systolic expression in two independent, inbred, homozygous ro- BPs diverge to a maximum of 70 mm Hg between SHR dent models of human essential hypertension: the sponta- and its WKY control, and to a maximum of 60 mm Hg neously hypertensive rat (SHR) and the blood pressure between the BPH and its BPL control. Studying prehyper- high mouse (BPH). tensive animals allowed us to minimize the effect of con- The SHR, the current paradigm for essential hypertension founding factors on gene expression, and therefore research, was developed in a breeding program based solely on increase the odds of detecting pathogenic mechanisms of selection by elevated blood pressure (BP) in the Wistar rat.10 hypertension, while decreasing the chance of detecting The Wistar/Kyoto (WKY) strain was established as a normo- consequential effects of BP elevation. However, the 42 tensive control strain for the SHR by inbreeding of the normo- mm Hg (120 78 mm Hg) difference in systolic BP tensive Wistar colony (from which the SHR originally emerged) between BPH and BPL mice, and the 18 mm Hg (125 by brother/sister mating.11 In addition to elevated BP, the SHR 107 mm Hg) difference between SHR and WKY rats,19 exhibits many of the co-morbidities observed in human hyper- indicate that we cannot completely exclude distinct tension, such as insulin resistance, hypertriglyceridemia, and responses to BP as determinants of differential gene ex- abdominal obesity.12–15 The BPH inbred, hypertensive mouse pression between the strains, even at these early ages.17,19 strain, developed by Schlager16 in a selection program based solely on elevated BP, also parallels human hypertension with Preparation of RNA elevated BP and co-morbidities such increased heart rate and Total RNA was extracted from isolated adrenal glands of early mortality.17 During the breeding program used to develop the SHR and WKY rats, as well as the BPH and BPL mice, the BPH strain, Schlager developed the hypotensive genetically/ with the RNAzol (guanidinium thiocyanate) kit (TelTest, hereditary low BP mouse (BPL) strain to serve as a control for Friendswood, TX), followed by RNase-free DNase I (Qia- the BPH.16 gen, Valencia, CA) treatment to eliminate residual geno- Adrenal gland secretory products, both medullary and mic DNA. Integrity of the RNA was confirmed through cortical, are logical candidates to study hypertension be- 28S and 18S rRNA profiles on Agilent (Palo Alto, CA) cause they can directly influence cardiovascular, endo- columns and ethidium bromide-stained gels (data not crine, and sympathetic function. The purpose of this study shown). is to compare adrenal gene expression in two independent rodent models of the same human disease, with the goal of Microarray Analysis uncovering shared, common mechanisms of hypertension across mammalian species. Gene expression in the adrenal gland of each animal (n 3 SHR, n 3 WKY, n 3 BPH, n 3 BPL) was Methods measured using standard Affymetrix protocols and Af- Rodent Strains fymetrix (Santa Clara, CA) GeneChips, as previously described.3 RG-U34A rat GeneChips were used for mea- Three juvenile ( 4-week-old) SHR and three juvenile ( 4- surement of SHR and WKY gene expression, and MG- week-old) normotensive WKY male rats were obtained from U74Av2 mouse GeneChips were used to assay BPH and colonies at the University of California, San Diego, in La BPL gene expression. The rat RG-U34A chip contains Jolla, CA.18 Juvenile SHR are “prehypertensive” with a sys- 8740 probe sets (excluding quality controls) correspond- tolic BP ( 125 mm Hg at 6 weeks19) slightly higher than ing to all full-length, annotated rat gene clusters ( 6000) juvenile WKY ( 107 mm Hg at 6 weeks19) but still not from the UniGene database (Build 34) as well as 3000 completely elevated. Upon maturity, the SHR exhibits a expressed sequence tag (EST) clusters. The mouse MG- systolic BP ( 200 mm Hg) approximately 70 mm Hg higher U74Av2 chip contains 12,422 probe sets (excluding than that of WKY ( 130 mm Hg). quality controls) corresponding to all functionally char- Three juvenile ( 5-week-old) BPH and three juvenile acterized sequences ( 6000) in the mouse UniGene ( 5-week-old) BPL male mice were obtained from colo- database (Build 74) and thousands of EST clusters nies at the Jackson Laboratory, Bar Harbor, ME. Juvenile ( 6000). Tab-delimited text files of all chip spot features BPH are prehypertensive with a systolic BP ( 120 mm and probe design information are publicly available on the Hg at 7 weeks17), already somewhat higher than juvenile Affymetrix website: http://www.affymetrix.com. BPL ( 78 mm Hg at 7 weeks17) but still not completely We compared the expression patterns of all orthologous elevated. The divergence in systolic BP reaches its maxi- genes on the rat and mouse chips. Orthologs were derived mum at 21 weeks of age, when BPH shows a systolic BP from the HomoloGene and UniGene databases at the Na- ( 130 mm Hg)17 approximately 60 mm Hg higher than tional Center for Biotechnology Information (NCBI) that of BPL ( 70 mm Hg).17 (http://www.ncbi.nlm.nih.gov). Orthologous probe set in- We chose juvenile animals ( 4 to 5 weeks old) because formation can be downloaded from the Affymetrix web- at that age, the SHR and BPH are prehypertensive—they site: http://www.affymetrix.com. 20

Real-time–polymerase chain reaction derexpressed in both SHR and BPH). Orthologs uniquely expressed exhibit one of the following patterns of expres- A commonly used method, real-time–polymerase chain sion: 1) overexpressed in BPH, underexpressed in SHR; 2) reaction (RT-PCR), to confirm microarray fidelity, was overexpressed in BPH, no change in SHR; 3) underex- performed on SHR, WKY, BPH, and BPL adrenal mRNA pressed in BPH, overexpressed in SHR; 4) underexpressed with the SuperScript first-strand synthesis system (Invitro- gen, Carlsbad, CA), an ABI-7700 (Applied Biosystems, in BPH, no change in SHR; 5) no change in BPH, under- Foster City, CA) thermal cycler and fluorescent plate expressed in SHR; or 6) no change in BPH, overexpressed reader, and the Amplifluor universal detection system (Se- in SHR. rologicals Corporation; Norcross, GA), as previously de- The following groups were chosen for functional scribed.20 Data normalization was performed by quan- clustering based on their known or purported role in BP tification of the endogenous 18S rRNA, and final nano- control or hypertension: adrenergic receptors, apopto- gram equivalents were determined with relative standard sis, catecholamines and sympathetic function (including curve analysis (Applied Biosystems). Statistical signifi- chromogranins/secretogranins), cholinergic systems, in- cance was computed with a two-tailed Student t test. flammation (leukotriene and prostaglandin synthesis), intermediary metabolism, neurotrophins, other vasocon- SHR Cd36 Mutation strictor/vasodilator systems, oxidative stress, proteases, To detect the dysfunctional chimeric Cd36 gene in the renin-angiotensin-aldosterone system, and steroid hor- SHR, we resequenced the gene and verified the presence mone biosynthesis/degradation and receptors. of the previously published polymorphisms.21 The rat Cd36 gene was resequenced in adrenal mRNA from 12- week-old SHR and WKY rats (three each). The RT-PCR Results was performed using PTC-200 DNA Engine thermal cy- Microarray Statistical Results clers (MJ Research, Watertown, MA) using the Qiagen one-step RT-PCR kit and the following gene-specific Statistical analysis of the rat and mouse microarray exper- primers: CD36F2, [226] 5‘-CAAAAACTGGGTGAA- iments yielded similar percentages of probe sets differen- AACGGG-3‘ [246]; and CD36B2, [912] 5‘-TCAAAC- tially expressed by strain: 13.9% for SHR/WKY v 16.9% ACAGCATAGATGGACCTG-3‘ [889]. First strand for BPH/BPL (Table 1). In both experiments (ie, SHR/ cDNA was prepared from 500 ng of total RNA template WKY and BPH/BPL), about half of the differentially ex- by reverse transcription (using Omniscript and Sensiscript pressed probe sets were overexpressed and the other half reverse transcriptases) at 54°C for 30 min, followed by were underexpressed (Table 1). PCR as described previously.22 As a negative control, The rat and mouse GeneChips contain probe sets for when RNA was pretreated with RNase A (Qiagen), no 5273 genes orthologous between the two species. Each product in the RT-PCR assay was detected after gel elec- chip contained a large portion of orthologous genes: trophoresis. As a second negative control, no PCR product 60.3% of the rat chip and 42.4% of the mouse chip. In both was obtained when water was taken instead of RNA the SHR and BPH, about half of the differentially ex- samples in the reaction mixture. The RT-PCR products pressed orthologs were overexpressed and about half were were then sequenced on ABI-3100 automated fluorescent underexpressed (Table 1). Orthologs designated as com- DNA sequencer. monly differentially expressed (ie, overexpressed in both BPH and SHR, or underexpressed in both BPH and SHR) Data Analysis comprised 10% of the differentially expressed orthologs Statistical analysis of the microarray data was performed (Table 1, Fig. 1). Approximately 90% of the orthologs with Cyber-T,23 a bayesian probabilistic framework de- differentially expressed in SHR or BPH were uniquely signed for microarray experiments without large numbers expressed (defined in Methods) (Table 1, Fig. 1). of replicates, as well as a standard t test. Probe sets were Did the directional patterns of ortholog differential ex- considered significantly differentially expressed at P .05 pression (Table 1) differ from those expected by chance for Cyber-T or t test to minimize false negatives and gain alone? In the rat, for example, 11.4% of the differentially a broad perspective on biochemical systems perturbed in expressed orthologs (93/815) shared a common direction, the hypertensive rodent strains. whereas in the mouse this value was 9.2% (93/1012). The All probe sets, regardless of statistical significance, expectation of differential expression by chance alone were sorted into orthologous and functional clusters. The might be stated: 1 : 1 : 1 :1 , or 25%:25%: orthologous clusters consisted of two distinct groups: or- 25%:25%. Thus, directionally shared differential expres- thologs with common expression, and orthologs with sion was observed to be substantially less than predicted unique expression. We define commonly expressed or- under random conditions (in the rat, 2 154, P .0001; thologs as being significantly differentially expressed in in the mouse, 2 224, P .0001). Therefore, such the same direction in both of the hypertensive rodent rat:mouse directional pairings appear to be a highly re- strains (ie, overexpressed in both SHR and BPH, or un- stricted subset of all differentially expressed genes. 21

Table 1. SHR and BPH ortholog comparison

Species Strains Rat SHR, WKY Mouse BPH, BPL GeneChip RG-U34A MG-U74Av2 Probe sets 8740 12422 Total differentially expressed probe sets 1217 (13.9%) 2108 (16.9%) Overexpressed probe sets 580 1059 Underexpressed probe sets 637 1049 Orthologous probe sets 5273 (60.3%) 5273 (42.4%) Total differentially expressed orthologs 815 (15.4% of 5273) 1012 (19.2% of 5273) Overexpressed orthologs 389 492 Common (shared by rat and mouse) 41 (10.5% of 389) 41 (8.3% of 492) Unique (unshared by rat and mouse) 348 (89.5% of 389) 451 (91.7% of 492) Underexpressed orthologs 426 520 Common (shared by rat and mouse) 52 (12.2% of 426) 52 (10% of 520) Unique (unshared by rat and mouse) 374 (87.8% of 426) 468 (90% of 520)

The number of significantly differentially expressed genes for the rat (SHR, WKY) and the mouse (BPH, BPL) is shown. “Common” expressed orthologs are differentially expressed in the same direction in SHR and BPH; ie, overexpressed in both SHR and BPH, or underexpressed in both SHR and BPH. “Unique” expressed orthologs can show six types of expression patterns (described in the Methods section). Probe sets were considered significantly differentially expressed if they achieved P . 05 by Cyber-T or by t test.

RT-PCR: Verification Discussion of Microarray Fidelity Microarray Statistical Results

Relative expression (SHR versus WKY; BPH versus BPL) The mouse GeneChip contains 3682 more probe sets than the of a subset of genes (n 25) was veriﬁed with RT-PCR rat GeneChip, yet both chips showed approximately the same (data not shown), a commonly used technique to quantify percentage of differentially expressed genes: 16.9% of the relative gene expression. Microarray and RT-PCR results mouse chip and 13.9% of the rat chip (Table 1). Such agreed over a large range of values. Linear regression widespread differential gene expression in two independent analysis for RT-PCR-fold change versus chip-fold change models of the same human disease reinforces the complex yielded Pearson correlation coefﬁcients of R 0.788 for and polygenic nature of hypertension, especially because the SHR/WKY experiment and R 0.739 for the BPH/ 66% of the differentially expressed genes exhibit subtle BPL experiment. Genes were picked from a spectrum of changes between twofold underexpressed and twofold functional categories so as to generalize our results to all Unique orthologs transcripts, rather than only a few particular systems. 100 451/492 348/389 468/520 90 374/426

Orthologous and Functional Clustering 80

Differentially expressed genes were sorted into ortholo- 70 gous and functional clusters. The cluster of differentially 60 expressed orthologs included 41 probe sets overexpressed 50 in both SHR and BPH (Table 2, Fig. 1) and 52 probe sets Percentage (%) 40 underexpressed in both SHR and BPH (Table 3, Fig. 1). 30 The actual number of genes overexpressed/underex- Common orthologs 20 pressed in common (28 overexpressed and 35 underex- 52/426 41/389 52/520 pressed) is less than the number of probe sets because of 10 41/492 redundancy in probe sets (ie, two probe sets representing 0 SHR BPH SHR BPH SHR BPH SHR BPH the same gene). Even the subset of differentially expressed Overexpressed Underexpressed Overexpressed Underexpressed orthologs with common directionality in the two species’ FIG. 1. Distribution of significantly differentially expressed or- genetically hypertensive models (Fig. 2) showed substan- thologs in SHR and BPH. The graph displays the percent of signifi- tial heterogeneity in biological processes represented. cantly (P .05) differentially expressed orthologs stratified by For the purposes of the current discussion, data for the ortholog classification (common versus unique; defined in Methods section) and direction of expression (overexpressed versus under- following functional clusters are presented: catecholamines expressed). Approximately 10% of the significantly differentially and sympathetic function (Fig. 3), steroid hormone biosyn- expressed orthologs show a “common” direction of expression, ie, thesis/degradation and receptors (Fig. 4), oxidative stress overexpressed in both SHR and BPH or underexpressed in both SHR and BPH. Conversely, 90% of the significantly differentially ex- (Table 4), and intermediary metabolism (Fig. 5). (See Sup- pressed orthologs lack the same direction of expression in SHR and plementary Tables 1 and 2 online.) BPH (ie, show “unique” expression). Table 2. Orthologous probe sets overexpressed in both SHR and BPH

Mouse Rat Fold Mouse Fold Functional Group Probe Set Gene Symbol Change Change Rat Probe Set ID ID Orthologous Gene Name (rat, mouse) (rat, mouse) (SHR/WKY) (BPH/BPL) Activation and detoxification of exogenous chemicals M26125_at 101587_at epoxide hydrolase 1, microsomal Ephx1 1.54 1.46 Adrenal tumor suppressor M32754cds_s_at 102266_at inhibin alpha Inha 1.51 1.59 Cellular adhesion AJ009698_g_at 101560_at Embigin Emb 3.37 2.64 Chaperones rc_AA818604_s_at 93875_at Hspa1b; 3.01 5.08 heat shock 70kD protein 1B; heat shock protein 1A Hspa1a Cysteine protease inhibitor AF090692_at 103245_at cystatin 8 Cst8 1.70 3.84 Glycosylation AF047707_g_at 96623_at UDP-glucose ceramide glucosyltransferase Ugcg 2.61 1.72 Inflammation S77528cds_s_at 92925_at CCAAT/enhancer binding protein (C/EBP), beta Cebpb 1.25 1.52 Intermediary metabolism rc_A1236284_s_at 102381_at fatty acid-Coenzyme A ligase, long chain 4 Facl4 1.36 1.28 M29249cds_at 99425_at 3-hydroxy-3-methylglutaryl-Coenzyme A reductase Hmgcr 2.56 3.11 rc_AA817685_at 98533_at cytochrome b-5 Cyb5 1.73 1.40 Nuclear hormone receptors X99470_at 93141_at nuclear receptor subfamily 0, group B, member 1 Nr0b1 1.81 1.47 U17254_at 102371_at immediate early gene transcription factor NGFI-B; Nr4a1 2.03 5.04 nuclear receptor subfamily 4, group A, member 1 L08595_at 92248_at nuclear receptor subfamily 4, group A, member 2 Nr4a2 4.10 3.92 Oxidative stress AB008807_at 97819_at glutathione S-transferase omega 1 Gsto1 1.51 1.28 U73525_at 98130_at thioredoxin 2 Txn2 1.26 1.20 Prohormone processing L07281_at 99643_f_at carboxypeptidase E Cpe 1.73 1.37 Signal transduction D14839_at 100346_at fibroblast growth factor 9 guanine nucleotide Fgf9 2.28 2.91 binding protein, S50461_s_at 97226_at alpha 12 Gna12 1.27 1.31 U35345_s_at 97823_g_at p21 (CDKN1A)-activated kinase 2 Pak2 1.99 1.27

D85183_s_at 103070_at protein tyrosine phosphatase, non-receptor type Ptpns1 4.47 1.86 22 substrate 1 23

overexpressed (data not shown). Furthermore, in the SHR Fold differs

mouse and BPH, genetic linkage analyses have estimated that 1 1 change l 6 5 d e activities d . . only a few major loci play a role in the pathogenesis of e an 1 1 3.07 1.66 1.09 1.67 1.39 1.27 fol 24,25 t t th hypertension (at least three in SHR, and four to ﬁve in Change symbo t ra ra 26 e , (BPH/BPL) Mous e BPH ). Therefore, much of the differential gene expres- 4) th abou gen n sion we noted is likely to reﬂect responses to these few h e r o

bot major BP-determining genes. In addition, allelic variation e n i (colum at other loci could produce gene expression differences l n 1 5 6 5 5 7 7 6 nam Fold 8 7 e knowledg entirely unrelated to BP. In both species, the number of . . t t 1 2 2.2 1.5 3.3 1.4 1.2 2.3 strai

gen differentially expressed genes is approximately divided in symbo e Change Ra e e half between overexpressed and underexpressed (Table 1), th (SHR/WKY) curren , gen n perhaps reﬂecting a complex response of activation and s o ogs d ou depression of both pressor and depressor mechanisms in

hypertensiv hypertension. base e orthol s l th n ortholog 1 i , Symbol mouse) 2 s 2 1 3)

, Orthologous Clusters e 1 a 1 severa 1 n t set 3 n a categorie I c e BC024558 l l s . (rat

Gen We compared expression of orthologs on the rat and S P DAP-4; Sdfr Nsep Zfp361 Vamp Hes prob (colum mouse chips to probe the possibility of shared or even ) e shown universal genetic mechanisms of hypertension across e BPL functiona nam l ar

e mammalian species. Intriguingly, just 10% of the differ- ) 6 entially expressed orthologs showed common directional n gen BPH 1 s genera

d patterns of expression (ie, overexpressed in both SHR and o n ) an int sequence BPH, or underexpressed in both SHR and BPH) (Table 1, (colum Y mouse) d ) 2 A , 1 Fig. 1). The set of orthologs with common patterns of WK n 1 protei e orthologou r place

, expression may represent conserved mammalian mecha- g cDN 1 (rat e 2 2) ;

e nisms of generation of, or response to hypertension, and n e r 4 s protei (SHR wer e (Drosophila a therefore may be particularly relevant to the study of d n s r e b fluorescence bindin type-lik e L recepto f m

t human essential hypertension. It is also possible that genes t 1 Nam (colum s H e r Gene n D e . I

m with common patterns of expression might not be related a protei C3 spli t r , t , f d se

3 to the hypertensive trait, and could reﬂect genetic drift facto o o e Gen n y elemen membran 36 r d i l overexpresse (with subsequent ﬁxation by strain inbreeding to homozy- i s ) e d n m prob m semicolon gosity) or random chance. Even the subset of differentially a e a fluorescence/BP .05 f e y a H expressed orthologs with common directionality in the two r derive n b i P e protei l r i d ( mous (BP

enhance species’ hypertensive models showed substantial hertero- r e r sensitiv x y r e s cel d a

e geneity of biological processes (Fig. 2). o l c h large-associate an Orthologou e

p Conversely, 90% of the differentially expressed or- finge chang t s y separate s c u d o e

l thologs exhibited unique expression patterns in SHR and Affymetri BC024558 h o , ar fol significantl s p stroma disk hair nucleas zin vesicle-associate s

e BPH (deﬁned in Methods). Initially, the gross lack of 1) e

n rat/mouse agreement in expression patterns of orthologs hav mous d t t appeared to cast doubt on the relevance of applying d t t t t t t an (colum Set a a an

e knowledge of either model to human treatment. If two D , _ _ I e D 3 5 t I 5) closely related rodents, both selected for the same trait and 3 9 names/symbol se n mous t Mouse 1 2

e inbred to homozygosity, showed 90% discordance in d 9 6 Prob 9 9 93043_a 104136_a 160887_a 93740_a 93324_a 98926_a an differential expression of their adrenal transcriptomes, prob t (colum t ra ) differen how relevant are they to human essential hypertension? In ra e o x

th fact, the difference in expression of orthologs between the tw n e i two strains may simply be a reflection of how different th g ; ID organisms respond to similar stress, or a reflection of strain Affymetri t fluorescence . t t t polymorphisms not related to BP. Because much of the Y t t Group ortholo a Se mouse a l _ n differential expression in orthologs could reflect responses t _ e d a 8 factors s 6 e an

t to hypertension or genotypes unrelated to hypertension, it _ t n 5 t 3 (continued) 0 hav ra

3 is perhaps not surprising that two independent hyperten- Prob products. 5 experiments e d 4 e 8 t y 5

th sive models would exhibit substantial differences in pat- 2. A 1 n liste A gen Ra e Functiona 0

fluorescence/WK terns of gene expression, especially when considering the s _ e B R c th D13417_a rc_A1072435_a rc_A1112516_a A M24105_a U67140_g_a r X99338cds_i_a substantial interspeciﬁc (rat/mouse) differences in factors f Gene Tabl Transcriptio Transport Unknown microarra (SH betwee o such as size, weight, lifespan, and the presence of other 24 Fold 9 7 3 3 0 4 5 1 8 7 6 6 9 7 7 9 4 4 5 6 2 6 8 8 6 4 2 6 6 4 5 5 e ...... 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.10 0.42 0.33 0.38 0.51 0.34 Change (BPH/BPL) Mous 1 7 0 3 3 9 8 1 0 6 7 5 2 4 6 7 1 9 1 3 3 1 Fold 1 5 1 3 6 7 2 6 7 1 5 7 6 5 0 4 ...... t 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0.1 0.2 0.5 0.5 0.6 0.3 Change Ra (SHR/WKY) 0 3 b 5 2 H2-Q1 Ccr H2-M Symbol mouse) ; a Cox8 ; ; , e Ldh ; a 5 l 1 b c 6 5 ; a c 1 a f h d 0 b a a 6 3 Cide l o g 5 l e d u (rat q 3 0 x x x r l s Gen 1 f ; n g p m d o o d e o 1 1 n n l t h i C S L M C C F A A C E Ldhb N RT1-M3 RT1Aw2 Cox8h E H C — C Cmkbr5 V, 2, e y cell ; A typ e alpha , 3 (heart/ factor, s mouse) n H , ) 1 subunit mRNA c , , death I 5 locu l A V r VIII- (rat n 1 e e t subcomponent, i t A cel d d e n i i n i t t r o u e procollagen lactate , q p p t histocompatibilit t b oxidase M3; regio e 1 ; e ; r o e ; u p s p 1 r recepto B t v Nam s i (ubiquinone fragmentatio chain y y subuni p ) l t l e c , a e 6 ,M 5 i flavoprotein, e e o o BPH e e A 1 g effecto s A 2 A e locu s p p g (LOC291541) n ,B n d simila s i e e a y n , n e 2 i 3 a s a i DN alph a d A d s t s Gen motif 10 t x i x a l an p n , e e i e t g i e x e l 5 s e s e s C V a b b R o oxidas n n d , n a r gene gene(Aw2) e componen , e n r n o c , n d d i n a t (C- e i a b b cytochrom SH CIDE- i n y t locu t r e e c m 1 1 ; c e e ; h r ; s r e typ e h t n a 6 transferrin g m 5 e o s s c , i e o l 1 subunit-lik dehydrogenas , o t y t h norvegicu r polypeptide n n y n l dehydrogenas r i n i bot n a a a p , i e x s g n h r x a x a i e clas clas H o n c n o regio t a 0 e e i s o b o s 3 n 0 n 1 1 n a Orthologou r r t m dehydrogenas alph subcomplex histocompatibilit Q muscle) VIIIb dehydrogenase lyase bet activato death-inducin alph a d o D 1 n n l p a y o u i S a c e c c l M A C f complemen lactat RT cytochrom NAD electro Rattu chemokin RT 3-hydroxy-3-methylglutaryl-Coenzym collagen t t t t t t a t a t t t t t _ _ D a i r a t t t t t t t t t t I Probe a _ a a a a _ _ _ a a t 7 _ _ 1 3 _ _ _ _ _ 8 e 6 3 8 3 5 8 1 4 7 3 8 underexpresse 4 4 0 8 4 0 5 5 0 1 8 Se s 1 6 3 5 1 3 0 2 1 0 6 0 3 6 0 0 0 7 2 9 4 2 set Mous 9 1 9 9 9 9 1 1 1 9 9 96020_a 160851_r_a 101990_a 102730_a 97201_s_a 161968_f_a 97869_a 101898_s_a 94324_f_a 99994_a 101080_a e prob s t t ID a t _ (voltage t t t t t t t s a a a a proteins s Group _ Se _ _ _ _ t t l g t t A t s s 6 4 metabolism e a a matrix N _ _ 0 6 t t t _ t t _ t s s y t t 5 8 R r a g a Orthologou 5 d d a a 2 2 _ _ c c _ m 9 _ _ 9 9 response 7 bindin channel 5 0 6 6 7 Prob 8 3 5 8 8 6 e 9 8 3 1 2 1 t 3. 3 7 A A 4 m m 0 2 4 4 5 5 4 0 A A 7 e 1 3 6 5 3 0 4 4 Ra _ _ Functiona 2 6 5 8 5 5 F 0 0 c c M X64827cds_s_a rc_A1237007_a J rc_A1171090_g_a U07181_g_a S X dependent) A r AJ005394_a J X71127_a X rc_AA800243_a X r D86215_a Y12009_a X U16025_g_a M10094_a Calciu Calciu Cytoskeleton Extracellula Inflammation Intermediar Immun Tabl 25 Fold 0 0 6 8 9 9 7 5 8 mouse; 7 5 4 6 6 4 1 3 7 e d ...... 0 0 0 0 0 0 0 0 0 0.37 0.44 0.54 0.73 an t experiments. Change ra y products. (BPH/BPL) Mous e e fluorescence/WKY th R n gen e (SH microarra th e f e o betwee 2 8 9 0 5 3 4 1 4 4 0 3 5 s n Fold 6 3 7 6 3 8 5 5 6 ...... chang t mous 0 0 0 0 0 0 0 0 0 0.1 0.4 0.6 0.5 d d differ Change l Ra fol functio an t t e (SHR/WKY) ra ra , th e symbo 4) e th n h about gen e r bot o n (colum e i l p s 1 nam set Symbol mouse) e Txni knowledg e symbo , e t ; 1 e Tieg 1 l 2 gen c 2 1 2 p ; prob e 3 r 1 5 s p n p gen ) i (rat a d r Gen r d p th p Serpina1d Csnk2a1-rs4 s b curren s c o c p , h m t P D S S A S P T Csnk2a1; Serpina1; Tieg C Vdup1 n BPL o d BPH orthologs base orthologou l d s , an clade 3) 1d Y , n TGFB D-3; r casein severa ; 4 thyroid n glycine- WK n ; I mouse) e ; categorie . d , l 1 (colum 1 an e e e homolog inhibitor membe e s (SHR non-receptor shown (rat , a e 4 nam d , e response protein r A e e ) sequenc u ar functiona 2 e h cysteine) t e ) n l 2 i d r a 6 r gen polypeptide protein v respons o s n n s cystein t e i e g i (o Nam SPOT1 h clad 3 ; e h d b t i a 1 e , e growt 2 genera e , e o proteinas h A relate s 9 o r y n n t (colum ) a i e p 5 underexpresse s ) t int responsiv alph growt Gen ) a phosphatase u g serin m n e a 1 d 1,25-dihydroxyvitami , i orthologou earl e s n y l y s ; e , m i m II .05 z a e y 2 interactin ( e 1 s d protei t inhibitor 2) i y n b e n r place n a alph n 1 d n i e responsiv h earl e m d P h e , cysteine o b ( e e l e e p C ric II r k y d s a i - i fluorescence tyrosin hormon wer m l r l o e e kinas 11 x L (o - a s e y (colum protei inducibl h d n o C p h o n e e r p D membe r h e I R B p e i , a z t d A r Gene p Orthologou i e a kinas A proteinas typ hormon inducibl ric thioredoxi P i e . se c t u significantl p s d s a S cystein protei thyroi TGF casei upregulate serin e e prob hav e t d t fluorescence/BP semicolon t t H an a t t t t D e a a _ mous t t t t t y a I (BP Probe g _ a a a _ b x e t 5 _ _ _ _ 9 d e 9 2 8 9 7 1 mous 3 0 5 4 5 3 Se d 0 2 9 2 8 0 chang an 4 8 4 0 7 6 d t Affymetri Mous 1 9 9 9 9 1 93550_a 102874_a 160306_a 160547_s_a 104546_g_a 93109_f_a 99602_a , separate fol ra e 1) e e n ar th s n i mous g d t (colum an ID D , I t t t t t t t t 5) ortholo a a n Group se Se _ _ n t l e t a s 1 factors a e names/symbol a _ 8 e t _ t t t _ s n t 5 s prob a s (continued) (colum d a 0 stress t hav _ _ ) c _ inhibitors _ 1 0 d 2 ra 1 7 Prob e 5 0 1 e 6 x 4 2 transduction t 3. 8 1 differen 1 5 0 4 l 5 liste A 6 o e 7 8 5 2 s Ra _ Functiona 7 2 6 5 tw 0 c J K01934mRNA#2_a S X rc_AA875506_a M U44948_a U rc_A1014169_a rc_A1010453_a r U57499_a rc_A1172476_a e Myelin Oxidativ Phosphorylation Proteas Signa Transcriptio Unknown Tabl Gene Affymetri fluorescence th 26

Activation/detoxification of exogenous chemicals Adrenal tumor suppressor magnifying the effect of Hmgcr excess to increase choles- Calcium binding proteins 2% 2% terol biosynthesis. 11% 3% 2% 2% Calcium channels (voltage-dependent) 2% Cellular adhesion Cholesterol is the precursor of glucocorticoid steroid 3% 2% 2% Chaperones hormones that play a profound but still incompletely un- 3% Cysteine protease inhibitor 6% 2% Cytoskeleton derstood role in BP homeostasis and hypertension in hu- Extracellular matrix 28–33 6% Glycosylation mans and rodents. Abnormalities in the gluco- Immune response 10% corticoid receptor have been proposed as a cause of glu- 3% Inflammation 34,35 Intermediary metabolism cocorticoid dependence in hypertension. The heat- 2% Myelin shock 70-kD protein 1a gene (Hspa1a), which encodes a 2% Nuclear hormone receptors 2% Oxidative stress protein with a crucial role in glucocorticoid receptor acti- 5% Phosphorylation 25% vation, is overexpressed 3.01-fold in SHR and 5.08-fold in 5% Prohormone processing 2% Protease inhibitors BPH. Aberrant Hspa1a function may contribute to the Signal transduction Transcription factors glucocorticoid dependence of hypertension. Its differential Transport expression has been implicated previously in hyperten- Unknown 36 FIG. 2. Functional classification of shared orthologous genes in sion. SHR and BPH. The orthologs differentially expressed in the same Another metabolic abnormality observed in hyperten- direction (n 63) in SHR and BPH (ie, overexpressed in both SHR sion is dyslipidemia. The SHR and BPH underexpressed and BPH, or underexpressed in both SHR and BPH) were sorted into functional groups based on known function of the gene products. hormone-sensitive lipase (Lipe) by 0.61-fold and 0.41- The pie chart shows the percent of orthologs that fall into the various fold, respectively. Lipe catalyzes the rate-limiting step in functional groups. lipolysis of stored triglycerides to form free fatty acids, an important source of energy in mammals. Lipe is regulated disease states (such as the metabolic syndrome in the by catecholamines and hormones (eg, insulin). The con- SHR). sistent alterations of Lipe observed here may thus contrib- Many of the orthologs differentially expressed in com- ute to metabolic and homeostatic abnormalities in mon between SHR and BPH are intriguing candidate hypertension common to mammalian species. genes for hypertension pathogenesis (Tables 2 and 3). Enhanced oxidative stress is another mechanism rap- Evidence is increasing that supports the role of inflamma- idly gaining support as both a pathogenic and an end- tion in pathogenesis and consequences of many cardiovas- organ damaging mechanism in hypertension. The cular diseases, including hypertension. The CCAAT/ decreased ability of superoxide dismutase to scavenge the enhancer-binding protein (Cebpb) (1.25-fold over- free radical superoxide (O2 ) in hypertensives has previ- expressed in SHR; 1.52-fold overexpressed in BPH) codes ously been reported.37–43 Superoxide dismutase 3 (Sod3) for a transcription factor responsive to interleukin 6, and is underexpressed 0.60-fold in SHR and 0.68-fold in BPH. may serve as a control point for widespread, global Such consistent underexpression may represent a funda- changes in the inflammatory cascade. The complement mental susceptibility of genetic hypertension to vascular component 1, q subcomponent, polypeptide (C1qb) and organ damage. (0.47-fold underexpressed in SHR; 0.57-fold underex- To identify common mechanisms causing or respond- pressed in BPH) is the first and major component of the ing to genetic hypertension in mammalian species, we complement cascade, an important mechanism in inflam- determined the orthologous genes differentially expressed mation. Mutations in Cebpb and C1qb could have pro- in the same direction in SHR and BPH (Tables 2 and 3). found consequences on inflammatory processes in SHR Even within this focused and specific set of orthologous and BPH. genes, the multifactorial and systemic nature of hyperten- Many metabolic abnormalities often accompany hyper- sion is apparent in the classification of the orthologs into tension in a disorder called the metabolic syndrome, or diverse functional groups (Fig. 2, Tables 2 and 3). syndrome X.2 Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (Hmgcr) is the rate-limiting en- Catecholamines and zyme in cholesterol biosynthesis and target of the statin Sympathetic Function drugs in humans.27 Its coordinate differential overexpression in two rodent models of genetic hypertension (2.56- Analysis of catecholamine biosynthetic gene expression fold overexpressed in SHR; 3.11-fold overexpressed in suggests fundamentally different patterns in the two mod- BPH) suggests the possibility that its overexpression els of genetic hypertension, with depressed synthesis of might be a systematic facet of hereditary hypertension in catecholamines likely in SHR, but enhanced synthesis in mammals, perhaps even contributing to the diverse meta- BPH (Fig. 3). Tyrosine hydroxylase, which catalyzes the bolic abnormalities associated with the human disease rate-limiting step of catecholamine biosynthesis, is not state. Underexpression of HMG-CoA lyase (Hmgcl) in differentially expressed in SHR but is overexpressed 1.61- both hypertensive strains (0.74-fold underexpressed in fold in BPH. GTP cyclohydrolase 1 (Gch), the rate-limit- SHR; 0.59-fold underexpressed in BPH) might increase ing enzyme in synthesis of the essential cofactor of the substrate availability of HMG-CoA to Hmgcr, perhaps tyrosine hydroxylase (tetrahydrobiopterin), is not differen- 27

A. SHR B. BPH Dbh, but the BPH overexpressed Pnmt 2.11-fold. Other 1.68 investigators have observed decreased Dbh activity in the GTP Gch 7,8-dtp GTP Gch 7,8-dtp SHR.44 Pts Pts 0.74 Phenylalanine Phenylalanine Transport of dopamine and monoamines into secretory 0.21 Phenylalanine hydroxylase 6-pt Phenylalanine hydroxylase 6-pt vesicles is mediated by Slc18a1 and Slc18a2, also known Spr Spr Tyrosine Tyrosine A. SHR Legend Tyrosine hydroxylase BH4 1.61 Tyrosine hydroxylase BH4 Overexpressed in SHR Not statistically significant Mineralocorticoid Pathway Underexpressed in SHR No data Dihydroxyalanine Dihydroxyalanine Cholesterol Cyp11a Dopa decarboxylase Dopa decarboxylase Glucocorticoid Pathway Androgen/Estrogen Pathway Pregnenolone Cyp17a1 17α-Hydroxypregnenolone Cyp17a1 Dehydroepiandrosterone Dopamine Dopamine Hsd3b1Hsd3b2 Hsd3b3 Hsd3b1Hsd3b2 Hsd3b3 Hsd3b1Hsd3b2 Hsd3b3 0.49 2.20 Slc18a1 Slc18a2 Slc18a1 Slc18a2 Hsd3b4 Hsd3b5 Hsd3b4 Hsd3b5 Hsd3b4 Hsd3b5 Hsd3b6 Hsd3b7 Hsd3b6 Hsd3b7 Hsd3b6 Hsd3b7 Dopamine Vesicles Dopamine 0.39 Progesterone Cyp17a1 17α-Hydroxyprogesterone Cyp17a1 Androstenedione Cyp19a1 Dopamine beta-hydroxylase Dopamine beta-hydroxylase Cyp21 Cyp21 Hsd17b1 Hsd17b2 Hsd17b3 Chga 0.67 Scg2 1.53 Chga 2.12 Scg2 Hsd17b4 Hsd17b12 Estrone Norepinephrine Norepinephrine 11-Deoxycorticosterone 11-Deoxycortisol Chgb Scg3 1.87 Chgb Scg3 Hsd17b10 Hsd17b7 0.40 Cyp11b1 Cyp11b1 Hsd17b1 Sgne1 Vgf Sgne1 Vgf Testosterone Hsd17b2 Hsd17b3 Corticosterone Cortisol Cyp19a1 0.49 Slc18a1 Slc18a2 2.20 Slc18a1 Slc18a2 Hsd17b4 Hsd17b12 Cyp11b2 0.57 Hsd17b10 Hsd17b7 Steroid degradation Estradiol Norepinephrine Norepinephrine 3.50 0.40 18-Hydroxycorticosterone Akr1c1Akr1c2 Akr1c6 Akr1c21 Akr1c13 Steroid receptors Phenylethanolamine N-methyltransferase 0.67 2.11 Phenylethanolamine N-methyltransferase 1.47 Cyp11b2 0.57 15.23 Sult4a1 Sult1b1 Sult2b1 Sth2 Ste 2.08 Ar Esr1 Esr2 Esrrb Esrra Hsd11b1 Hsd11b2 1.40 Nr3c1 (GR) Nr3c2 (MR) Pgr Epinephrine Epinephrine Aldosterone B. BPH Legend Adrenergic receptors 0.42 Adrenergic receptors Overexpressed in BPH Not statistically significant Adra1a Adra1b Adra2a Adra2b Adra2c Adrb1 Adra1a Adra1b Adra2a Adra2b Adra2c Adrb1 Mineralocorticoid Pathway Underexpressed in BPH No data Cholesterol Adrb2 Adrb3 Drd1a Drd2 Drd3 Drd4 Adrb2 Adrb3 Drd1a Drd2 Drd3 Drd4 Cyp11a 0.45 0.51 0.75 Glucocorticoid Pathway Androgen/Estrogen Pathway Pregnenolone Cyp17a1 17α-Hydroxypregnenolone Dehydroepiandrosterone Catecholamine degradation Catecholamine degradation Cyp17a1 Hsd3b1Hsd3b2 Hsd3b3 Hsd3b1Hsd3b2 Hsd3b3 Hsd3b1Hsd3b2 Hsd3b3 37.39 Comt Slc6a2 Slc6a3 Maoa 0.68 Comt Slc6a2 Slc6a3 Maoa Hsd3b4 Hsd3b5 Hsd3b4 Hsd3b5 Hsd3b4 Hsd3b5 0.52 Maob Sult1a1 Sult1a2 Maob Sult1a1 Sult1a2 2.38 Hsd3b6 Hsd3b7 2.38 Hsd3b6 Hsd3b7 2.38 Hsd3b6 Hsd3b7 0.53 Legend Legend Progesterone Cyp17a1 17α-Hydroxyprogesterone Cyp17a1 Androstenedione Cyp19a1 0.23 Overexpressed in SHR Not statistically significant Overexpressed in BPH Not statistically significant Hsd17b1 Hsd17b2 Hsd17b3 Underexpressed in SHR No data Underexpressed in BPH No data Cyp21 Cyp21 0.56 Hsd17b4 Hsd17b12 0.39 Estrone 11-Deoxycorticosterone 11-Deoxycortisol FIG. 3. Catecholamines and sympathetic function in SHR and BPH. Hsd17b10 Hsd17b7 Cyp11b1 Cyp11b1 Hsd17b1 0.39 Gene expression of the catecholamine biosynthetic and target (re- Testosterone Hsd17b2 Hsd17b3 Corticosterone Cortisol ceptor) pathway is shown in the SHR hypertensive rat strain (A ) and 0.23 Cyp19a1 0.56 Hsd17b4 Hsd17b12 Cyp11b2 Hsd17b10 Hsd17b7 BPH hypertensive mouse strain (B ). Red indicates an overexpressed Steroid degradation Estradiol gene, blue indicates an underexpressed gene, white indicates no 18-Hydroxycorticosterone Akr1c1Akr1c2 Akr1c6 Akr1c21 Akr1c13 1.81 Steroid receptors data (ie, no probe on chip), and gray indicates lack of statistical Cyp11b2 0.56 Sult4a1 Sult1b1 Sult2b1 Sth2 Ste Ar Esr1 Esr2 Esrrb Esrra 0.56 Hsd11b1 Hsd11b2 Nr3c1 (GR) Nr3c2 (MR) Pgr significance. The bold number listed next to significantly (P .05) Aldosterone differentially expressed genes is the fold change (BPH/BPL or SHR/ WKY). Abbreviations: 6-pt 6-pyruvoyl-tetrahydropterin; 7,8-dtp FIG. 4. Steroid hormone biosynthesis/degradation and receptors. 7,8-dihydroneopterin triphosphate; Adra1a adrenergic receptor, Gene expression of steroid hormone biosynthetic enzymes and re- alpha 1a; Adra1b adrenergic receptor, alpha 1b; Adra2a adren- ceptors is shown for SHR (A ) and BPH (B ). Red indicates an over- ergic receptor, alpha 2a; Adra2b adrenergic receptor, alpha 2b; expressed gene, blue indicates an underexpressed gene, white Adra2c adrenergic receptor, alpha 2c; Adrb1 adrenergic recep- indicates no data (ie. no probe on chip), and gray indicates lack of tor, beta 1; Adrb2 adrenergic receptor, beta 2; Adrb3 adren- statistical significance. The bold number listed next to significantly (P .05) differentially expressed genes is the fold change (SHR/ ergic receptor, beta 3; BH4 tetrahydrobiopterin; Chga chromogranin A; Chgb chromogranin B; Comt catechol-O- WKY or BPH/BPL). Abbreviations: Akr1c1 aldo-keto reductase methyltransferase; Drd1a dopamine receptor 1a; Drd2 dopa- family 1, member c1 (20-alpha-hydroxysteroid dehydrogenase); mine receptor 2; Drd3 dopamine receptor 3; Drd4 dopamine Akr1c2 aldo-keto reductase family 1, member c2 (3-alpha-hy- receptor 4; Gch GTP cyclohydrolase 1; GTP guanosine triphos- droxysteroid dehydrogenase); Akr1c6 aldo-keto reductase family phate; Maoa monoamine oxidase A; Maob monoamine oxidase 1, member C6; Akr1c13 aldo-keto reductase family 1, member B; Pts 6-pyruvoyl-tetrahydrobiopterin synthase; Scg2 sececre- C13; Akr1c21 aldo-keto reductase family 1, member C21; Ar togranin II; Scg3 secretogranin III; Sgne1 secretory granule androgen receptor; Cyp7a1 cytochrome P450, 7a1; Cyp11a neuroendocrine protein 1; Slc6a2 solute carrier family 6 (neuro- cytochrome P450, subfamily 11a; Cyp11b1 cytochrome P450, transmitter transporter, noradrenalin), member 2; Slc6a3 solute subfamily 11b, polypeptide 1; Cyp11b2 cytochrome P450, sub- carrier family 6 (neurotransmitter transporter, dopamine), member family 11b, polypeptide 2; Cyp17a1 cytochrome P450, subfamily 3; Slc18a1 solute carrier family 18 (vesiclular monoamine trans- 17a; Cyp19a1 cytochrome P450, family 19, subfamily a, polypep- porter) member 1; Slc18a2 solute carrier family 18 (vesicular tide 1; Cyp21 cytochrome P450, subfamily 21a; Esr1 estrogen monoamine transporter) member 2; Spr sepiapterin reductase; receptor 1; Esr2 estrogen receptor 2; Esrra estrogen-related Sult1a1 sulfotransferase family 1A, phenol-preferring, member receptor ; Esrrb estrogen-related receptor ; Hsd3b hydrox- 1; Sult1a2 sulfotransferase family 1A, member 2; Vgf VGF ysteroid dehydrogenase, delta 5 -3- ; Hsd11b1 hydroxysteroid nerve growth factor-inducible. 11- dehydrogenase 1; Hsd11b2 hydroxysteroid 11- dehydrogenase 2; Hsd17b hydroxysteroid 17- dehydrogenase; Nr3c1 nuclear receptor subfamily 3 group C member 1 (glucocorticoid tially expressed in SHR but is overexpressed 1.68-fold in receptor); Nr3c2 nuclear receptor subfamily 3 group C member 2 BPH. In contrast, the SHR underexpressed dopamine (mineralocorticoid receptor); Pgr progesterone receptor; Ste sulfotransferase, estrogen preferring; Sth2 sulfotransferase, hy- hydroxylase (Dbh) and phenylethanolamine-N-methyl- droxysteroid preferring 2; Sult1b1 sulfotransferase family 1B, transferase (Pnmt) 0.39-fold and 0.67-fold, respectively member 1; Sult2b1 sulfotransferase family, cytosolic, 2B, mem- (Fig. 3). The mouse chip did not contain a probe set for ber 1; Sult4a1 sulfotransferase family 4A, member 1. 28 Fold A A A A A A / / / / / / e — — — — N N N N N N/A N N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.73 1.26 0.67 1.28 1.48 Change (BPH/BPL) Mous 9 A A A A A A A A A A Fold / / / / / / / / — — — — — — — — — — — — t N N N N N N N N N/A N/A N/ N/ 1.72 0.84 3.13 0.4 1.62 1.20 0.11 Change Ra (SHR/WKY) l o b 3 m 1 2 3 y 2 1 2 4 1 5 3 a S 1 m m m 1 2 4 1 c m o x x x t x i x h t t t l r s b f f f e p s s s s c p p p s g p d p c c c y d n G G G G G G G G X G G G G G M C N N N — Nox4 Gst13-1 Gsta3 Gsta4 Gstk1 Rac1 Rac2 Cybb Gcl Gsta Gsta e G 1 r t o t t i i n b l i u h b 2 n 1 2 u i e subuni s e d e e r n i 8 c t ) i o type 3 4 1 1 t i t p t y a e a a a a l 1 2 3 a i p a c 1 t u u u y e l o a e modifie alph o m m m mitochondria alph alph alph s substrat substrat c 4 1 2 m s subuni kapp specific online) s p , , , , , , , , , i a r r r a e polypeptid e e e e e y r m m d a o o o s 2 1 4 5 3 e N s s s t t t e a h a s f e 1 e a a a c c c e e e e e e p t e g r r r s a l i a a a s s s s s s e l a f f f e e e ligase n n n a bet s a a a a a a f f f h e a t e e c c c a s s s , , d d d d d e Tabl (kidne r i i i p i i i i i t g e l l l n t n n n 5 G s i l c x x x x x o h y o o o botulinu botulinu a a a 4 e o r t e y u o o o o o s s s r r r s t 2 r r r r r t t t d h n e 4 3 3 d o o o - s a m - - - e e e e e y t t t y p e C C i y b b-245 d a S S S r p p p s p p S-transferase S-transferas S-transferase S-transferase S-transferas S-transferase y y y h i t c d cystein c c c d d e x e e - u e e e e e e e e e e e e e e e e l l l l d o e e e i i i n n n n n n n n n n m r t g n h h h oxidas o o o o o o o o o o - i e o e a i i i i i i i i i i p p p r s a n p h h h h h h h h h h i H online) o o o m o h t t t t t t t t t t o r r r h m l c a n a a a a a a a a a a t t t t t e t t t t t t t t t t o a u u u m n t Supplementar u y u u u u u u u u u u e 2 l u e e e l l l l l l l l l a l a y e g g g g g x G g g g g m g g glutathion glutathion G n n c n NADP glutathion glutathion glutathion RAS-relate RAS-relate cytochrom Glutamat glutathion glutathione-S-transferase se Tabl e y t t t t a a t a t t t t t t t t t t t t _ a _ a _ D a r a f s a a a a t t t t t t t t t t _ _ (Pleas I Probe f _ r _ a a a _ a a _ _ a _ _ _ _ a t _ 7 _ _ 6 5 _ _ _ _ 5 6 4 _ 2 9 _ 6 e e 2 4 3 5 0 2 0 9 2 7 8 8 1 5 3 2 6 3 cluster 4 3 8 6 5 3 1 9 4 4 9 0 4 6 0 1 3 6 S l 5 1 6 8 0 1 1 8 9 6 1 0 0 3 3 0 7 2 chain) 3 7 6 9 6 0 7 9 4 4 0 6 0 0 7 0 0 7 Mous 9 1 9 1 9 9 9 9 1 93015_a 96085_a 96670_a 9 1 — — 1 1 — 1 101555_a 103579_a 1 9 1 100300_a — 9 101872_a 160063_i_a 160335_a t Supplementar e se functiona e s transpor n ID (Pleas stres t t e t t t Group t t t Se a a a a l a _ _ (electro _ e _ _ P450s t t t s 9 7 t s 7 t a a a _ a 8 8 _ e 4 a t t systems _ s _ s _ t 5 1 2 _ a s a Oxidativ d s g d s a 3 0 2 oxidase Prob e _ oxidase c _ _ c _ _ _ _ 9 0 7 t e 9 9 5 2 5 6 5 2 1 8 8 1 H 2 2 6 0 1 9 1 4. 9 8 A A 1 7 2 3 4 4 Ra 6 8 Functiona 5 1 defense sources A A A e 8 4 7 4 1 4 8 2 5 _ _ _ defens 1 0 0 7 0 S S 2 3 0 0 c c c X J E r L S83436_i_at X62660mRNA_at X78848cds_f_a S72506_s_a — — — Xanthin r J rc_A1233261_i_a — X r D00680_at L — — Other — — — — Mitochondri Cytochrom NADP U — X rc_AA892258_at — RO Glutathione-dependent RO Tabl 29 Fold A A A A / / / / e — — N N N N N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 1.14 0.73 0.61 0.71 1.38 1.40 1.35 1.20 4.15 1.54 0.68 0.55 0.29 1.19 1.28 Change (BPH/BPL) Mous 6 0 4 2 7 1 A A A A A A A A A A Fold / / / / / / / / / — — — — — — — — — — — — t N N N N N N N N N N/ 1.2 0.25 0.6 1.34 1.42 1.66 3.4 1.2 0.2 1.5 1.74 0.61 Change Ra (SHR/WKY) l o b 1 2 3 6 5 m 1 2 1 d d d 1 3 6 2 4 p 2 2 1 y t h 1 2 3 1 2 3 r i l r r 2 l 1 p t t m p m x x x x x s t t t t t t d d d S t n n n n n n n n n n n d d d d d g s s s s s s a o o o x x x x x x x x r r r r o o r o e T T T S T S T S T T P P P P M T G G P C P G P G G P Gstz1 Mif Mgst3 Mgst2 Ccs Hag Gsto1 G n e G e 1 3 2 r e e e s 1 a dismutas r facto e 1 2 a e e f y a a 6 5 s t t 1 2 n n e e u u 1 i e i i a h h e r p a p t t m m omeg t t m , , , , , , , - o a hydrolas e r e e e e S S-transferas S-transferas zet s N p s s s s inhibitor 3 1 2 e isomerase) ) 1 2 3 a e e e a a a a e superoxid r g n e r r r r n e e e D e e e e r e n e e e e s s s n o k i f s s s f f f f i t a a a e s fo 2 a a s s s a s h t t t c t t t n 3 t n n n n G u u u e a c c c ( 2 a a a a a a r r t u u u r r r r m m m e e e t 1 2 3 6 4 t t t t d d d u t s s s - 1 3 2 l - - - - k k i i i migratio e e e i i glutathion n n n n n n S l l g glutathion glutathion S S S S r r r 2 i 1 transferas S-transferase d d d S-transferase i i i i i e e e l - - - e l l l x x x x x s s s e e e e e e e e e e e n n n n n n n n a o o o o o i i i i i i i i a a a d d d n n n n n i i i x x x x x x x x d d d d d n n n m o o o o o chaperon e x x x o o o o o o o o i i i i i e e e e e o o o o s r r r r r r o o o d d d d d d d d h h h h h x x x i i i i i s a r r r t t t t t e e e e e e e e l x x x x x o o o o e e e a a a a a r r r r r r r r r a a a a o o o o o t t t t t p p p o o o o o o o o t c r r r r r r r r i i i i i i i i i u u u u u (maleylacetoacetat a u u u e e e e a e l l l a a l l h h h h h h h h t t t S t t t p p p S S t t p C p p p g g g m p macrophag g g microsoma coppe microsoma glutathion glutathion glutathion hydroxyacy t t t a a a t t t t t t t t t t t t _ _ _ D a a r a a f s a a a t t t t t t t t t t t t t t t t t I Probe _ a _ _ _ a a a _ a a a a _ _ a a a a a _ a a _ _ t _ 7 5 _ _ _ 8 3 _ 7 _ _ _ 2 7 ______3 8 _ 7 e e 5 3 1 4 2 0 6 8 6 2 3 2 7 4 8 7 5 6 0 5 3 9 0 7 3 1 8 4 5 0 4 0 3 5 9 5 5 6 8 9 0 0 9 8 6 2 4 1 3 6 S 9 0 0 9 1 6 0 1 0 0 7 6 2 0 1 8 8 0 4 5 0 4 4 9 4 9 6 6 6 4 8 5 7 6 0 0 6 2 6 6 6 9 3 3 3 9 5 0 0 0 Mous 1 9 1 1 1 9 9 9 1 9 9 9 1 1 9 9 9 103909_a 9 9 9 9 96258_a — 9 1 104742_a 1 1 — 160350_a 100629_a 97819_a 100042_a defense ID specific t t c t e a a _ _ t t t t Group t t Se t a a g g a a a l systems a _ _ _ _ _ systems e _ _ t t t _ t n systems t t s s 6 0 0 9 3 specifi e a g a a a _ _ 9 8 5 4 8 n peroxid t t t _ t _ _ e s s _ _ t 2 3 6 1 0 s a a a a (continued) 5 9 s s d d a 1 5 9 6 n 0 Prob _ _ _ _ c c _ 6 0 _ _ _ 7 9 9 2 1 6 t 5 9 6 1 4 4 8 0 4 7 2 8 8 7 9 0 2 9 2 5 4 0 5 2 4 0 9 4. 5 A A 1 A A 0 5 0 8 0 9 6 7 1 4 4 Ra Functiona 7 A A A A A 0 e 3 6 4 8 2 7 0 0 0 0 3 _ _ _ _ _ defense 1 7 9 0 6 0 6 F F 0 0 0 c c c c c — U — r — r A Y Y X Thioredoxi — Peroxiredoxi r U r — A Superoxid — — — S73424_s_at Hydroge r Paraoxonas U rc_A1012802_a J — — U86635_g_a — AB008807_a rc_A1012589_s_at X X D — Tabl 30 Fold e — — N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 0.29 0.15 1.87 0.73 0.31 2.12 Change (BPH/BPL) Mous A A A A A A Fold / / — — — — — — t N N N/ N/ N/ N/A N/A N/ N/A N/A 0.38 1.41 1.70 Change Ra (SHR/WKY) l o b 1 0 b a 1 m 1 1 6 7 9 1 4 1 5 6 8 9 1 2 3 x y 2 3 1 c c c b c c c c b b b b b b b o S c c c c c c c c c c c c c c c p p p m c c c b b b b b b b b b b b b b b b e H U U U A A A A A A A A A A A A A A A n e G C C C B B B B B B C B B C C C y y y y y y y y y y y y y y y l l l l i i i i m m m m e a a a a f f f f m - - - - a b b b b 0 1 A B 6 8 9 1 2 3 5 u u u u N 1 1 6 7 9 1 4 1 r r r r r r r s s s s sub-famil sub-famil sub-famil sub-famil sub-famil sub-famil sub-famil sub-famil sub-famil sub-famil sub-famil r r r r r r r r e e e e e e e e , , , , , , , , , , , , , , , e e e e e e e e b b b b b b b n e e e e 1 2 3 b b b b b b b b t t t t e m m m m m m m t t t t 1 n n n m m m m m m m m e e e e e e e e e e e i i i G e e e e e e e e s s s s e e e e m m m m m m m t t t s s s s s m m m m m m m m , , , , , , , o o o a a a a a ) ) ) ) ) ) ) r r r , , , , , , , , c c c cassette cassette c cassette cassette cassette cassette cassette cassette cassette cassette cassette n ) ) ) ) ) ) ) ) P P P P P P P p p p e g g g g g g g g g g g g g g g P P P P P P P P R R R R R R R g g g g n n n n A A A A A A A A i i i i M M M M M M M y n n n T T T T T T T T i i i / / / / / / / d d d d l l l x / / / / / / / / n n n n R R R R R R R o p p p i i i i R R R R R R R R T T T T T T T u u u b b b b e D D D D D D D D F F F F F F F - - - - o o o c c c C C M C C C C C M M M M M M M P P P P m ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( n n n T T T T e U U A h A A U ATP-bindin A ATP-bindin ATP-bindin ATP-bindin ATP-bindin ATP-bindin ATP-bindin ATP-bindin ATP-bindin ATP-bindin ATP-bindin t t t t t t t t a D a a a t t t t t t t t t _ I Probe _ s a a a _ a _ t _ _ _ 4 _ _ 1 0 e e 0 2 2 9 7 2 7 1 2 7 0 9 9 1 2 9 S 7 3 3 0 3 1 5 2 2 3 0 6 9 7 9 0 Mous 1 9 9 9 9 9 1 — 103800_a 103300_a 92418_a 104394_a 94733_a — 1 93414_a 95283_a 103689_a 93407_a cell) m ID of t fro t pathology t Group Se t t t S s t a l a systems a _ e ROS) _ t _ r 0 RO proteins s y s 1 t _ (expor b t t t d _ t g 6 stres a 3 a a a (continued) t 3 a d 9 Prob _ 4 _ _ _ 6 _ e 7 8 t 1 4 4 8 1 4 1 9 5 9 9 1 0 2 4. 1 5 0 3 8 6 3 Ra 6 Functiona A 3 e 0 3 6 6 0 0 _ (vasomoto affecte detoxifie 8 0 9 B F 6 4 c Uncouplin X A A AB010467_s_a — AB010466_s_a L r — AF010597_s_at X D86086_s_a D — AF106563_s_at — — Transpor S — Oxidativ Tabl 31

the as the vesicular monoamine transporters (Vmat1 and r (SHR probe Fold

fo Vmat2). The SHR underexpressed Slc18a1 0.49-fold and e A A A d / / / e e a — — overexpressed Slc18a2 2.20-fold, but the mouse chip did N N N N/A chang

Change not have probe sets for Slc18a1 and Slc18a2. Underex- d becaus (BPH/BPL) Mous pression of Slc18a1, the transporter speciﬁcally located in expresse a fol y t adrenal chromafﬁn granules, is consistent with a decrease dat ra o ,

n of vesicular catecholamine storage in SHR. Slc18a2 is s 4) i

e thought to function primarily in the brain and other nor- n A A A A Fold / / / differentiall adrenergic neurons. The effect of Slc18a2 overexpression lum ther t N N N N/A N/ y t 0.29 in the adrenal gland of SHR is unclear. (co Change Ra tha l

s Chromogranins and secretogranins are the major solu- (SHR/WKY) ble proteins in neurotransmitter and catecholamine secre- symbo significantl 45,46 l t

e tory vesicles, and have roles in vesiculogenesis and indicate o 47 no b regulation of catecholamine secretion. The SHR exhibits s gen i 2 2 “—” m , e x x no differential expression of chromogranin A (Chga) and y 1 2 3 3) o o s s S gen n m m o o chromogranin B (Chgb), but 0.67-fold underexpression of e e N N H H — Nos symbol n th secretogranin 2 (Scg2) (Fig. 3). The overexpression of e e e Th (colum G chromogranin A (Chga), chromogranin B (Chgb), and . e secretogranin 2 (Scg2), 1.53-fold, 1.87-fold, and 2.12-fold becaus nam shown

d (Fig. 3), respectively, in BPH further supports enhanced e e

ar catecholamine storage in BPH. An increase in catechol- gen type. d

, amine synthesis would tend to increase the steady-state reporte an l 2) t ,

bold level of catecholamines, perhaps eventuating in an in- d 6) no cel n s in an l i crease in the number or size of vesicles. n e l

e Expression patterns of catecholamine degradation en- l s 1 a alternative b n (colum i , show o

e zymes are inconsistent in SHR but suggest decreased ) c chang e r u d u m ar

d catecholamine degradation in BPH. Catechol-O-methyl- e a ) fol (column n n i endothelia e N s transferase (Comt), the initial enzyme responsible for cat- region , , , .05 th = ID e 2 1 3

t echolamine degradation upon reuptake from the synapse, n e (5 e e e P fluorescence e ( s s tha is overexpressed 37.39-fold in SHR (Fig. 3), which is 2 3 L a a s G s prob h h e e e 2

t t actually the largest fold change observed in SHR. Such s e n n a y y

n large fold changes may indicate major quantitative or even s s synthas change e indicate mous d e e e g

” qualitative gene mutations that could have profound ef- ) y d d d fol i i g x t

x x fects in other organs. In contrast, SHR exhibits underex- n N/A an o oxygenas oxygenas i “ o o oxid t c fluorescence/BP i e e e pression of two other enzymes with catecholamine l c c c ra i i H p r r m x t t s degradation function: monoamine oxidase B (Maob, 0.52- e i i (BP h n n hem hem nitri symbol significan e fold underexpressed) and sulfotransferase family 1a, mem- e y

Th ber 1 (Sult1a1, 0.53-fold underexpressed) (Fig. 3). Comt is . Affymetri chang . d underexpressed 0.68-fold in BPH (Fig. 3), which may t t fol D a a t t exaggerate the prohypertensive effects of enhanced cate- Statisticall e I Probe _ a _ . stress t 2 _ 0 GeneChip cholamine biosynthesis by increasing steady-state levels e e e 6 5 2 e mous 0 6 4 S of catecholamines. th under d 1 3 4 n d 0 8 0 an o Overall, gene expression patterns suggest that that SHR Mous 1 9 1 — — 94167_a , t oxidativ 5) liste has depressed catecholamine production and that BPH n i n s exis i t d

t might suffer from increased catecholamine action. The i ) no effect of decreased adrenal catecholamine biosynthetic en- d (colum di )

involve zyme transcripts in SHR is not completely understood, but e mouse y ID

r may serve as a compensatory mechanism for high BP. An t t gen o a t g overproduction of adrenal catecholamines in BPH would Group _ Se ra s l , _ e likely contribute to BP elevation in the strain. Changes in t fluorescence t (ie A functionall a Y

s expression of catecholamine biosynthetic genes, however, N t _ e R s a (continued) ar

Prob could have unpredictable and counterintuitive effects in _ _ m d 1 t 9 correspondin 5 specie 9 0 tissues other than the adrenal gland, such as the brain. e 4. r 0 5 3 Ra liste Functiona th 4 4 e 7 Nonetheless, aberrant (albeit discordant) adrenal catechol- r 5 s 4 6 0 fo

J S81433_at AF058787_at U D AJ011115_a amine production may underlie hypertensive pathology in t

Tabl Gene fluorescence/WK se particula both SHR and BPH. 32

Mitochondrial Long Chain-Fatty Acid β-Oxidation Patterns of expression of SHR steroid degradation en- A. SHR Long Chain Fatty Acid B. BPH Long Chain Fatty Acid zymes suggest an increase in progesterone catabolism. Fatty Acid CoA Ligase 2 Fatty Acid CoA Ligase 2 0.51 Fatty Acid CoA Ligase 3 Fatty Acid CoA Ligase 3 Fatty Acid CoA Ligase 4 Fatty Acid CoA Ligase 4 1.27 Aldo-keto reductase family 1, member c1 (Akr1c1) and Fatty Acid CoA Ligase 5 1.33 Fatty Acid CoA Ligase 5 0.43 Fatty Acid CoA Ligase 6 Fatty Acid CoA Ligase 6 aldo-keto reductase family 1, member c2 (Akr1c2) code Long chain Acyl-CoA Long chain Acyl-CoA for enzymes that specifically degrade progesterone49–51 Carnitine O-palmitoyltransferase Ia Carnitine O-palmitoyltransferase Ia 1.32 Carnitine O-palmitoyltransferase Ib Carnitine O-palmitoyltransferase Ib (Akr1c2 can also degrade androstenedione and dihydrotes- Cytosol Long chain acyl-carnitine Cytosol Long chain acyl-carnitine Carnitine/acylcarnitine translocase 1.26 Carnitine/acylcarnitine translocase 0.46 tosterone), and are overexpressed in SHR 15.23-fold (the Mitochondrion Long chain acyl-carnitine Mitochondrion Long chain acyl-carnitine Carnitine O-Palmitoyltransferase II 1.44 Carnitine O-Palmitoyltransferase II 0.53 second largest fold change in SHR) and 3.50-fold, respec- Long chain Acyl-CoA Long chain Acyl-CoA tively. The large increase in expression of progesterone Acyl-CoA dehydrogenases: Acyl-CoA dehydrogenases: Very Long Chain Very Long Chain 2.99 degradation enzymes has at least three possible implica- Long Chain Long Chain Acyl-CoA (n-2) Medium Chain Acyl-CoA (n-2) Medium Chain Short Chain 1.26 Short Chain 0.57 tions for glucocorticoid activity in SHR. 1) Because pro- unsaturated saturated unsaturated saturated 2,4 Dienoyl-CoA trans-Δ2-enoyl-CoA 2,4 Dienoyl-CoA trans-Δ2-enoyl-CoA gesterone is a precursor of corticosterone, degradation of Delta Isomerase Enoyl-CoA Hydratase Delta Isomerase Enoyl-CoA Hydratase 0.75 2,4 Dienoyl-CoA reductase 1.45 2,4 Dienoyl-CoA reductase 0.56 progesterone may be a compensatory mechanism to re- Acetyl-CoA cis-Δ3-enoyl-CoA 3-L-Hydroxyacyl-CoA Acetyl-CoA cis-Δ3-enoyl-CoA 3-L-Hydroxyacyl-CoA short chain 3-OH-acyl-CoA Dehydrogenase 0.63 short chain 3-OH-acyl-CoA Dehydrogenase 1.27 Thiolase 3-Ketoacyl-CoA long chain 3-OH-acyl-CoA Dehydrogenase 0.66 Thiolase 3-Ketoacyl-CoA long chain 3-OH-acyl-CoA Dehydrogenase duce excessive glucocorticoid levels or activity, a theory Legend Legend supported by enhanced Hsd11b2 activity and decreased Overexpressed in SHR Not statistically significant Overexpressed in BPH Not statistically significant Underexpressed in SHR No data Underexpressed in BPH No data Cyp11b2 activity; 2) progesterone has been shown to have FIG. 5. Differences in metabolic gene expression patterns between anti-glucocorticoid effects in vitro52,53 and in vivo54; mu- SHR and BPH. Gene expression of the mitochondrial long chain fatty acid -oxidation pathway is shown for SHR (A) and BPH (B). Red tations that lead to abnormally high rates of progesterone indicates an overexpressed gene, blue indicates an underexpressed degradation could be a facet of the enhanced sensitivity of gene, white indicates no data (ie, no probe on chip), and gray SHR to glucocorticoids; and 3) previous studies have indicates lack of statistical significance. The bold number listed next to significantly (P .05) differentially expressed genes is the fold shown that progesterone can bind to the glucocorticoid change (BPH/BPL or SHR/WKY). receptor with high affinity in vitro, and induce nuclear translocation and binding of the receptor complex.55 If, in Steroid Hormone Biosynthesis/ the SHR, progesterone exerts a higher than normal pro- Degradation and Receptors portion of its genomic actions through the glucocorticoid receptor, we may predict overactivity of glucocorticoid Hypertension in the SHR may be glucocorticoid depen- receptor signaling in effector tissues. Interestingly, we dent.28–32 A trait that is also observed in human essential have previously observed overexpression of the glucocor- hypertensives33 but unexplored in BPH. The glucocorti- ticoid receptor in the microcirculation of the SHR.34 coid dependence of the SHR appears to hinge on an enhanced sensitivity to rather than an increase in circulat- Oxidative Stress ing levels of the hormone, with an enhanced level of Glucocorticoids are also implicated in hypertension as a glucocorticoid receptors in peripheral tissue.34 We exam- trigger of increased reactive oxygen species (ROS) and ined adrenal steroid hormone biosynthetic pathways in therefore oxidative stress. Increased levels of ROS are SHR and BPH for glucocorticoid abnormalities (Fig. 4). detectable in the circulation in both human56,57 and rodent The steroid hormone pathways we investigated are based hypertension,58–61 and glucocorticoids can induce expres- on human metabolism because steroid synthesis pathways sion of xanthine oxidase (a source of superoxide) in the in rodents are not as well understood as in humans. A kidney62 and increase oxidative stress in the microvascu- crucial distinction to make for this discussion is that the lature.63 At this point, the specific role of glucocorticoids most abundant active glucocorticoid is corticosterone in in production of ROS is unclear, but strong evidence is rodents and cortisol in humans. emerging in support of oxidative stress as a pathologic An important observation in the steroid synthesis by mechanism of microvascular damage and end-organ injury SHR is underexpression of cytochrome P450, subfamily in hypertension.35 The oxidative stress observed in hyper- 11, polypeptide 2 (Cyp11b2) (commonly known as aldo- tensives can be attributable to an increase in ROS produc- sterone synthase) 0.57-fold (Fig. 4). Cyp11b2 converts tion, a decrease in ROS scavenging, or a combination of corticosterone (the primary glucocorticoid) to aldosterone both. (the primary mineralocorticoid). Underexpression of Cyp11b2 could lead to corticosterone excess or aldoste- ROS Production Under normal conditions, the most rone deficiency. Furthermore, the mineralocorticoid recep- abundant source of ROS within cells is the electron trans- tor (Nr3c2) can bind both glucocorticoids and miner- fer processes of the mitochondria.64 Other ROS sources alocorticoids. Receptor-binding specificity is conferred by include the cytochrome P450 electron transferring enzyme hydroxysteroid dehydrogenase 11 2 (Hsd11b2), the en- systems,65,66 as well as xanthine oxidase and NADPH zyme that protects the receptor from glucocorticoid acti- oxidase, two important superoxide-producing enzyme sys- vation by degrading glucocorticoids to inactive tems67–69 implicated in hypertension,70–76 which are ex- metabolites.48 In what may be a compensatory mechanism pressed in almost all cells of the microcirculation.77 for excess glucocorticoid activity, the SHR overexpressed The SHR and BPH show global perturbations in ex- the gene coding for Hsd11b2 by 1.40-fold. pression of the electron transport chain of the mitochon- 33 dria (Supplementary Table 1), an observation discussed fundamental defect of hypertensives to effectively scav- later (see subheading Intermediary Metabolism) that is enge ROS. perhaps more relevant to intermediary metabolic function Export of Detoxified ROS From Cell Reactive oxy- than to production of ROS. Twenty-two cytochrome P450 gen species that are detoxified by the cellular defense genes are differentially expressed in SHR and BPH: some mechanisms are exported from the cell through a variety genes are upregulated and some downregulated (Supple- of transport proteins, such as the multidrug resistance mentary Table 2). Analysis of expression of these cyto- proteins, also known as the ATP-binding cassettes. Nei- chrome P450 genes at the transcript level does not provide ther the SHR nor the BPH show consistent patterns of strong evidence for or against the role of the genes in ROS differential expression of the ATP-binding cassette production. genes. The NADPH oxidase system is best known for its role Although vasoconstriction (with increased arteriolar in superoxide production as part of the bactericidal mech- tone) does increase BP, an increase in arterial pressure per anism of neutrophils and other phagocytic cells. Recent se may not be entirely sufficient to account for the vascular evidence has shown that NADPH oxidase activity medi- 35 70 lesions and end-organ injury observed in hypertension. ates endothelial dysfunction in hypertension and that the The observation of global perturbations (Table 4) in gene enzyme activity is elevated in the kidney of hyperten- 72 expression patterns of the oxidative stress defense systems sives. Proteins of the NADPH oxidase complex are in two independent models of human essential hyperten- coded for by seven genes: Cyba, Cybb, Ncf1, Ncf2, Ncf4, sion (ie, SHR and BPH) further suggests that oxidative Rac1 (in macrophages), and Rac2 (in neutrophils). Genes stress is a common mammalian mechanism activated in of the NADPH oxidase system are not differentially ex- hereditary hypertension, and likely contributes to vascular pressed in either SHR or BPH, but many of the genes are and end-organ injury in this setting. Investigation into such not represented on the rat microarray (Table 4). heretofore unexplained links between ROS, glucocorti- Xanthine oxidase, coded by the gene xanthine dehydro- coids, and microvascular damage is likely to provide novel genase (Xdh), is a source of superoxide, and elevated 74–76 insights into both the pathogenesis and consequences of activity of the enzyme is implicated in hypertension. hypertension. Endothelial cells of the microvasculature, but not those in larger vessels, are a major source of xanthine oxidase in Intermediary Metabolism the body.78 The SHR underexpressed Xdh 0.49-fold, whereas BPH overexpressed Xdh 1.48-fold. If the major- Hypertension in humans is often observed as part of a ity of the Xdh activity occurs within the endothelial cells condition known as the cardiovascular dysmetabolic of the microvasculature; homogenates of the entire adrenal syndrome, or syndrome X. In addition to increased BP, glands may not be ideal for measuring xanthine oxidase the metabolic syndrome is characterized by a variety of activity. Nonetheless, our data suggest opposing roles of risk factors, such as central obesity, dyslipidemia, dys- xanthine oxidase as a source of oxidative stress in SHR glycemia, insulin resistance, and elevated levels of the and BPH. circulating inflammatory mediator C-reactive protein, which are risk factors for cardiovascular disease and ROS Scavenging Defense against ROS includes a mul- type 2 diabetes. We investigated intermediary metabo- titude of enzymes: catalase (Cat), which specifically at- lism transcripts of the SHR and BPH and observed tacks hydrogen peroxide (H2O2), superoxide dismutase widespread derangements in metabolic gene expression (Sod), which targets superoxide (O2 ), and the glutathione, patterns (Fig. 5, Supplementary Tables 1 and 3), sug- paraoxonase, peroxiredoxin, and thioredoxin defense sys- gesting that the SHR and BPH strains sustain rather tems that protect against a variety of ROS.A consistent global metabolic abnormalities in addition to increased pattern of overexpression or underexpression does not BP. In fact, abnormalities of the intermediary metabo- exist in the ROS defense systems of SHR and BPH, as lism of the SHR are well known21 and the strain also some of the genes are upregulated and others are down- serves as a model of the metabolic syndrome. The regulated (Table 4). The global perturbations in the ROS metabolism of BPH has yet to be investigated in great enzyme defense systems do, however, suggest that the detail. adrenal glands of both SHR and BPH are subjected to The SHR shows derangements in expression of genes enhanced oxidative stress. Previous studies suggest that involved in fatty acid degradation and synthesis, glu- hypertensive organisms may suffer from an impaired abil- coneogenesis, glycolysis, and the tricarboxylic acid ity to scavenge ROS and thus protect against increases in (TCA) cycle (Supplementary Tables 1 and 3). Genes oxidative stress. The SHR, for example, has reduced involved in the electron transport chain, fatty acid deg- mRNA levels as well as reduced enzyme activity of su- radation and synthesis, glycolysis, gluconeogensis, and peroxide dismutase (Sod) in many but not all tissues.37–43 the TCA cycle are almost globally underexpressed in Superoxide dismutase 3 (Sod3) is one of the orthologous BPH (Supplementary Tables 1 and 3). In general, the genes commonly underexpressed in both SHR (0.60-fold) entire gene network of the intermediary metabolism of and BPH (0.68-fold), and may thus be the source of a the BPH is underexpressed, whereas the gene network 34 of the SHR is globally differentially expressed, but not Conclusion uniform in direction of expression. The very different The SHR rat strain and BPH mouse strain are two perturbation patterns in intermediary metabolism of independent genetic models of human essential hyper- SHR and BPH suggest that, although each strain suffers tension. Because a diverse set of potential mechanisms from metabolic abnormalities, the mouse BPH strain can lead to development of hypertension in humans, it is has sustained a more unidirectional alteration of such unlikely a priori that elevation of BP in the SHR and functions. BPH results from precisely the same mechanisms. The Because the SHR and BPH models of human hyper- question arises then as to which hypertensive rodent tension are oligogenic, involving about three to five 24 –26 strain is more appropriate as a model of human essential major loci cosegregating with BP, the widespread hypertension. metabolic transcript changes we observed are likely to The SHR and BPH strains have each reached more than be downstream effects resulting from a few primary 50 generations of inbreeding and, in the case of the SHR, genetic causes.A candidate for one of these primary different stocks of the strain exist in institutions throughout genetic defects is 3-hydroxy-3-methylglutaryl-coen- the world. The existence of multiple colonies and such zyme A reductase, an ortholog overexpressed in com- extensive inbreeding may lead to mutations that did not mon by the SHR and BPH (Hmgcr; Table 2). Hmgcr is contribute to the hypertensive phenotype during the selec- the rate-limiting enzyme in cholesterol biosythesis and tion process. At least one such mutation exists in SHR. A also the target of the statin drugs in dyslipidemic hu- mutation in the Cd36 gene of the SHR leads to undetect- mans.27 Alterations in expression of this crucial “bot- able protein levels of the transcribed gene and has been tleneck” enzyme could serve as a primary genetic defect proposed as a cause of insulin resistance, defective fatty that disrupts many facets of the intermediary metabo- acid metabolism, and hypertriglyceridemia in the SHR,21 lism of SHR and BPH. In that case, then, the metabolic yet the mutation does not exist in all substrains of SHR, differential gene expression patterns in SHR and BPH including the original stock developed in Japan.79 Muta- might reflect the ways in which the two organisms tions in Cd36 have not been well investigated in BPH; respond to the same primary stress. Cd36 mRNA is well expressed in the BPH (Supplemen- A previous study by Aitman et al21 marshaled data tary Table 3). from transcript expression microarrays, congenic strain We found 1217 genes differentially expressed in SHR mapping, and radiation hybrid mapping to identify a (13.9% of the microarray) and 2108 genes differentially mutation in the Cd36 gene, a fatty acid transporter, that expressed in BPH (16.9% of the microarray), yet genetic may account for one of the defective glucose and fatty analyses have estimated that only about three major loci contribute to hypertension in SHR,24,25 whereas about four acid metabolism, hypertriglyceridemia, and hyperten- 26 sion quantitative trait loci (QTLs) on SHR chromosome or five major loci contribute to hypertension in BPH. 4.A genomic deletion event is thought to result in a Clearly, then, much if not most of the differential gene chimeric Cd36 gene that encodes a dysfunctional pro- expression observed here must be secondary to or com- pensating for a much smaller number of primary genetic tein undetectable in SHR adipocyte plasma mem- 21 defects. It is also likely that a subset of differentially branes. The global gene expression derangements that expressed genes exists that results from polymorphisms or we observe in the SHR metabolism might, in part, result interspecies and interstrain differences unrelated to hyper- from this primary defect in Cd36, and the defect could tension. Our current technique cannot distinguish between also partially explain the differences we observe in differentially expressed genes resulting from hypertension metabolic expression patterns between SHR and BPH. and those resulting from interspecies and interstrain dif- It is unknown whether the BPH genome contains a 79 ferences not related to hypertension. Furthermore, the mi- defective copy of the Cd36 gene. Another study croarrays we used contain probe sets for only several showed that the Cd36 mutation is not present in the thousand genes each and therefore provide limited views original SHR colony developed by Okamoto in Japan, of the rat and mouse genomes, which likely harbor up indicating that the mutation did not play a role in the to 25,000 genes each (estimate from the Ensembl selection for high BP in the SHR, nor the insulin resis- project, www.ensembl.org). Microarray analyses of the tance observed in the original stock. The SHR strain we entire rat and mouse genomes would likely produce hun- 18 used did harbor the Cd36 mutation. dreds if not thousands of additional differentially ex- The fact that the SHR and BPH animals suffer from pressed genes. metabolic maladies although the two strains were selected Because the SHR exhibits several phenotypes of the solely on the basis of BP indicates a clear, yet unresolved metabolic syndrome and the BPH may also suffer from link between metabolic abnormalities and increases in BP. similar metabolic abnormalities, altered gene expression in The question is unresolved as to whether metabolic abnor- the SHR and BPH may be affected by or causative of malities arise from or contribute to the BP elevation in the metabolic abnormalities in addition to hypertension. The hypertensive strain. vast number of differentially expressed genes and the 35 presence of other associated or confounding phenotypes author of this paper and the following co-authors clearly make it difficult to identify the primary genetic directed and supervised the research: Daniel T. defects in hypertension, based solely on microarray stud- O’Connor, Geert W. Schmid-Schönbein, Nicholas J. ies. The small number of genes that cause hypertension in Schork, Sushil K. Mahata, Nitish R. Mahapatra, and SHR and BPH may or may not lie in our dataset of Payam Mahboubi. We appreciate the assistance of the differentially expressed genes, depending on whether the University of California San Diego (UCSD) Veterans underlying mutations confer quantitative or qualitative Medical Research Foundation (VMRF) and Center for changes in gene expression. Nonetheless, our strategy to AIDS Research Genomics Core for performing the compare differentially, commonly expressed orthologs real-time PCR experiments and the UCSD VMRF may be key to determining shared, fundamental gene Microarray Center for performing the GeneChip expression mechanisms across mammalian species, and, experiments. Dr. Morton Printz provided the SHR and therefore, may be particularly relevant to the study of WKY rat tissues. human essential hypertension. Our dataset of differentially expressed orthologs un- References shared in 90% of cases by SHR and BPH (Table 1) 1. O’Connor DT, Insel PA, Ziegler MG, Hook VY, Smith DW, shows that the same selection paradigm in two different Hamilton BA, Taylor PW, Parmer RJ: Heredity and the autonomic inbred, homozygous rodent species creates patterns of nervous system in human hypertension. Curr Hypertens Rep 2000; gene expression that are fundamentally and quantitatively 2:16–22. 2. 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38 39

ABSTRACT

The genetic basis of hypertension in the Spontaneously Hypertensive

Rat (SHR) remains unknown. At this point, researchers have published ~80

SHR blood pressure Quantitative Trait Loci (QTLs) that provide many clues to the genetic underpinnings of hypertension in the SHR, yet discussion of candidate genes has traditionally failed to exceed conjecture. The advent of cost-effective and high-throughput DNA sequencing technology now allows researchers to identify polymorphisms within candidate genes and determine the effects of discovered polymorphisms on gene function. The gap in physiology between the immediate effects of candidate gene mutations and the development of hypertension, however, is quite large, so adrenal mRNA abundance is proposed as an intermediate phenotype between gene mutations and hypertensive disease phenotypes. In the current study, a novel method that integrates adrenal gland microarray data with QTL data is used to identify candidate genes for hypertension in the SHR. The underlying hypothesis of the method is that the nucleotide mutations in the genes responsible for hypertension in the SHR manifest as changes in mRNA abundance in the adrenal gland, even before penetrance of classical hypertensive phenotypes. Seven candidate genes were identified

(chromogranin A, catechol-O-methyltransferase, dopamine beta-hydroxylase, endothelin receptor type B, electron-transferring-flavoprotein dehydrogenase,

40 neuropeptide Y, and phenylethanolamine-N-methyltransferase) and resequenced. Polymorphisms discovered in the promoter and/or 3- untranslated regions of the chromogranin A (Chga) and phenylethanolamine-

N-methyltransferase (Pnmt) genes are strong candidates to explain the mechanism of differential expression of Chga (1.73-fold overexpressed) and

Pnmt (0.67-fold underexpressed) mRNA observed in the SHR adrenal gland.

Functional studies on the effect of the Chga and Pnmt gene polymorphisms on mRNA abundance are presented in Chapter 4.

INTRODUCTION

The use of linkage analysis and QTL mapping to dissect the genetic basis of human essential hypertension, a highly complex and polygenic trait1, has thus far generated poor and weakly tractable results. In contrast, linkage analysis of monogenic, Mendelian disorders such as Huntington disease2,3 and cystic fibrosis4-6 has conclusively pinpointed susceptibility loci responsible for the traits. Diseases, such as essential hypertension, are defined as complex traits because development of the disease phenotype depends on multiple genes acting independently or acting interactively with themselves and the environment—it is this polygenic and intricate nature of complex traits that proves problematic for linkage analysis1,7. Furthermore, dissection of the genetic basis of human essential hypertension is confounded by the

41 uncontrolled and dominant impact of the environment on the ultimate disease phenotypes. Indeed, genes are estimated to be responsible for only ~20-30% of the elevated blood pressure phenotype in human essential hypertension8.

The SHR/WKY hypertensive/normotensive rat strains are an ideal model in which to dissect the genetic basis of essential hypertension because the disease has a purely genetic basis in these strains, i.e., differences in SHR and WKY genotypes are responsible for 100% of the difference in hypertensive phenotypes. The power to detect susceptibility loci for essential hypertension is improved in the SHR/WKY model since the environment can be strictly controlled and genetics completely dictate disease progression. Yet, the highly complex and polygenic nature of essential hypertension in humans is mirrored in the SHR9,10 and continues to prove problematic in genetic analyses.

Historically, identification of novel blood pressure QTLs in the SHR was rarely, if ever, followed by positional candidate polymorphism discovery or functional testing. With the recent advances in genomic sequencing technology, it is now possible for researchers to couple SHR blood pressure

QTL mapping with polymorphism discovery in positional candidate genes, and the scientific literature is beginning to reflect this paradigm shift. While polymorphism discovery in positional candidates is a crucial step, the ultimate

42 goal of elucidating the mechanism whereby mutations in the DNA sequence of candidate genes can cause hypertension requires additional investigation.

The objective of the current study is to begin to bridge the rather wide gap between identification of polymorphisms in candidate genes and the ultimate development of hypertension. A novel method that integrates adrenal gland microarray data with SHR QTL data is used to identify 7 candidate genes for hypertension in the SHR. The hypothesis underlying the method is that the nucleotide mutations in the genes responsible for hypertension in the

SHR manifest as changes in mRNA abundance in the adrenal gland, even before penetrance of hypertensive disease phenotypes. Each of the 7 candidate genes was resequenced to identify polymorphisms that could contribute to the differential expression of the genes in the SHR adrenal gland.

Creating the link between gene polymorphisms and changes in mRNA transcription constitutes a first step in elucidating a genetic mechanism whereby the candidate genes contribute to the pathogenesis of hypertension in the SHR. Functional studies determining the effects of the candidate gene polymorphisms are described in Chapter 4.

METHODS

Candidate gene identification strategy

Initially, candidate genes were drawn from the population of genes that displayed differential expression of mRNA between the adrenal glands of the

SHR and the WKY rat strains. Differential mRNA expression was determined by two methods: (1) microarray analysis of SHR and WKY adrenal glands

(described in Chapter 2); and (2) review of the scientific literature. A literature review was performed in addition to the microarray analysis in order to maximize the set of genes known to be differentially expressed in the SHR adrenal gland. From the set of differentially expressed genes in the SHR adrenal gland, a gene was selected as a candidate gene for hypertension if it showed a dramatic change in adrenal mRNA expression and it had a plausible and compelling biological role in hypertensive pathophysiology.

Two criteria were then applied to the set of genes showing adrenal gland differential expression in order to identify additional candidate genes:

Criteria 1: The gene is a positional candidate for a blood pressure

Quantitative Trait Locus (QTL) in the SHR.

Positional candidates for SHR blood pressure QTLs were identified through alignment of the chromosomal position of genes differentially expressed in the SHR adrenal gland with the chromosomal position of peak markers for all publicly available SHR blood pressure QTLs.

A flat file containing an annotated list of current (as of April 7, 2004)

Rattus norvegicus QTLs was downloaded from the Rat Genome Database

FTP server (http://rgd.mcw.edu/pub/) and parsed to identify the peak markers for all SHR or SHR-stroke prone blood pressure QTLs. Next, the chromosomal position (in base pairs) of the QTL peak markers was determined using the

University of California, Santa Cruz, rat genome browser

(http://genome.ucsc.edu/)11,12. Similarly, the chromosomal position of genes differentially expressed in the SHR adrenal gland was determined using

Affymetrix RG-U34A microarray annotation (date = 12/15/2003; downloaded from the Affymetrix webpage: http://www.affymetrix.com/) and/or the University of California, Santa Cruz, rat genome browser. A gene differentially expressed in the SHR adrenal gland was identified as a positional candidate for a SHR blood pressure QTL if its chromosomal base pair position was close to (within

~5 Mb) the chromosomal position of a QTL peak marker and the gene was also a biologically plausible candidate for hypertensive pathology.

Criteria 2: The gene is a positional candidate for a Quantitative Trait

Locus (QTL) in the SHR recombinant inbred (RI) strains.

Positional candidates for SHR RI strain QTLs were identified through alignment of the chromosomal position of genes differentially expressed in the

SHR adrenal gland with the chromosomal position of peak markers for the RI

45 strain QTLs. Alignment of gene and QTL peak marker base pair positions was performed as described above.

A collaborative effort yielded the QTLs in the SHR RI strains (technically referred to as the HXB/BXH strain set13-15). The RI strains were developed from the SHR and Brown Norway (BN) rat strains as a tool to map the genetic basis of complex traits, particularly hypertension. Investigation of the SHR RI strains forms the foundation of the doctoral dissertation of our collaborator, and a complete discussion of the methodology and results can be found in the doctoral dissertation of Martin Jirout (Molecular Pathology Graduate Program,

University of California, San Diego).

Resequencing of candidate genes

The nucleotide sequence of each candidate gene was downloaded from the University of California, Santa Cruz, rat genome browser

(http://genome.ucsc.edu/)11,12. The Primer3 program

(http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi)16 was used to design polymerase chain reaction (PCR) primers to amplify the proximal promoter

(~1500 base pairs), the 3- and 5- untranslated regions (UTR), exons, and all intron/exon border regions of the candidate genes from genomic DNA of the

Spontaneously Hypertensive Rat (SHR), Wistar-Kyoto (WKY) (Charles River

Laboratories, Wilmington, MA), or Brown Norway (BN)13,14 rat strains.

Primers were designed to produce 500-700 bp amplicons, the ideal amplicon length to achieve high quality sequencing reads using the Applied

Biosystems 3100 Genetic Analyzer. Genomic DNA PCR was performed using

HotStar Master Mix (Qiagen, Valencia, CA) and 25 ng of genomic DNA.

Shrimp Alkaline Phosphatase and Exonuclease I (Fermentas, Hanover, MD) were used to purify genomic DNA PCR amplicons through inactivation (i.e. phosphate removal) and degradation of unincorporated nucleotides and primers.

Sequencing PCR was performed using Big Dye Terminator Version 3.1

(Applied Biosystems, Foster City, CA), which contains AmpliTaq Gold DNA

Polymerase and dideoxynucleotides. Sequencing PCR product was column purified using multi-screen 96-well plates (Fisher Scientific) and Sephadex G-

50 beads (Sigma-Aldrich), and subsequently sequenced with the Applied

Biosystems 3100 Genetic Analyzer. Polymorphisms were identified and visually confirmed in the sequencing chromatograms using EditView 1.0.1 software for Mac OS 9 (Applied Biosystems). A list of the PCR and sequencing primers can be found in Appendix A. Nucleotide sequence of the resequenced genes can be found in Appendix B.

RESULTS

Candidate gene identification strategy

The starting set of genes from which to select candidate genes for hypertension in the SHR consisted of 1217 genes differentially expressed in the SHR adrenal gland (SHR mRNA vs. WKY mRNA) (Figure 3.1), as determined by microarray analysis (Chapter 2) and a review of gene expression results published in the scientific literature. Microarray analysis failed to show differential expression of the chromogranin A (Chga) gene, but the literature review revealed a Northern blot experiment that showed a 1.73- fold overexpression of Chga mRNA in the SHR adrenal gland (vs. WKY)17.

Among the 1217 differentially expressed genes, one gene was selected as a candidate gene because it showed a dramatic 50-fold change in adrenal mRNA abundance and was also a compelling biological candidate for hypertension pathophysiology.

The set of 1217 adrenal differentially expressed genes was constrained with two criteria in order to identify additional candidate genes (Figure 3.1).

Application of the first criteria (the gene is a biologically interesting positional candidate for a blood pressure QTL in SHR) led to selection of 5 candidate genes, and imposition of the second criteria (the gene is a biologically interesting positional candidate for a QTL in the RI strains) led to selection of 3 candidate genes. Two candidate genes were independently identified using

48 both the first and second criteria, so a total 6 distinct candidate genes (not 5

+3 = 8) were identified (Figure 3.2).

Candidate gene selection

In total, 7 genes were selected as candidate genes (1 selected based only on adrenal gland differential expression; 6 selected based on both adrenal differential expression and QTL data) (Table 3.1, Figure 3.2).

The electron-transferring-flavoprotein dehydrogenase (Etfdh) gene was selected since it showed a dramatic 50-fold underexpression in SHR adrenal gland (see Chapter 2) and its biological function, transfer of electrons from flavin-containing dehydrogenases to the electron transport chain of the mitochondria, makes it a logical and compelling candidate for the oxidative stress component of hypertensive pathology18.

The catechol-O-methyltransferase (Comt), dopamine beta-hydroxylase

(Dbh), endothelin receptor type B (Ednrb), neuropeptide Y (Npy), and phenylethanolamine-N-methyltransferase gene (Pnmt) genes were selected because they showed adrenal gland differential expression and were also biologically interesting positional candidates for SHR blood pressure QTLs

(Table 3.1, Table 3.2, Figure 3.2). Comt was overexpressed 37.39-fold in SHR adrenal gland and lies within 5.4 Mb (megabases; 1 x 106 bases) of the

Bp10419 QTL peak. Dbh was underexpressed 0.39-fold in SHR adrenal gland

49 and lies within 0.3 Mb of the Bp1520 QTL peak. Ednrb was overexpressed

2.09-fold in SHR adrenal gland and lies within 0.1 Mb of the Bp12621 QTL peak. Npy was underexpressed 0.67-fold in SHR adrenal gland and lies within

0.1 Mb of the Bp13522 QTL peak. Pnmt was underexpressed 0.67-fold in SHR adrenal gland and lies within 13.6 Mb of the Bp123 QTL peak.

The chromogranin A (Chga) gene was selected as a candidate because it showed adrenal gland differential expression and was also a biologically interesting positional candidate for a SHR RI strain QTL (Table 3.1, Figure

3.2). Chga was overexpressed 1.73-fold in SHR adrenal gland17 and was a positional candidate for an adrenal gland Chga protein QTL in the SHR RI strains (Figure 3.3). The QTL peak has a suggestive LOD score of 2.58 and its location was confirmed by permutation-based bootstrapping methods. It was estimated that the QTL accounts for ~35% in the variance of the adrenal Chga protein trait. The SHR genotype is associated with elevated adrenal Chga protein abundance.

As described above, adrenal gland differential expression and proximity to the Bp1520 and Bp123 QTL peaks led to selection of Dbh and Pnmt as candidate genes (Table 3.1, Figure 3.2). The selection of Dbh and Pnmt as candidate genes is further supported by several SHR RI strain QTLs. The Dbh gene is a positional candidate for adrenal Dbh mRNA and for adrenal Dbh enzyme activity (whole tissue) RI strain QTLs (unpublished data, not shown).

The SHR genotype is associated with decreased adrenal Dbh mRNA and decreased adrenal Dbh enzyme activity (whole tissue). Similarly, the Pnmt gene is a positional candidate for adrenal Pnmt mRNA and for adrenal Pnmt enzyme activity (whole tissue) RI strain QTLs (unpublished data, not shown).

The SHR genotype is associated with decreased adrenal Pnmt mRNA and decreased adrenal Pnmt ezyme activity (whole tissue).

The final list of candidate genes for hypertension in the SHR consists of

7 genes: Chga, Comt, Dbh, Ednrb, Etfdh, Npy, and Pnmt. Genes were selected as candidates based on different combinations of three criteria: (1) differential expression of mRNA in SHR adrenal gland (all candidate genes satisfied this criteria); (2) proximity and biological relevance to a SHR blood pressure QTL; (3) proximity and biological relevance to a SHR RI strain QTL.

Candidate gene polymorphisms discovery

Each candidate gene was resequenced in the SHR hypertensive rat strain and the appropriate normotensive control rat strain (Table 3.1):

• Etfdh was identified solely on the basis of adrenal mRNA differential

expression in SHR versus WKY and, therefore, was resequenced in SHR

and WKY.

• Ednrb and Npy displayed adrenal mRNA differential expression in SHR

versus WKY and were also positional candidates for blood pressure QTLs

generated from SHR x WKY crosses. Therefore, Ednrb and Npy were

resequenced in SHR and WKY.

• Even though the Comt gene was a positional candidate for the Bp104 QTL

generated from a SHR x Wild (not WKY) cross, the gene was resequenced

in the SHR and WKY strains since its mRNA was differentially expressed in

the adrenal glands of SHR versus WKY.

• Chga was differentially expressed in the adrenal gland of SHR versus WKY

and was a positional candidate for a SHR RI strain QTL, and, therefore,

was resequenced in SHR, WKY, and BN.

• Dbh and Pnmt were differentially expressed in the adrenal gland of SHR

versus WKY, positional candidates for SHR blood pressure QTLs, and

positional candidates for SHR RI strain QTLs. Dbh and Pnmt were

resequenced in SHR and BN by a collaborator. Sequencing data for Dbh is

not presented.

The proximal promoter (~1500 bp), 5- and 3- untranslated regions

(UTR), exons, and intron/exon borders of the 7 candidate genes were resequenced in genomic DNA from the SHR, WKY, and/or BN strains (Table

3.3). Single nucleotide polymorphisms (SNPs) discovered in the candidate genes are named in such a way as to describe both the position at which the

SNP occurs and the change in nucleotide from normotensive strain to

52 hypertensive strain. For example, the name “T-450C” would describe a SNP discovered at position -450 in the gene, wherein a “T” is found in the normotensive strain (WKY or BN) and a “C” is found in the hypertensive strain

(SHR). The term “In/Del” is used to describe an insertion/deletion polymorphism.

Comt, Ednrb, Etfdh: No polymorphisms were found between the Comt (Figure

3.4), Ednrb (Figure 3.5), or Etfdh (Figure 3.6) genes of the SHR and WKY.

Npy: Resequencing of the Npy gene in SHR and WKY revealed a “TC” dinucleotide repeat length polymorphism at position -1025 in the promoter

(SHR = 22 “TC” dinucleotide repeats, WKY = 23 “TC” dinucleotide repeats

(Figure 3.7).

Chga: Multiple polymorphisms were discovered in the promoter, coding, and

3UTR regions of the Chga gene in the SHR, WKY, and BN strains (Figure

3.8). The nucleotide sequence of the Chga promoter and exons in WKY and

BN was identical. Five polymorphic sites were discovered in the promoter

(-1694 In/Del -/G; A-1616T; -753 “A” repeat: WKY = 11, SHR = 15; C-177T;

C-59T), two amino acid repeat length polymorphisms were found in the coding region (Exon 5 glutamine repeat: WKY = 20, SHR = 12; Exon 6 glutamate

53 repeat: WKY = 16, SHR = 15), and 1 SNP was identified in the 3UTR. Several polymorphisms were also discovered in the introns.

Pnmt: Resequencing of Pnmt in SHR and BN revealed 5 polymorphisms in the promoter (T-529C; -457 In/Del A/-; T-404C; C-396T; C-351T) and 2 SNPs in the coding region (A+209G; T+1475C) (Figure 3.9). The A+209G coding region polymorphism is a silent, synonymous SNP that does not change the predicted amino acid (alaninealanine). In contrast, the T+1475C coding SNP does result in an amino acid change, and is predicted to change the last residue in the Pnmt protein from valine (in BN) to alanine (in SHR). 3-D modeling of the Pnmt protein structure shows that the valinealanine residue is part of the carboxy terminal tail that is not crucial for catalytic function of the enzyme (data not shown). One intronic SNP was identified: A+320G.

DISCUSSION

Candidate gene identification strategy

The method used to select candidate genes forms an integrative and novel approach to identify hypertension susceptibility genes in the SHR. The underlying hypothesis of the selection strategy is that the genetic mutations responsible for hypertension in the SHR manifest as changes in mRNA transcript abundance in the adrenal gland, even before penetrance of

54 hypertensive disease phenotypes. The use of mRNA transcript abundance as an intermediate phenotype might prove to be even more powerful than traditional biochemical intermediate phenotypes (e.g. plasma catecholamines) since mRNA abundance is a more proximal and, perhaps, accurate reflection of gene action than post-translational, multifactorial biochemical parameters.

In accordance with the hypothesis described above, the first step in candidate gene selection was to identify the genes that show a difference in mRNA transcript abundance in the SHR adrenal gland (Figure 3.1). From the set of adrenal differentially expressed genes, a gene could be selected as a candidate gene for hypertension in the SHR if it showed dramatic differential expression and was a compelling biological candidate for hypertension pathophysiology.

The second step used to select candidates was to isolate genes from the set of adrenal differentially expressed genes that satisfy at least one of the following two criteria: (1) the gene must be a positional candidate for a blood pressure QTL in the SHR; or (2) the gene must be a positional candidate for a

QTL in the SHR RI strains (Figure 3.1). After selection of all candidates, the genes were resequenced in the SHR and normotensive control strains to identify polymorphisms that could explain the observed differences in adrenal mRNA abundance in vivo.

Candidate gene selection

Differentially expressed genes in the SHR adrenal gland

The vast majority of adrenal mRNA differential expression was determined using microarray analysis (see Chapter 2). Only one gene, chromogranin A (Chga), was identified as differentially expressed during review of the scientific literature. In total, 1217 genes were identified as differentially expressed in the SHR adrenal gland.

Electron-transferring-flavoprotein dehydrogenase (Etfdh) showed a dramatic 50-fold underexpression in SHR adrenal gland (see Chapter 2) and was selected as a candidate gene since its biological function, transfer of electrons from flavin-containing dehydrogenases to the electron transport chain of the mitochondria, makes it a compelling candidate for the enhanced oxidative stress phenotype observed hypertension18.

SHR blood pressure QTLs

Linkage analysis and QTL mapping of elevated blood pressure in the

SHR has implicated many loci in development of the trait. Indeed, 79 SHR blood pressure QTLs are deposited in the public databases (as of April 7,

2004). The multitude of SHR blood pressure QTLs provides many leads into dissecting the genetic basis of hypertension in the SHR, yet definitive links

56 between candidate genes and development of hypertension have not been reported.

Alignment of the chromosomal position of all genes differentially expressed in SHR adrenal gland with the chromosomal position of peak markers for all published SHR blood pressure QTLs revealed a novel set of positional candidates for these QTLs. Positional candidates were selected as candidate genes for hypertension if they were located close to the QTL peak

(within ~5 Mb) and had plausible biological roles in hypertensive pathophysiolgy. Catechol-O-methyltransferase (Comt), dopamine beta- hydroxylase (Dbh), endothelin receptor-type B (Ednrb), neuropeptide Y (Npy), and phenylethanolamine-N-methyltransferase (Pnmt) were selected as candidate genes since they were differentially expressed in the SHR adrenal gland and were also positional candidates for previously published SHR blood pressure QLTs (Table 3.2, Figure 3.2).

SHR RI strain QTLs

Development of the SHR RI (recombinant inbred) strains from the SHR and BN progenitors in 1982, marked the beginning of a new tool with the potential to greatly increase the ability to dissect the genetics of hypertension13-15. Yet, ~25 years after development of the RI strains commenced, linkage analysis and QTL mapping of elevated blood pressure in

57 the RI strains has not conclusively pinpointed any genes involved in the pathogenesis of hypertension in the SHR. The complex and polygenic nature of hypertension continues to prove problematic for genetic analysis, even in the RI strains.

The emergence of “genetical genomic” strategies has made it possible to integrate highthroughput, large-scale microarray data sets into linkage analysis by treating mRNA abundance as a quantitative trait24. It appears advantageous to use mRNA abundance as a quantitative trait since it could more accurately reflect gene action than the distant, complex elevated blood pressure trait. A collaborative effort that utilized genetical genomic techniques produced intriguing Chga and Pnmt physiological and expression (i.e., based on mRNA abundance) QTLs in the SHR RI strains.

The chromogranin A (Chga) and phenylethanolamine-N- methyltransferase (Pnmt) genes are logical and strong positional candidates for SHR RI strain QTLs. Chga is a positional candidate for an adrenal Chga protein QTL, wherein the SHR genotype is associated with increased adrenal

Chga protein (Figure 3.3). Pnmt is a positional candidate for an adrenal Pnmt mRNA abundance eQTL and an adrenal Pnmt enzyme activity (whole tissue)

QTL (data not shown). The SHR genotype is associated with reduced adrenal

Pnmt mRNA and reduced adrenal Pnmt enzyme activity (data not shown).

Since Chga and Pnmt are differentially expressed in SHR adrenal gland and

58 are positional candidates for QTLs in the SHR RI strains, both genes were selected as candidate genes.

Candidate gene polymorphism discovery

Each candidate gene was resequenced in order to identify polymorphisms within the gene that could contribute to its adrenal mRNA differential expression in vivo. The proximal promoter and untranslated (5- and 3-UTR) regions of each gene are logical locations to search for polymorphisms that could alter transcription and mRNA abundance, but it is also conceivable that polymorphism within the coding region (i.e., exons) and intron/exon splice sites could affect mRNA levels. While it is well-known that introns contain transcriptional regulatory motifs, the introns were not specifically resequenced because these regions are poorly conserved and are usually not crucial for gene function, suggesting that introns might be more likely to harbor innocuous mutations. Therefore, the promoter, 5- and 3-

UTRs, exons, and intron/exon splice sites of each candidate gene were resequenced.

Catechol-O-methyltransferase (Comt)

No polymorphisms were discovered between the Comt genes in the

SHR and WKY strains (Table 3.3, Figure 3.4).

Endothelin receptor type B (Ednrb) and

No polymorphisms were found in the Ednrb gene of the SHR and WKY strains (Table 3.3, Figure 3.5).

Electron transferring flavoprotein dehydrogenase (Etfdh)

No polymorphisms were discovered between the Etfdh genes in SHR and WKY (Table 3.3, Figure 3.6), which is somewhat surprising since microarray analysis showed a 50-fold difference in mRNA levels between the

SHR and WKY adrenal glands. Reanalysis of the microarray gene expression data, however, revealed a discrepancy in the specificity of the Etfdh probe set in the microarray annotation used in the initial analysis (annotation date =

December 15, 2003) and the microarray annotation used in the reanalysis

(annotation date = March 9, 2007) (Table 3.4). The target of the

“rc_AI237007_at” probe set, which initially seemed to be Etfdh (based on annotation from December 15, 2003), apparently targets Znf324_predicted

(based on annotation from March 9, 2007), not Etfdh. Based on the reanalysis,

Etfdh would not have been selected as a candidate gene.

Uncertainty in probe set targets, as illustrated with Etfdh and

Znf324_predicted, is a problem of the utmost importance in microarray research since it can completely invalidate the results. Microarrays for species

60 with incomplete and newly sequenced genomes may suffer most from probe set target uncertainly, as was the case with the rat genome microarray back in

2003. Uncertainty in probe set targeting, however, is not likely to be a problem for the set of well-defined, functionally verified genes that forms the foundation of most microarray research.

Neuropeptide Y (Npy)

A “TC” dinucleotide repeat polymorphism was discovered in the promoter region of the Npy gene (Table 3.3, Figure 3.7) that could explain the

0.67-fold underexpression of Npy mRNA in the adrenal gland of SHR. Even though Npy is a biologically intriguing candidate gene for hypertension in the

SHR, the effect of the Npy promoter polymorphism was not pursued in further functional studies because it seemed unlikely that a 2 base pair deletion in a string of 46 bases (SHR = 22 “TC” dinucleotide repeats, WKY = 23 “TC” dinucleotide repeats) could have a large impact on mRNA transcription.

Chromogranin A (Chga)

Chga is of particular interest as a candidate gene since much data suggests it has a pathogenic role in human essential hypertension25. Multiple polymorphisms were discovered in the promoter, coding, and 3UTR regions of the Chga gene in the SHR, WKY, and BN strains (Table 3.3, Figure 3.8). The

61 nucleotide sequence of the Chga promoter and exons in WKY and BN was identical. The promoter and/or 3UTR polymorphisms could explain the 1.73- fold overexpression of Chga mRNA in the SHR adrenal gland, and could also explain the elevated levels of SHR adrenal Chga protein mapped in the SHR

RI strains (Figure 3.3). A 2.21-fold increase of Chga protein in the adrenal gland of the SHR has also been reported in the literature17.

Coding region mutations that change amino acid (or, in this case, delete amino acids) are primarily thought to affect protein function, not mRNA transcription—though it is conceivable that a change in protein function could feedback on mRNA transcription. The effect of the coding region mutations on

Chga protein function is investigated in Chapter 4. Polymorphisms were also discovered in the introns of the Chga locus but their impact on transcription was not investigated further. The role of Chga in formation and exocytosis of catecholamine storage vesicles in chromaffin cells of the adrenal medulla26 makes the gene a compelling candidate for hypertension in the SHR.

Phenylethanolamine-N-methyltransferase (Pnmt)

Polymorphisms were discovered in the promoter and coding regions of the SHR and BN Pnmt genes (Table 3.3, Figure 3.9). The promoter polymorphisms could contribute to the decreased adrenal Pnmt mRNA and decreased adrenal Pnmt enzyme activity (whole tissue) of the SHR, which was

62 mapped in the SHR RI strains (data not shown). Furthermore, if the WKY

Pnmt gene has the same sequence as the BN Pnmt gene, the promoter polymorphisms could also explain the 0.67-fold underexpression of Pnmt mRNA observed in the SHR adrenal gland (compared to the WKY adrenal gland). Effects of the promoter polymorphisms on Pnmt transcription are elucidated in Chapter 4.

The A+209G coding region polymorphisms is a nonsynonymous SNP that does not change the predicted amino acid and, therefore, is not expected to have an effect on Pnmt enzymatic function. In contrast, the T+1475C coding region SNP does change the amino acid of the last residue of the Pnmt protein from valine (in BN) to alanine (in SHR). 3-D structure models of the Pnmt protein show that the last amino acid residue is located in a carboxy terminal tail that is not crucial for catalytic activity of the protein (data not shown). valinealanine residue is part of the carboxy terminal tail that is not crucial for catalytic function of the enzyme (data not shown). One polymorphism was discovered in an intron but it is not investigated further. The role of Pnmt in converting norepinephrine to epinephrine makes the gene an intriguing candidate for hypertension in the SHR.

CONCLUSION

Traditional linkage analysis and QTL mapping of blood pressure in the

SHR has implicated many loci in development of hypertension. Indeed, 79

SHR blood pressure QTLs have been reported in the scientific literature. In the past, most publications that present SHR blood pressure QTLs and speculate about positional candidates do not couple QTL identification with positional candidate polymorphism discovery and functional testing. Completion of the rat genome sequence and the availability of reliable, cost-effective DNA sequencing, however, has begun to shift the paradigm of SHR blood pressure

QTL mapping to include resequencing of positional candidate genes. Still, functional testing of polymorphisms within candidate genes remains unreported.

A novel method that integrates adrenal gland microarray data, previously reported SHR blood pressure QTLs, and linkage analysis/QTL mapping in the SHR RI strains was used to identify 7 candidate genes for hypertension in the SHR. The underlying hypothesis of the method is that the genetic mutations responsible for hypertension in the SHR manifest as changes in mRNA transcript abundance in the adrenal gland, even before penetrance of hypertensive disease phenotypes. Abundance of mRNA is a more proximal and, perhaps, more accurate reflection of gene action than distant post-translational and multifactorial biochemical parameters. Candidate

64 genes were chosen, in part, because they were biologically interesting candidates for hypertensive pathophysiology that showed differences in mRNA level in the SHR adrenal gland.

The candidate genes were resequenced in the SHR hypertensive strain and the BN or WKY normotensive control strains. Two of the candidate genes,

Chga and Pnmt, harbor polymorphisms in the promoter and/or 3UTR that could contribute to the differences in Chga and Pnmt mRNA abundance observed in the SHR adrenal gland, and could also contribute to Chga and

Pnmt QLTs in the SHR RI strains. The role of Chga and Pnmt in adrenal catecholamine secretion and biosynthesis make the genes compelling candidates for functional testing in Chapter 4.

ACKNOWLEDGEMENTS

The text of Chapter 3, in part or in full, will be submitted for publication.

The dissertation author was the primary researcher and author, and the following co-authors directed and supervised the research: Daniel T.

OConnor, Geert W. Schmid-Schönbein, Nitish R. Mahapatra, and Martin

Jirout. Vafa Mahboubi and Kenton Murthy provided vital instruction and advice on resequencing of candidate genes. Martin Jirout generously contributed the

Pnmt sequencing data and the Chga and Pnmt SHR RI strain QTLs. Ted Kurtz provided genomic DNA from the SHR and WKY rat strains. Thank you all!

Figure 3.1: Candidate gene identification strategy

The strategy to select candidate genes for hypertension in the SHR is depicted. The initial set of genes from which candidates genes were selected consisted of 1217 genes showing differential expression of mRNA between SHR and WKY adrenal glands. One candidate gene for hypertensioin was selected from the set of 1217 genes because it showed a dramatic 50-fold difference in adrenal mRNA abundance and it had a compelling role in hypertensive patholophysiology. An additional 8 candidate genes were identified by requiring that the adrenal differentially expressed genes were also positional candidates for either a SHR blood pressure QTL (5 genes) or a SHR RI strain QTL (3 genes). 66

FIGURE 3.1

SHR (Rattus norvegicus) genome

1 2 3 4 5 6 7 8 9 10 11

12 13 14 15 16 17 18 19 20 X ~25,000 genes

Genes differentially expressed in SHR adrenal gland

mRNA

SHR or WKY abundnace mRNA strain

1,217 genes (1 candidate identified)

Positional candidate for Positional candidate for blood pressure QTL in SHR QTL in RI strains

gene A gene B LOD score LOD score

Chromosomal position Chromosomal position 5 candidate genes identified 3 candidate genes identified 67

FIGURE 3.2

SHR adrenal gland differential expression

Etfdh

Comt Ednrb Chga Npy Dbh Pnmt

SHR blood pressure SHR RI strain QTL QTL positional candidate positional candidate

Figure 3.2: Rationale for candidate gene selection The rationale for selection of each candidate gene is depicted. Gene symbols: Chga (chromogranin A); Comt (catechol-O-methyltransferase); Dbh (dopamine beta-hydroxylase); Ednrb (endothelin receptor, type B); Etfdh (electron transferring flavoprotein dehydrogenase); Npy (neuropeptide Y); Pnmt (phenylethanolamine-N-methyltransferase). 68

Figure 3.3: The Chga Gene is a Positional Candidate for a RI Strain Adrenal Chga Protein QTL

Mapping of Chga protein as a quantitative trait in the SHR RI strains resulted in a linkage peak on chromosome 6 directly over the position of the Chga gene (shown with a red arrow). The QTL peak has a suggestive LOD score of 2.58 and its location was confirmed by permutation-based bootstrapping methods. It was estimated that the QTL accounts for ~35% in the trait variance. The SHR genotype is associated with increased adrenal Chga protein.

69 Bootstrap counts Bootstrap bootstrap counts 40 30 20 10 0 70 60 50 145 137 129 Chga 113 LOD = 2.58 105 97 89 81 73 Figure 3.3 65 57 49 Rat chromosome 6 (cM) 41 33 25 17 Bootstrap LOD plot 9 Legend

1 LOD score LOD 0.5 0.0 1.0 2.0 1.5 2.5 3.0 FIGURE 3.4

Exon 1 Exon3'UTR 4

6000 -1000 -500 0 +500 +1000 +1500 +2000 +2500 +3000 +3500 +4000

Legend Exon

Untranslated region of exon (UTR)

No sequencing data. Region not resequenced.

Figure 3.4: Polymorphism discovery in the Comt gene The region of the Comt gene that was resequenced is depicted as a horizontal line. Exons are represented as red boxes with the untranslated regions shown in blue. Intronic regions where resequencing was not performed are shown as white boxes. The Comt mRNA cap site (transcriptional start site) is depicted as position “0”. Base pair position of the promoter is numbered negatively in descending order upstream of the cap site. Base pair position of the exonic and intronic regions are numbered in ascending order downstream of the cap site. No polymorphisms

were discovered. 70 FIGURE 3.5

-2000 -1000 0 +1000 +2500 +3500 +4500

Legend Exon

Untranslated region of exon (UTR)

No sequencing data. Region not resequenced.

Figure 3.5: Polymorphism discovery in the Ednrb gene The region of the Ednrb gene that was resequenced is depicted as a horizontal line. Exons are represented as red boxes with the untranslated regions shown in blue. Intronic regions where resequencing was not performed are shown as white boxes. The Ednrb mRNA cap site (transcriptional start site) is depicted as position “0”. Base pair position of the promoter is numbered negatively in descending order upstream of the cap site. Base pair position of the exonic and intronic regions are numbered in ascending order downstream of the cap site. No polymorphisms were discovered. 71 FIGURE 3.6

-1000 0 +1000 +3000 +5000 +7000 +9000 +11000 +13000 +15000 +17000 +19000 +21000

Legend Exon

Untranslated region of exon (UTR)

No sequencing data. Region not resequenced.

Figure 3.6: Polymorphism discovery in the Etfdh gene The region of the Etfdh gene that was resequenced is depicted as a horizontal line. Exons are represented as red boxes with the untranslated regions shown in blue. Intronic regions where resequencing was not performed are shown as white boxes. The Etfdh mRNA cap site (transcriptional start site) is depicted as position “0”. Base pair position of the promoter is numbered negatively in descending order upstream of the cap site. Base pair position of the exonic and intronic regions are numbered in ascending order downstream of the cap site. No polymorphisms were discovered. 72 FIGURE 3.7

-1025 "TC" repeat WKY = 23 SHR = 22

-1000 0 +1000 +5000 +6000 +7000 Legend Exon

Untranslated region of exon (UTR)

No sequencing data. Region not resequenced.

Figure 3.7: Polymorphism discovery in the Npy gene The region of the Npy gene that was resequenced is depicted as a horizontal line. Exons are represented as red boxes with the untranslated regions shown in blue. Intronic regions where resequencing was not performed are shown as white boxes. The Npy mRNA cap site (transcriptional start site) is depicted as position “0”. Base pair position of the promoter is numbered negatively in descending order upstream of the cap site. Base pair position of the exonic and intronic regions are numbered in ascending order downstream of the cap site. The “TC” dinucleotide repeat length polymorphism discovered in the promoter at position -1025 is indicated with an arrow. 73 FIGURE 3.8

T+12339C C-177T A+1196T C+3168T T+413C C+885T A+8388G G+11177T C+3863T C+6587T -1694 In/Del -/G G+3033T -1000 bp

+2000 bp +5000 bp +7000 bp +10000 bp +12000 bp A-1616T C-59T A+1113G T+3961C Gln repeat Glu repeat SHR = 12 SHR = 15 G+10882A G+12611T "A" repeat +3386 In/Del C/- WKY = 20 WKY = 16 SHR = 11 "TC" repeat WKY = 15 Legend Exon

Untranslated region of exon (UTR)

No sequencing data. Region not resequenced.

Figure 3.8: Polymorphism discovery in the Chga gene The region of the Chga gene that was resequenced is depicted as a horizontal line. Exons are represented as red boxes with the untranslated regions shown in blue. Intronic regions where resequencing was not performed are shown as white boxes. The Chga mRNA cap site (transcriptional start site) is designated as position “0”. Base pair position of the promoter is numbered negatively in descending order upstream of the cap site. Base pair position of the exonic and intronic regions are numbered in ascending order downstream of the cap site. The location of the 5 promoter, 2 coding , and 1 3’UTR SNPs are shown with vertical lines. The positions of the intronic SNPs are also

indicated with vertical lines. 74 FIGURE 3.9

A+209G T+1475C -457 In/Del A/- (Ala Ala) (Val Ala) T-529C C-396T

-500 +500 C-351T 0 +1000 +1500 T-404C A+320G

Legend Exon

Untranslated region of exon (UTR)

No sequencing data. Region not resequenced.

Figure 3.9: Polymorphism discovery in the Pnmt gene The region of the Pnmt gene that was resequenced is depicted as a horizontal line. Exons are represented as red boxes with the untranslated regions shown in blue. Intronic regions where resequencing was not performed are shown as white boxes. The Pnmt mRNA cap site (transcriptional start site) is depicted as position “0”. Base pair position of the promoter is numbered negatively in descending order upstream of the cap site. Base pair position of the exonic and intronic regions are numbered in ascending order downstream of the cap site. The promoter and coding regions polymorphisms are indicated with vertical lines. The position of the one of intronic SNP that was 75 discovered is also indicated with a vertical line. Table 3.1 Candidate genes for hypertension in the SHR

The 7 genes selected as candidates for hypertension in the SHR are listed. Candidate gene: The symbol of the candidate gene—Chga (chromogranin A); Comt (catechol-O-methyltransferase); Dbh (dopamine beta-hydroxylase); Ednrb (endothelin receptor type B); Etfdh (electron-transferring-flavoprotein, dehydrogenase); Npy (neuropeptide Y); Pnmt (phenylethanolamine-N-methyltransferase). Rationale: The rationale for selection of each candidate gene. mRNA = differential expression in the SHR adrenal gland (compared to WKY). SHR QTL = positional candidate for a SHR blood pressure QTL. RI QTL = positional candidate for a SHR RI strain QTL. mRNA fold change (SHR/WKY): The fold change of the candidate gene mRNA observed in the SHR adrenal gland (SHR expression level/WKY expression level). SHR QTL: The name of the SHR blood pressure QTL for which the candidate gene is a positional candidate. RI QTL: “Yes” indicates that the candidate gene is a positional candidate for a SHR RI strain QTL. “No” indicates that the candidate gene is not a positional candidate for a SHR RI strain QTL.

mRNA FC SHR RI Candidate gene Rationale (SHR/WKY) QTL QTL Strains sequenced Chga mRNA; RI QTL 1.73 n/a YES SHR/WKY/BN

Comt mRNA; SHR QTL 37.39 Bp104 NO SHR/WKY

Dbh mRNA; SHR QTL; RI QTL 0.39 Bp15 YES SHR/BN

Ednrb mRNA; SHR QTL 2.09 Bp126 NO SHR/WKY

Etfdh mRNA 0.02 n/a NO SHR/WKY

Npy mRNA; SHR QTL 0.67 Bp135 NO SHR/WKY

Pnmt mRNA; SHR QTL; RI QTL 0.67 Bp1 YES SHR/BN 76 Table 3.2. Positional candidates for SHR blood pressure QTLs

Differentially expressed genes in the SHR adrenal gland that are positional candidates for SHR blood pressure QTLs are listed. QTL: Name of the SHR blood pressure QTL. Cross: The rat strains used to generate the QTL. Chr: Chromosome number of the QTL. QTL peak marker: Name of the QTL peak marker. Peak marker position (Mb): Chromosomal physical position of the QTL peak marker in units of mega-bases (1 x 106 bases). Positional candidate: Symbol of the SHR adrenal differentially expressed gene that is proposed as a positional candidate for the QTL. Gene symbols: Comt (catechol-O-methyltransferase); Dbh (dopamine beta-hydroxylase); Ednrb (endothelin receptor, type B); Npy (neuropeptide Y); Pnmt (phenylethanolamine-N-methyltransferase). Positional candidate position (Mb): Chromosomal physical position of the candidate gene in units of mega-bases (1 x 106 bases). Positional candidate fold change (SHR/WKY): The mRNA fold change of the positional candidate in the SHR adrenal gland. Fold change was calculated as the quotient of SHR expression to WKY expression.

Peak Positional Positional QTL marker candidate candidate peak position Positional position fold change QTL Cross Chr marker (Mb) candidate (Mb) (SHR/WKY) SHR x Bp104 11 Sst 79.2 Comt 84.6 37.39 Wild

SHRSP x Bp15 3 D3Mgh16 6.3 Dbh 6.0 0.39 WKY

SHRSP x Bp126 15 Ednrb 87.9 Ednrb 87.9 2.09 WKY

SHR x Bp135 4 Npy 78.3 Npy 78.3 0.67 WKY

SHRSP x 77 Bp1 10 Gh1 95.7 Pnmt 82.1 0.67 WKY Table 3.3. Polymorphism discovery in candidate genes

Polymorphisms discovered in the promoter, 5 UTR, coding, 3 UTR, and intron/exon splice site regions of candidate genes are listed. The specific strains in which the candidate genes were resequenced are also listed. Abbreviations: Gln = glutamine, Glu = glutamate, Ala = alanine, Val = valine. Gene symbols: Chga (chromogranin A); Comt (catechol-O-methyltransferase); Ednrb (endothelin receptor type B); Etfdh (electron-transferring-flavoprotein, dehydrogenase); Npy (neuropeptide Y); Pnmt (phenylethanolamine-N-methyltransferase).

Intron/Exon Gene Promoter 5 UTR Coding 3 UTR splice sites Strains 1) -1694 In/Del -/G 2) A-1616T 1) Exon 5, Gln repeat 3) -753 “A” repeat (WKY = 20, SHR = 12) Chga None G+11177T None SHR/WKY/BN (WKY = 11, SHR = 15) 2) Exon 6, Glu repeat 4) C-177T (WKY = 16, SHR = 15) 5) C-59T

Comt None None None None None SHR/WKY

Ednrb None None None None None SHR/WKY

Etfdh None None None None None SHR/WKY

-1025 “TC” repeat Npy None None None None SHR/WKY (WKY = 23, SHR = 22)

1) T-529C 1) Exon 1, A+209G 2) -457 In/Del A/- (Ala Ala) Pnmt 3) T-404C None None None SHR/BN 2) Exon 3, T+1475C 4) C-396T (Val Ala) 5) C-351T 78 Table 3.4. Lack of specificity of the Etfdh probe set

Identification of electron-transferring-flavoprotein dehydrogenase (Etfdh) as a candidate gene for hypertension in the SHR was based on adrenal differential expression of the “rc_AI237007_at” probe set (see Chapter 2). Reanalysis of the probe set data with updated annotation showed that the target gene of the “rc_AI237007_at” probe set changed from Etfdh to Znf324_predicted. Based on analysis of the “rc_AI237007_at” probe set with annotation from 3/9/2007, Etfdh would not be selected as a candidate gene. RG-U34A Probe Set ID: name of the probe set on the Affymetrix RG-U34A microarray. Annotation Date: the date of microarray annotation used in the analysis. Target Gene: the name of the gene targeted by the microarray probe set. mRNA fold change (SHR/WKY): fold change of target gene mRNA (SHR intensity divided by WKY intensity) as indicated by the microarray probe set.

RG-U34A Annotation mRNA fold change Probe Set ID Date Target Gene (SHR/WKY) Candidate gene? Electron-transferring-flavoprotein rc_AI237007_at 12/15/2003 0.05 Yes dehydrogenase (Etfdh)

Zinc finger protein 324, predicted (Znf324_predicted) rc_AI237007_at 03/09/2007 0.05 No Hypothetical protein LOC691759 (LOC691759) 79 80

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21. Kato, N, Mashimo, T, Nabika, T, Cui, ZH, Ikeda, K & Yamori, Y. Genome-wide searches for blood pressure quantitative trait loci in the stroke-prone spontaneously hypertensive rat of a Japanese colony. J Hypertens 21, 295-303 (2003).

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Chapter 4: Promoter Polymorphisms Contribute to Adrenal mRNA Differential Expression of the Chromogranin A and Phenylethanolamine-N- methyltransferase Genes in the Spontaneously Hypertensive Rat

84 85

ABSTRACT

In chapter 3, candidate genes for hypertension in the SHR were identified and resequenced. Polymorphisms discovered in the chromogranin A

(Chga) and phenylethanolamine-N-methyltransferase (Pnmt) candidate genes were hypothesized to contribute to (1) the differential expression of Chga

(1.73-fold overexpressed) and Pnmt (0.67-fold underexpressed) mRNA in the

SHR adrenal gland, and to (2) the adrenal Chga protein, adrenal Pnmt mRNA, and adrenal Pnmt enzymatic activity QTLs in the SHR RI (recombinant inbred) strains. The biological function of Chga and Pnmt make the genes compelling candidates for hypertension in the SHR: Chga is the most highly abundant protein secreted from catecholamine storage granules, is crucial for formation of secretory granules, and is a prohormone that is proteolytically processed into bioactive peptides capable of modulating catecholamine secretion, vasodilation, glucose homeostasis, and vascular inflammation; the Pnmt enzyme catalyzes conversion of norepinephrine to epinephrine in the final step of catecholamine biosynthesis. In the current chapter, the results of luciferase assays performed with Chga and Pnmt luciferase reporter constructs indicate that polymorphisms within the Chga and Pnmt promoters of the SHR result in increased Chga promoter activity and decreased Pnmt promoter activity, and are consistent with the in vivo adrenal mRNA phenotypes and RI strain QTLs.

However, the differential transcriptional responses of the Chga and Pnmt

86 promoters were only observed after stimulation with nicotine (a nicotinic, cholinergic receptor agonist) and dexamethasone (a synthetic glucocorticoid), respectively. Experiments to elucidate the specific molecular mechanism whereby the SHR Pnmt promoter responds differentially to dexamethasone revealed a possible complex interaction of the five SNPs within the promoter.

The objective of the current study is to build evidence that the Chga and Pnmt genes in the SHR harbor polymorphisms that make pathogenic contributions hypertensive disease phenotypes. The physiological gap between DNA mutations in candidate genes and hypertensive disease processes is quite large, and the current study begins to bridge that gap by demonstrating that polymorphisms in the Chga and Pnmt promoters lead to changes in Chga and

Pnmt mRNA transcription.

INTRODUCTION

In chapter 3, candidate genes for hypertension in the SHR were selected based on the hypothesis that nucleotide mutations in the gene or genes responsible for hypertension in the SHR manifest as changes in mRNA transcript abundance in the adrenal gland, even before penetrance of classic hypertensive disease phenotypes. Each candidate gene displayed differential expression of mRNA in the SHR adrenal gland, and was resequenced in order to identify polymorphisms within the genes that could contribute to the adrenal

87 differential expression. The chromogranin A (Chga) and phenylethanolamine-

N-methyltransferase (Pnmt) genes emerged as the strongest candidate genes that contained polymorphisms that could contribute to the adrenal differential expression (Chga, 1.73-fold overexpressed; Pnmt, 0.67-fold underexpressed).

In the current chapter, the effects of the Chga and Pnmt polymorphisms were investigated through quantification of luciferase and embryonic alkaline phosphatase (EAP) reporter vector expression in PC12 chromaffin cells.

The PC12 chromaffin cell line is derived from a pheochromocytoma isolated from the adrenal medulla of a male, New England Deaconess Hospital strain, white rat (Rattus norvegicus)1. Since the Chga and Pnmt genes are normally highly expressed in adrenal chromaffin cells in vivo, PC12 cells are a valid and appropriate system in which to evaluate the expression of Chga and

Pnmt reporter constructs.

Historically, identification of candidate genes for hypertension in the

SHR has rarely been followed with resequencing of candidate genes or functional studies to elucidate the effects of polymorphisms discovered during resequencing. While polymorphism discovery in Chga and Pnmt in Chapter 3 was a crucial step, functional studies of identified polymorphisms are required to demonstrate a pathogenic role of the genes in hypertension of the SHR.

The ultimate goal of pinpointing the specific gene or genes that cause hypertension in the SHR and elucidating the mechanism whereby mutations in

88 the nucleotide sequence of these genes leads to physiological disease processes will require much investigation.

The objective of the current study is to begin to bridge the rather wide physiological gap between polymorphisms in candidate genes and development of hypertension in the SHR. To accomplish this objective, the relationship between promoter polymorphisms in the Chga and Pnmt genes and abnormal adrenal mRNA levels will be investigated. Creating the link between gene polymorphisms and changes in mRNA transcription is an important step to determine if the mutations within Chga and Pnmt do indeed make pathogenic contributions to hypertension in the SHR.

METHODS

Construction of the Chga and Pnmt promoter/luciferase reporter plasmids

Primer32 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi) was used to design polymerase chain reaction (PCR) primers to amplify a ~1.8 kb fragment of the chromogranin A (Chga) proximal promoter or a ~1 kb fragment of the phenylethanolamine-N-methyltransferase (Pnmt) proximal promoter from genomic DNA of the SHR (Chga and Pnmt) , WKY (Chga), or BN (Pnmt) rat strains. Computational sequence analysis of the Chga promoter revealed the absence of KpnI and HindIII restriction enzyme sites, and, therefore, a

KpnI (5-GGTACC-3) or HindIII (5-AAGCTT-3) site was inserted in the 5-end of the forward (5- TAAAGGTACCAGCACACCTAAACTGTGAC-3) or reverse

(5-GAGCAAGCTTGTGCGGAAAGAGAG-3) PCR primer, respectively.

Computational sequence analysis of the Pnmt promoter revealed the absence of SacI and XhoI restriction enzyme sites, and, therefore, a SacI (5-GAGCTC-

3) or XhoI (5-CTCGAG-3) restriction enzyme site was inserted in the 5-end of the forward (5- GTGAGAGCTCGCAGGGTCCTCTACCTGTGA -3) or reverse (5- CACGCTCGAGGCTCCTGTTG-3) PCR primer, respectively.

Amplified Chga and Pnmt promoter fragments were digested with the appropriate restriction enzymes and subsequently inserted into the polylinker region of the firefly luciferase reporter vector, pGL3-Basic (Promega, Madison,

WI), with T4 DNA ligase (Invitrogen). The pGL3-Basic vector lacks eukaryotic promoter and enhancer sequences, and contains the cDNA for firefly luciferase. Creation of single nucleotide polymorphism (SNP) variants of the

Pnmt promoter/luciferase reporter constructs was accomplished using the

QuickChange Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA). A list of the mutagenesis primers can be found in Appendix C. Correct insertion and/or mutation of the Chga and Pnmt promoter fragments was confirmed by

DNA sequencing. Plasmid DNA was prepared and purified for transfection using the QIAfilter Plasmid Midi Kit (Qiagen, Valencia, CA).

Construction of the Chga 3-UTR/luciferase reporter plasmids

Primer32 was used to design PCR primers to amplify the 333 bp 3- untranslated region (3-UTR) of Chga from SHR and WKY genomic DNA.

Computational sequence analysis of the Chga 3-UTR revealed the presence of the XbaI restriction enzyme site, so the XbaI-compatible NheI site, which is absent in the Chga 3-UTR of SHR and WKY, was inserted into the 5-end of both the forward (5-ACGGGCTAGCGGCACTGGCTGGTGGGGTCCGGCCA-

3) and reverse (5-AAAGGCTAGCGAAGAGCCCAAAGCAGGTTTATTCT-3)

PCR primers. Digestion of the PCR amplified 3-UTR fragments with NheI was followed with T4 DNA ligase (Invitrogen) catalyzed insertion of the fragments into the XbaI site of the firefly luciferase reporter vector, pGL3-Promoter

(Promega, Madison, WI), which contains the SV40 promoter and the cDNA for firefly luciferase. Correct insertion of the Chga 3-UTRs was confirmed by DNA sequencing. Plasmid DNA was prepared and purified for transfection using the

QIAfilter Plasmid Midi Kit (Qiagen).

Construction of the Chga cDNA/EAP reporter plasmids

Primer32 was used to design PCR primers to amplify the 1389 bp rat

Chga cDNA (without the 5- and 3-UTR sequences) from a Norway rat (Rattus

Norvegicus) cDNA clone (NM_021655). The XhoI and Kozak (5-GCCACC-3) sequences were included in the 5-end of the forward primer (5-

ATGCCTCGAGGCCACCATGCGCTCCTCCGCGGCTTTGG-3'), and the KpnI site was inserted in the 5-end of the reverse primer (5-

ATGCGGTACCCTCCCCGTCGCTAAGCCTGCAG-3. T4 DNA ligase

(Invitrogen) was used to insert the rat Chga cDNA between the XhoI and KpnI sites in the polylinker region of our custom embryonic alkaline phosphatase

(EAP) reporter vector, pEAP-N2, which contains the CMV promoter and cDNA for EAP.

After insertion into the pEAP-N2 vector, the Norway rat Chga cDNA

(which was identical to the WKY Chga cDNA) was mutated so that the length of the glutamine repeat region matched the length of the region found in SHR.

The SHR Chga cDNA-EAP reporter was constructed by PCR amplifying the

WKY cDNA-EAP construct with mutagenesis primers (designed to delete 8 glutamine residues or 24 bp), treating the PCR product with Dpn1 to digest the

WKY template DNA, and ligating the linear SHR PCR product with the Rapid

DNA Ligation Kit (Roche, Indianapolis, IN). Sequence of the SHR and WKY

Chga cDNA was confirmed by DNA sequencing. Plasmid DNA was prepared and purified for transfection using the QIAfilter Plasmid Midi Kit (Qiagen).

Chga and Pnmt luciferase reporter transfection and luciferase activity assays

Rat PC12 pheochromocytoma cells [grown in DMEM high glucose

(Invitrogen) with 5% heat-inactivated fetal bovine serum (Gemini Bioproducts,

Woodland, CA), 10% heat-inactivated horse serum (Gemini Bioproducts), penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine (0.292 mg/ml)] were transfected (at 50-60% confluence, 1 day after splitting 1:4) with Chga

(promoter or 3-UTR) or Pnmt (promoter) reporter plasmid DNA [1 μg supercoiled DNA per well; 12-well polystyrene plates (coated with poly-L- lysine; Sigma), 2.2-cm diameter wells, Corning Inc., Corning, NY] using the liposome method (Superfect; Qiagen). Cells were incubated with or without dexamethasone (Calbiochem), pituitary adenylate cyclase-activating peptide

(PACAP) (ovine; Calbiochem), or nicotine (Sigma). Cells were lysed 16 hours after transfection with lysis buffer (300 μL per well) [0.1 M phosphate buffer

(K2HPO4 + KH2PO4) (pH 7.8), 1 mM DTT, and 0.1% Triton-X 100].

The bioluminescent activity of luciferase in 80 μL of cell lysate was determined using the AutoLumat LB 953 luminometer (EG&G Berthold,

Nashua, NH) to measure light emission (incubation time = 0 seconds, measure time = 10 seconds, temperature = 25˚C) after addition of assay buffer

[100 μl per sample; 100 mM Tris-acetate (pH 7.8), 10 mM Mg-acetate, 1 mM

EDTA (pH 8.0), 3 mM ATP, and 100 μM luciferin (Sigma-Aldrich)]. As a control

93 for varying cell number within individual wells, the total protein content was measured in the cell lysate using the Bio-Rad Protein Assay (coomassie blue dye absorbance shift; based on the Bradford method) (Bio-Rad, Hercules,

CA). Luciferase activity in the cell lysate is expressed as the normalized ratio of (luciferase activity)/(total protein content) or (RLU/μg protein).

Chga cDNA/EAP reporter EAP secretion and activity assay

Rat PC12 pheochromocytoma cells (from ATCC) [grown in F-12K

(Invitrogen) with 2.5% heat-inactivated fetal bovine serum (Gemini

Bioproducts, Woodland, CA), 15% heat-inactivated horse serum (Gemini

Bioproducts), penicillin (100 U/ml), streptomycin (100 μg/ml), and L-glutamine

(0.292 mg/ml)] were transfected (at ~50% confluence, 1 day after splitting 1:3) with Chga cDNA/EAP reporter plasmid DNA [2.5 μg supercoiled DNA per well;

6-well polystyrene plates (coated with poly-L-lysine (Sigma) and rat collagen

(Upstate)), 3.48-cm diameter wells, Corning Inc., Corning, NY] using the

GenePorter2 Transfection Reagent (cationic lipid) (Genlantis, San Diego, CA).

Two hours before performing the EAP secretion activity assays, the cells were incubated with tritiated norepinephrine (3H-NE; 0.5 μCi per 1 mL cell media). The assay was performed by stimulating the cells with 700 μL of

calcium (150 mM NaCl, 5 mM KCl, 2 mM CaCl2, 10 mM HEPES pH 7.4) or

barium (150 mM NaCl, 5 mM KCl, 2 mM BaCl2, 10 mM HEPES pH 7.4)

94 secretion buffer, and collecting cellular supernatant and lysate fractions after a

15-minute incubation. Tritiated norepinephrine (read for 2 minutes) and chemiluminescent EAP activity (read for 10 seconds) were measured in both cell supernatant and cell lysate fractions (Phospha-Light System, Applied

Biosystems). Final EAP activity is expressed as the normalized ratio of (EAP activity)/(3H-NE counts per minute) or (RLU/cpm).

Chga protein conservation and coiled-coil prediction

Human (Homo sapiens), cow (Bos taurus), rat (Rattus norvegicus), and mouse (Mus musculus) Chga amino acid sequence was downloaded from the

University of California, Santa Cruz, genome browser

(http://genome.ucsc.edu/)3,4 and aligned with the ClustalW algorithm (using

MacVector 9.0 software for Mac OSX) to determine conservation of the Chga protein across the species.

The probability of SHR and WKY Chga protein to assume a 3-D coiled- coil conformation was computed with the COILS algorithm

(http://www.ch.embnet.org/software/COILS_form.html)5. In brief, the algorithm searches for strongly amphipathic regions that display a pattern of alternating hydrophilic and hydrophobic residues repeated every 7 residues (heptad repeat residues ag, with a & d being hydrophobic). The minimum window was two such heptads, over 14 residues. The probability of coiled-coil

95 structure is presented as a function of amino acid position in the mature CHGA protein.

RESULTS

Chga promoter/reporter luciferase assays

Luciferase assays were performed on PC12 cells transfected with SHR and WKY Chga promoter/luciferase reporter constructs (Figure 4.1, Figure

4.2) to determine if differential transcriptional responses occur under basal (i.e. untreated cells) or stimulated [with 100 nM dexamethasone, 1 mM nicotine, or

100 nM pituitary adenylate cyclase-activating peptide (PACAP)] conditions

(Table 4.1, Figure 4.3).

The basal level (i.e. under no stimulation) of luciferase expression derived from the SHR Chga promoter was 0.73-fold lower than the expression derived from the WKY promoter (p = 0.002). Dexamethasone stimulation modestly increased expression of the WKY and SHR constructs but the expression of SHR remained 0.72-fold less than WKY (p = 0.003). PACAP stimulation dramatically increased expression of both the SHR and WKY constructs and also caused the relative difference in expression between the constructs to diminish (0.84-fold less in SHR, p = 0.102). Nicotine stimulated expression of the SHR Chga promoter construct so much that the expression increased to levels higher than WKY (1.35-fold increased in SHR, p = 0.001).

Chga 3-UTR/reporter luciferase assays

Luciferase assays were performed on PC12 cells transfected with SHR and WKY Chga 3-UTR/luciferase reporter constructs (Figure 4.4, Figure 4.5) to determine if the G+174T SNP within the 3-UTR affects luciferase expression (Figure 4.6). No difference in luciferase activity was detected among the control, SHR, and WKY constructs (ANOVA, p = 0.633).

Chga cDNA/EAP reporter secretion and activity assays

Nucleotide sequencing of the Chga locus (see Chapter 3), revealed the presence of glutamine (Gln or Q) and glutamate (Glu or E) repeat length polymorphisms in SHR (12 Gln, 15 Glu) and WKY (20 Gln, 16 Glu) Chga protein (Figure 4.7). The conservation of the glutamine and glutamate repeat regions across human (Homo sapiens), cow (Bos taurus), rat (Rattus norvegicus), and mouse (Mus musculus) was determined (Figure 4.8). The glutamine repeat is found only in rat and mouse. The glutamate repeat is present only rat.

Since human Chga protein is predicted to assume a 3-D coiled-coil structure (unpublished), the probability of the WKY and SHR proteins to also assume a coiled-coil structure was computationally predicted (Figure 4.9). The glutamine repeat polymorphism is predicted to change the probability of (by

~0.25) and number of residues involved in (8 residue change) a coiled-coil,

97 while polymorphism within the glutamate repeat is not predicted to alter coiled- coil conformation.

Embryonic alkaline phosphatase (EAP) secretion and activity assays were performed on PC12 cells transfected with SHR and WKY Chga cDNA/EAP reporter constructs (Figure 4.10) to determine if the glutamine repeat polymorphism affects Chga protein trafficking through or secretion from the regulated secretory pathway. After allowing sufficient time for the Chga-

EAP chimera to be transcribed, translated, and shuttled through the Golgi into regulated secretory vesicles (30 hours), exocytosis of PC12 cells was stimulated with barium (a potent secretory stimulus) or calcium (a negative control). The luminescent activity of EAP in the supernatant and cell lysate was then measured (Figure 4.11) and used to compute “relative secretion” and “sorting index” (Figure 4.12).

Relative secretion (Rs) is defined as the fraction of total EAP activity that is found in the supernatant: Rs = (EAP activity in supernatant)/(EAP

activity in supernatant + EAP activity in lysate). Sorting index (SI) is defined as the difference between relative secretion of barium and calcium treated cells

normalized by the relative secretion of calcium treated cells: SI = (Rsbarium –

Rscalcium)/Rscalcium. No statistical difference exists between the basal level (i.e. cells treated with calcium) of SHR and WKY Chga-EAP relative secretion in

PC-12 cells (unpaired t-test, p = 0.0933) or between the relative secretion of

SHR and WKY Chga-EAP in PC-12 cells stimulated with barium (unpaired t- test, p = 0.0664). No statistical difference exists between the sorting index of

SHR and WKY.

Pnmt promoter/reporter luciferase assays

Luciferase assays were performed on PC12 cells transfected with SHR and BN Pnmt promoter/luciferase reporter constructs (Figure 4.13, Figure

4.14) to determine if differential transcriptional responses occur under basal

(i.e. untreated) or stimulated [with 1 mM nicotine, 100 nM pituitary adenylate cyclase-activating peptide (PACAP), or dexamethasone (1, 10, or 100 nM)] conditions. The SHR and BN Pnmt promoters lack a differential response to

PACAP and to nicotine (Table 4.2, Figure 4.15), but the response of the SHR promoter to dexamethasone is blunted compared to the BN response (Table

4.3, Figure 4.16). The SHR promoter shows significantly less expression than the BN promoter at 10 nM (0.746-fold less in SHR, p = 0.016) and at 100 nM

(0.769-fold less in SHR, p = 0.004) doses of dexamethasone. There is no difference in the basal activity (i.e. in unstimulated cells) of SHR and BN Pnmt promoters.

Since the T-529C Pnmt promoter SNP lies adjacent to a near consensus glucocorticoid response element (GRE) (Figure 4.17), its role in the blunted response of the SHR promoter to dexamethasone was studied. SNP

99 variants of the SHR/BN Pnmt promoters were constructed to reflect all possible combinations of individual SNPs on the SHR or BN background

(Table 4.4). Constructs SNP1-SNP5 were formed by systematically mutating each of the 5 SNPs within the BN Pnmt promoter to the corresponding SHR variant. Constructs SNP1b-SNP5b were formed by systematically mutating each of the 5 SNPs within the SHR Pnmt promoter to the corresponding BN variant. Luciferase assays on dexamethasone treated PC12 cells transfected with the SNP variants revealed many differences in expression between the constructs (Figure 4.18, Table 4.5, Table 4.6). The SNP1 construct, which varies at the T-529C SNP, shows significantly reduced expression compared to both the BN (p < 0.0001) and SHR constructs (p < 0.0001). The SNP1b construct, which also varies at the T-529C SNP, shows a significantly reduced expression compared to the BN (p = 0.0015) construct but not the SHR construct.

DISCUSSION

The principle of the luciferase assays

The transcriptional effects of polymorphisms within the proximal promoter regions of the Chga and Pnmt genes (Figure 4.1, Figure 4.13) were probed with luciferase assays performed on PC12 cells transfected with promoter/luciferase reporter constructs. The constructs were made by

100 inserting PCR amplified fragments of the Chga/Pnmt promoters into vectors that lack promoters but contain the cDNA for luciferase (Figure 4.2, Figure

4.14). The luciferase assay is a highly sensitive and robust technique that can indirectly characterize the transcriptional activity of the Chga/Pnmt promoters through the quantification of the bioluminescent activity of luciferase protein.

Since the constructs differ only in the sequence of the inserted promoters and are transfected into PC12 cells that contain identical transcriptional and translational machinery, any differences detected in the bioluminescent activity of luciferase protein are directly proportional to differences in transcription of luciferase mRNA.

The effect of the Chga 3-UTR G+174T SNP (Figure 4.4) on mRNA transcript stability was investigated by performing luciferase assays on PC12 cells transfected with 3-UTR/luciferase reporter constructs (Figure 4.5). The constructs were made by inserting a PCR amplified fragment of the SHR and

WKY Chga 3-UTRs into a vector that contains the SV40 promoter and cDNA for luciferase. The luciferase assay is used to indirectly characterize the stability of a luciferase mRNA that contains a Chga 3-UTR, by quantifying the bioluminescent activity of luciferase protein translated from the mRNA. Since mRNA transcription of each SHR and WKY construct is driven by identical

SV40 promoters and the constructs are transfected into PC12 cells that contain identical transcriptional and translational machinery, any differences

101 detected in the bioluminescent activity of luciferase protein are directly proportional to the effect of the G+174 SNP on mRNA stability.

The principle of the EAP assays

The effect of the glutamine repeat polymorphism (Figure 4.7) on the ability of SHR and WKY Chga proteins to be trafficked through and secreted from the regulated secretory pathway was studied by performing EAP assays on barium (a potent stimulus of regulated secretion) or calcium (a negative control for barium) treated PC12 cells transfected with Chga cDNA/EAP reporter constructs (Figure 4.10). The constructs contain a CMV promoter that drives expression of Chga cDNA with embryonic alkaline phosphatase (EAP) cDNA fused, in frame, directly downstream. The EAP assay is used to characterize the trafficking and secretory ability of the Chga protein by quantifying the chemiluminescent activity of the Chga-EAP chimera in the supernatant and lysate fractions of PC12 cells. Since mRNA transcription of the Chga cDNA/EAP constructs is driven by identical CMV promoters and the constructs are transfected into PC12 cells that contain identical transcriptional and translational machinery, any differences detected in the chemiluminescent activity of the Chga-EAP chimeras are directly proportional to the effect of the glutamine repeat polymorphism on Chga protein function. An important assumption inherent to the EAP assay is that the glutamine repeat

102 polymorphism does not affect mRNA transcription or stability. It is also important to note that transfection of a SHR or WKY Chga/EAP chimera, rather than simply SHR or WKY Chga, is necessary in order to distinguish the

SHR/WKY Chga protein from the endogenous PC12 cell Chga protein.

Chga promoter transcriptional activity

In Chapter 3, it was hypothesized that the polymorphisms discovered within the promoter region of the SHR Chga gene could explain the 1.73-fold elevation of Chga mRNA observed in the SHR adrenal gland (compared to the

WKY adrenal gland)6 as well as the adrenal Chga protein Quantitative Trait

Locus (QTL) in the SHR RI strains (Figure 3.3). Luciferase assays performed on PC12 cells transfected with Chga promoter/luciferase reporter constructs demonstrated that the basal activity of the SHR Chga promoter is actually

0.73-fold less than the WKY promoter (p=0.002), a result inconsistent with the adrenal mRNA abundance and QTL data. However, since the adrenal medulla is innervated by the splanchnic nerve and enveloped by the adrenal cortex, chromaffin cells in vivo are repeatedly stimulated by acetylcholine7,8, pituitary adenylate cyclase-activating peptide (PACAP)9,10, and glucocorticoid11—all of which induce transcription of Chga mRNA. Therefore, luciferase assays were also performed on PC12 cells stimulated with nicotine (a nicotinic cholinergic receptor agonist and acetylcholine analog), PACAP, and dexamethasone (a

103 synthetic glucocorticoid) (Figure 4.3, Table 4.1) at doses consistent with in vivo concentrations.

While dexamethasone modestly induced activation of the SHR and

WKY Chga promoters, the SHR variant remained less active (by 0.72-fold, p =

0.003) than the WKY variant. PACAP treatment dramatically induced activation of both the SHR and WKY Chga promoters and reduced the difference between the two variants to a statistically insignificant 0.84-fold

(p=0.102). Surprisingly, nicotine stimulation caused the SHR Chga promoter to increase its activity to a level 1.35-fold higher than the WKY Chga promoter (p

= 0.001). Perhaps, the effects of acetylcholine acting alone, or acting additively or synergistically with PACAP, are partially responsible for the 1.73-fold increase of Chga mRNA observed in the SHR adrenal gland in vivo6 and the

SHR RI strain adrenal protein QTL.

Nicotine and PACAP are thought to induce Chga mRNA transcription through two seemingly disparate pathways that ultimately act through comparable mechanisms: binding of the CREB transcription factor to the cyclic-AMP response element (CRE) within the Chga promoter. Activation of nicotinic cholinergic receptors by nicotine activates a signaling cascade that is thought to involve Na+ influx, membrane depolarization, Ca2+ influx through L- type voltage-gated calcium channels, increase in intracellular Ca2+, activation of protein kinase C (PKC), and, finally, binding of CREB to CRE to induce

104

Chga mRNA transcription7. The induction of Chga transcription by nicotine has been shown to occur specifically through the nicotinic cholinergic receptors, not the muscarinic cholinergic receptors7. The G-protein coupled PACAP

receptor is coupled to Gs and its activation results in cAMP mediated activation of protein kinase A (PKA), and subsequent binding of CREB to CRE to induce Chga mRNA transcription9. Computational analysis of the Chga promoter sequences revealed the presence of CREs within the SHR and WKY promoters but none of the CREs contained nor was nearby a polymorphism

(data not shown). It is possible that the differential response of the SHR and

WKY Chga promoters involves the interaction of other motifs and transcription factors.

Chga mRNA stability

The results of the 3-UTR/luciferase reporter assays indicate that the

G+174T SNP in the Chga 3-UTR does not change the stability of the SHR or

WKY mRNA transcripts (Figure 4.6) and, therefore, does not contribute to the

1.73-fold elevation of SHR adrenal Chga mRNA in vivo nor to the SHR RI strain adrenal Chga protein QTL.

105

Chga protein trafficking and secretion

Although it is conceivable that coding region polymorphism could feedback on transcription, the effects of the Chga glutamine and glutamate repeat length polymorphisms (Figure 4.7) were hypothesized to affect the trafficking and regulated secretion of the Chga protein, not Chga mRNA transcription. Examination of the conservation of Chga protein sequence across human (Homo sapiens), cow (Bos taurus), rat (Rattus norvegicus), and mouse (Mus musculus) revealed that the glutamate repeat does not exist in species other than the rat and that the glutamine repeat does not exist outside of rodents (rat and mouse) (Figure 4.8), suggesting that both repeat regions might not have crucial roles in protein function.

In addition to inter-species Chga protein sequence alignment, computational predictions of the effect of the glutamine and glutamate repeats on 3-D coiled-coil structure were used to infer the importance of these repeat domains. We previously predicted that the human Chga protein exhibits a coiled-coil 3D-structure (unpublished), so the impact of the glutamine and glutamate repeats on the probability of SHR and WKY Chga proteins to also assume a coiled-coil structure was determined (Figure 4.9). Only the glutamine repeat polymorphism is predicted to have any probability of (by

~0.25) changing the coiled-coil structure of SHR Chga protein. Both the glutamine and glutamate repeats lie in regions of high coiled-coil probability,

106 however, so limitations of the COILS algorithm might give a false negative result for the glutamate repeat. A crystal structure of Chga protein does not exist.

EAP assays were used to determine the effect of the glutamine repeat polymorphism on the ability of SHR and WKY Chga proteins to be trafficked through and secreted from the regulated secretory pathway (Figure 4.11).

There was no difference in the basal/barium-induced relative secretion of SHR and WKY Chga-EAP (Figure 4.12) nor in the “Sorting Index” for SHR and WKY

Chga-EAP (Figure 4.12). The EAP assays suggest that the glutamine repeat polymorphism does not change the ability of the SHR and WKY Chga protein variants to be trafficked through or secreted from the regulated secretory pathway.

Pnmt promoter transcriptional activity

In Chapter 3, it was hypothesized that the polymorphisms discovered within the promoter region of the SHR Pnmt gene could contribute to the adrenal Pnmt mRNA downregulation and adrenal Pnmt QTLs in the SHR. In the Pnmt QTLs, the SHR genotype was associated with a reduction in adrenal

Pnmt mRNA and a reduction in adrenal Pnmt whole-tissue enzyme activity. It was also hypothesized that if the WKY Pnmt promoter is identical to the BN

Pnmt promoter (which requires experimental validation), then the promoter

107 polymorphisms could also explain the 0.67-fold underexpression of Pnmt mRNA in the adrenal gland of SHR (vs. WKY).

Luciferase assays performed on PC12 cells transfected with Pnmt promoter/luciferase reporter constructs demonstrated that the basal activity of the SHR Pnmt promoter is equal to the basal activity of the BN Pnmt promoter

(Figure 4.15). So, following the same rationale used in the Chga promoter luciferase assays, the activity of the Pnmt promoters was tested during stimulation with nicotine, PACAP (Figure 4.15, Table 4.2), or dexamethasone

(Figure 4.16, Table 4.3). Enhanced activity of the SHR/BN Pnmt promoters in response to nicotine and PACAP is in agreement with previously reported studies12-14, however, the activity level of the two promoter variants remained equal. Induction of SHR and BN Pnmt promoter activity by dexamethasone is also consistent with the scientific literature15-19, but the dampened response of the SHR promoter was unexpected. Computational analysis of the SHR/BN

Pnmt promoter sequence revealed a SNP (T-529C) that is adjacent to a glucocorticoid response element (GRE) (Figure 4.17) and, therefore, might contribute to the blunted response of the SHR promoter to dexamethasone.

Luciferase assays were performed with SNP variant constructs of the

SHR/BN Pnmt promoters (Table 4.4) in order to elucidate the role of each individual SNP, especially T-529C, in the blunted response of the SHR promoter to dexamethasone (Figure 4.18, Table 4.5, Table 4.6). Even though

108 the SNP1 construct, which contains the T-529C SNP, appears to have the largest impact on dexamethasone induction, a distinct and independent contribution of any single SNP to the blunted glucocorticoid response phenotype is unclear. Similar complex effects of promoter SNPs on transcription have recently been reported for the KRT1 gene20, and it has been suggested that other transcription factors, including Egr-1, AP2, Sp1, and

MAZ18,21, are important for Pnmt promoter activation. Nonetheless, the dampened glucocorticoid response of the SHR Pnmt promoter could partially explain the adrenal Pnmt QTLs in the SHR RI strains and the 0.67-fold underexpression of adrenal Pnmt mRNA (compared to the WKY).

Chga, Pnmt, and hypertension of the SHR

The luciferase assays performed on the Chga and Pnmt reporter constructs provide strong evidence that polymorphisms within promoter regions of these genes contribute to elevated adrenal Chga mRNA, the adrenal Chga protein QTL, reduced adrenal Pnmt mRNA, and the adrenal

Pnmt mRNA and enzymatic activity QTLs in the SHR. But since two different normotensive controls, the WKY and BN, were used to generate the Chga and

Pnmt data, further experiments must be done to integrate all 3 strains (SHR,

WKY, and BN) and formulate a comprehensive understanding of the Chga and

Pnmt promoter SNPs. Further experiments are also required to determine the

109 specific molecular mechanisms whereby nicotine and dexamethasone induce differential responses of the SHR Chga and Pnmt promoters, respectively.

If the promoter polymorphisms do in fact lead to elevated adrenal Chga mRNA and reduced adrenal Pnmt mRNA, what is the effect on Chga and

Pnmt protein function, and, ultimately, on the development of hypertension?

Alterations in mRNA quantity do not necessarily translate to coordinate 1:1 changes in protein quantity. In the case of Chga, however, evidence has been presented that shows coordinate and consistent elevations of adrenal Chga mRNA (1.73-fold), adrenal Chga protein (2.21-fold), and even plasma Chga protein (2.54-fold) in the SHR (compared to WKY)6. Coordinate decreases in adrenal Pnmt mRNA (0.67-fold) and adrenal (whole-tissue) Pnmt enzyme actively (0.70-fold) have also been shown in the SHR (compared to BN)

(unpublished).

The biological function of Chga and Pnmt make them compelling candidate genes for hypertension in the SHR. In chromaffin cells of the adrenal medulla, Chga is the most highly abundant protein secreted from catecholamine storage granules22, and Chga also plays a crucial role in formation of the secretory granules23,24. In addition, Chga is a prohormone that is proteolytically cleaved into bioactive peptides capable of modulating catecholamine secretion (through the catestatin fragment)22,25-27, vasodilation

(through the vasostatin fragment)28, glucose homeostasis (through the

110 pancreastatin fragment)29-34, and vascular inflammation (through the vasostatin fragment)35-38. The Pnmt enzyme catalyzes the final step of catecholamine biosynthesis: conversion of norepinephrine into epinephrine. The idea that adrenal catecholamines and Pnmt might be involved in the pathogenesis of hypertension in the SHR is not new. In fact, the first investigations that demonstrated reductions in SHR adrenal Pnmt expression appeared in the scientific literature in 198139 and 198340. Studies have also shown an elevation of catecholamine storage in6,41 and secretion from42 the SHR adrenal gland.

Investigation of Chga and Pnmt as candidate genes for hypertension in the SHR is of particular importance because humans with essential hypertension display Chga and Pnmt aberrations similar to those observed in the SHR. Plasma Chga is elevated in both the SHR6 and in human essential hypertensives43, and polymorphisms within the human Chga locus result in differences in Chga mRNA transcription44 and Chga protein function25.

Humans with essential hypertension also show reductions in plasma catestatin45, the catecholamine-release inhibitory peptide, indicating the possibility that altered proteolytic processing of the Chga protein might contribute to hypertension. Polymorphism within the promoter region of the human Pnmt locus has been associated with hypertension in African American subjects46.

111

CONCLUSION

The effects of polymorphisms within the Chga and Pnmt genes on mRNA transcription and/or protein function were studied using luciferase and embryonic alkaline phosphates (EAP) reporter constructs. Luciferase and EAP assays demonstrated that the polymorphisms in the promoter region of Chga and Pnmt are likely to contribute to the differential adrenal mRNA expression of these genes observed in vivo, and could also explain the Chga and Pnmt

QTLs in the SHR RI strains. The crucial role of Chga and Pnmt in the biosynthesis and exocytosis of catecholamines makes them strong candidate genes for hypertension in the SHR.

ACKNOWLEDGEMENTS

The text of Chapter 4, in part or in full, will be submitted for publication.

The dissertation author was the primary researcher and author, and the following co-authors directed and supervised the research: Daniel T.

OConnor, Geert W. Schmid-Schönbein, Nitish R. Mahapatra, and Martin

Jirout. The rat chromogranin A cDNA clone was generously provided by Lee

Eiden. DNA sequencing was performed by the DNA Sequencing Shared

Resource, UCSD Cancer Center, which is funded in part by NCI Cancer

Center Support Grant #2 P30CA23100-18.

112

FIGURE 4.1

-1694 In/Del -/G C-177T

-1500 -1000 -500 0

A-1616T -753 “A" repeat C-59T SHR = 11 WKY = 15

Figure 4.1: Polymorphism within the Chga promoter Resequencing of the Chga locus in the SHR and WKY rat strains revealed 5 polymorphic sites in the promoter region. The promoter region is depicted as a horizontal line and is numbered relative to the transcriptional 5’ cap site (position “0”). Nucleotides upstream of the 5’ cap site are numbered negatively in descending order from right to left (e.g., -5, -4, -3, -2, -1, 0) in terms of base pair position. Polymorphic sites are named in the following convention: WKY nuclotide, position of the polymorphism, SHR nucleotide. For example, “C-59T” denotes that at position -59 in the Chga promoter, a “C” is found in the WKY strain and a “T” is found in the SHR strain. The term “-1694 In/Del -/G” indicates an insertion/deletion mutation wherein at position -1694 in the Chga promoter, a nuclotide is missing in the WKY strain but a “G” exists in the SHR strain.

113

Figure 4.2: Chga promoter constructs

The effect of the SHR Chga promoter polymorphisms on mRNA transcription was elucidated using promoter/luciferase reporter constructs. The SHR Chga promoter (A) and the WKY Chga promoter (B) were inserted into promoter- less vectors containing the cDNA for luciferase. Transcription of luciferase mRNA within the SHR and WKY constructs is dependent upon the nucleotide sequence of the inserted promoters. Differential effects of Chga promoter polymorphisms on transcription can be measured through quantification of luciferase expression. 114

FIGURE 4.2

SH A R C r) h p 0 g m a (A 6000 p r e o s 1000 m a o m t e a t r

a l

- Chga

a SHR Construct

t e

b 2000

4000 3000 se ra cife SV40 late poly(A) signal lu

WK B Y C r) h pm 0 g mA a ( 6000 (eA p r n o ee s 1000 m g a o e m t s e a t r m

l c

- WKY Chga Construct

a t

b 2000

4000 3000 se ra cife SV40 late poly(A) signal lu 115

Figure 4.3: Transcriptional response of SHR/WKY Chga promoters to dexamethasone, nicotine, and PACAP

Luciferase assays were performed on PC12 cells transfected with SHR or WKY Chga promoter/luciferase reporter constructs. Transcriptional activity was investigated under basal conditions (i.e., under no stimulation) (A), or during stimulation with 100 nM dexamethasone (dex) (A), 1 mM nicotine (A), or 100 nM pituitary adenylate cyclase-activating peptide (PACAP) (B). The bioluminescent activity of luciferase is presented as a normalized intensity (mean ± SD), wherein luciferase fluorescent intensity (in Relative Light Units or RLU) is normalized by total protein (μg of protein). The control group is the pGL3-Basic luciferase reporter vector without a Chga promoter insert. Replicates: n = 3 mock and n = 4 control for all conditions; n = 4 SHR and n = 4 WKY for no stimulation, dexamethasone, and PACAP treated cells; n = 6 SHR and n = 6 WKY for nicotine treated cells. 116

FIGURE 4.3

A p = 0.001 4000 p = 0.003 p = 0.002 g protein)

μ 3000

2000

1000

Normalized intensity (RLU/ 0 mock mock mock no stimulation 100 nM dex 1 mM nicotine

25000 p = 0.102 B g protein)

μ 20000

15000

B 10000

p = 0.002 5000

0 mock mock

Normalized intensity (RLU/ no stimulation 100 nM PACAP

Legend Control (no promoter) WKY Chga promoter construct SHR Chga promoter construct 117

FIGURE 4.4

G+174T

0 +100 +200 +300

Figure 4.4: Polymorphism within the Chga 3’-UTR Resequencing of the Chga locus in the SHR and WKY rat strains revealed 1 polymorphism within the 3‘-untranslated region (UTR). The 3’-UTR is depicted as a horizontal line and is numbered relative to the stop codon of the last exon (position “0”). Nucleotides downstream of the stop codon are numbered in ascending order in terms of base pair position. One polymorphism was discovered at position +174 of the 3’-UTR, wherein a “G” is found in WKY and a “T” is found in SHR. 118

Figure 4.5: Chga 3-UTR constructs

The effect of the G+174T Chga 3-UTR polymorphism on mRNA stability was elucidated using 3-UTR/luciferase reporter constructs. The SHR Chga 3-UTR (A) and the WKY Chga 3-UTR (B) were inserted into vectors containing the SV40 promoter and the cDNA for luciferase. Transcription of luciferase mRNA is driven by the SV40 promoter in both the SHR and WKY constructs, so differences in luciferase expression are associated with the effect of G+174T SNP in the 3-UTR. 119

FIGURE 4.5

A SV40 promoter

1000 luciferase Ampr SHR Chga 4000 construct

luciferase 2000 stop codon 3000 SHR Chga 3’-UTR SV40 late poly (A) signal

SV40 promoter B 0

1000 luciferase Ampr WKY Chga 4000 construct

luciferase 2000 stop codon 3000 WKY Chga 3’-UTR SV40 late poly (A) signal 120

FIGURE 4.6

ANOVA p = 0.633 2000

g protein) 1500 μ

1000 n = 3 n = 6 n = 6

500 Normalized intensity (RLU/ 0 Mock Control WKY SHR Chga 3’UTR construct

Figure 4.6: Chga 3’-UTR luciferase assays Luciferase assays were performed on PC12 cells transfected with SHR or WKY Chga 3’-UTR/luciferase reporter constructs. The bioluminescent activity of luciferase is presented as a normalized intensity (mean ± SD), wherein the fluorescent intensity (in Relative Light Units or RLU) is normalized by total protein (μg of protein). The control group is the pGL3-Promoter luciferase reporter vector without a Chga 3’-UTR insert. No difference in luciferase activity was detected among the control, SHR, and WKY constructs (ANOVA, p = 0.633). Replicates: n = 3 mock, n = 3 control, n = 6 WKY, and n = 6 SHR. 121

Figure 4.7: Sequence of SHR and WKY Chga protein

An alignment of SHR and WKY Chga protein sequences (predicted from the mRNA nucleotide sequence) is shown. Amino acid residues are identical at all locations except in the glutamine (Q) and glutamate (E) repeat length polymorphisms, which are highlighted in red. Differences in the glutamine and glutamate repeats are enclosed in black boxes. 122

FIGURE 4.7

SHR 1 MRSSAALALLLCAGQVFALPVNSPMTKGDTKVMKCVLEVISDSLSKPSPM 50 WKY 1 MRSSAALALLLCAGQVFALPVNSPMTKGDTKVMKCVLEVISDSLSKPSPM 50

SHR 51 PVSPECLETLQGDERVLSILRHQNLLKELQDLALQGAKERAQQQQQQQQQ 100 WKY 51 PVSPECLETLQGDERVLSILRHQNLLKELQDLALQGAKERAQQQQQQQQQ 100

SHR 101 QQQ------HSSFEDELSEVFENQSPAAKHGDAASEAPSKDTVEKRED 142 WKY 101 QQQQQQQQQQQHSSFEDELSEVFENQSPAAKHGDAASEAPSKDTVEKRED 150

SHR 143 SDKGQQDAFEGTTEGPRPQAFPEPKQESSMMGNSQSPGEDTANNTQSPTS 192 WKY 151 SDKGQQDAFEGTTEGPRPQAFPEPKQESSMMGNSQSPGEDTANNTQSPTS 200

SHR 193 LPSQEHGIPQTTEGSERGPSAQQQARKAKQEEKEEEEE-KEEEEEEKEEK 241 WKY 201 LPSQEHGIPQTTEGSERGPSAQQQARKAKQEEKEEEEEEKEEEEEEKEEK 250

SHR 242 AIAREKAGPKEVPTAASSSHFYSGYKKIQKDDDGQSESQAVNGKTGASEA 291 WKY 251 AIAREKAGPKEVPTAASSSHFYSGYKKIQKDDDGQSESQAVNGKTGASEA 300

SHR 292 VPSEGKGELEHSQQEEDGEEAMAGPPQGLFPGGKGQELERKQQEEEEEEE 341 WKY 301 VPSEGKGELEHSQQEEDGEEAMAGPPQGLFPGGKGQELERKQQEEEEEEE 350

SHR 342 RLSREWEDKRWSRMDQLAKELTAEKRLEGEDDPDRSMKLSFRARAYGFRD 391 WKY 351 RLSREWEDKRWSRMDQLAKELTAEKRLEGEDDPDRSMKLSFRARAYGFRD 400

SHR 392 PGPQLRRGWRPSSREDSVEARGDFEEKKEEEGSANRRAEDQELESLSAIE 441 WKY 401 PGPQLRRGWRPSSREDSVEARGDFEEKKEEEGSANRRAEDQELESLSAIE 450

SHR 442 AELEKVAHQLQALRRG 457 WKY 451 AELEKVAHQLQALRRG 466

123

Figure 4.8: Inter-species conservation of Chga protein sequence

The conservation of the Chga protein across human (Homo sapiens), cow (Bos taurus), rat (Rattus norvegicus), and mouse (Mus musculus) is shown. Highly conserved amino acid residues (conserved across three of the four species) are highlighted in red. Extended stretches of glutamine (Q) and glutamate (E) repeats found in only in rat (E), or in both rat and mouse (Q, E) are enclosed in black boxes. 124

FIGURE 4.8

Human 1 MRSAAVLALLLCAGQVTALPVNSPMNKGDTEVMKCIVEVISDTLSKPSPM 50 Cow 1 MRSAAVLALLLCAGQVIALPVNSPMNKGDTEVMKCIVEVISDTLSKPSPM 50 Rat 1 MRSSAALALLLCAGQVFALPVNSPMTKGDTKVMKCVLEVISDSLSKPSPM 50 Mouse 1 MRSTAVLALLLCAGQVFALPVNSPMTKGDTKVMKCVLEVISDSLSKPSPM 50

Human 51 PVSQECFETLRGDERILSILRHQNLLKELQDLALQGAKERAHQ------93 Cow 51 PVSKECFETLRGDERILSILRHQNLLKELQDLALQGAKERTHQ------93 Rat 51 PVSPECLETLQGDERVLSILRHQNLLKELQDLALQGAKERAQQ---QQQ- 96 Mouse 51 PVSPECLETLQGDERILSILRHQNLLKELQDLALQGAKERAQQPLKQQQP 100

Human 94 ------QKKHSGFEDELSEVLENQSSQAELKEAVEEPSSKDVM 130 Cow 94 ------QKKHSSYEDELSEVLEKPNDQAEPKEVTEEVSSKDAA 130 Rat 97 --QQQQQQQQQQQQQQHSSFEDELSEVFENQSPAAKHGDAASEAPSKDTV 144 Mouse 101 PKQQQQQQQQQQQEQQHSSFEDELSEVFENQSPDAKHRDAAAEVPSRDTM 150

Human 131 EKREDSKEAEKS--GEATDGARPQALPEPMQESKAEGNNQAPGEEEEEEE 178 Cow 131 EKRDDFKEVEKS--DEDSDGDRPQASPGLGPGPKVEEDNQAPGEEEE--- 175 Rat 145 EKREDSDKGQQDAFEGTTEGPRPQAFPEPKQESSMMGNSQSPGED----- 189 Mouse 151 EKRKDSDKGQQDGFEATTEGPRPQAFPEPNQESPMMGDSESPGED----- 195

Human 179 EATNTHPPASLPSQKYPGPQAEGDSEGLSQGLVDREKGLSAEPGWQAKRE 228 Cow 176 APSNAHPLASLPSPKYPGPQAKEDSEGPSQGPASREKGLSAEQGRQTERE 225 Rat 190 TANNTQSPTSLPSQEHGIPQTTEGSE---RGPSAQQQARKAK---QEEKE 233 Mouse 196 TATNTQSPTSLPSQEHVDPQATGDSE---RGLSAQQQARKAK---QEEKE 239

Human 229 EEEE------EEEEAEAGEEAVPEEEGP-TVVLNPHPSLGYKEIRKGE 269 Cow 226 EEEE------KWEEAEAREKAVPEEESPPTAAFKPPPSLGNKETQR-- 265 Rat 234 EEEEEKEEEEEEKEEKAIAREKAGP-KEVP-TAASSSHFYSGYKKIQKDD 281 Mouse 240 EEE------EEEAVAREKAGP-EEVP-TAASSSHFHAGYKAIQKDD 277

Human 270 -SRSEALAVDGAGKPGAEEAQDPEGKGEQEHSQQKE-EEEEMAVVPQGLF 317 Cow 266 --AAPGWPEDGAGKMGAEEAKPPEGKGEWSHSRQ---EEEEMARAPQVLF 310 Rat 282 DGQSESQAVNG--KTGASEAVPSEGKGELEHSQQEEDGEEAMAGPPQGLF 329 Mouse 278 -GQSDSQAVDGDGKTEASEALPSEGKGELEHSQQEEDGEEAMVGTPQGLF 326

Human 318 R-GGKSGELE------QEEERLSKEWEDSKRWSKMDQLAKELTAEKRLE 359 Cow 311 R-GGKSGEPE------QEEQ-LSKEWEDAKRWSKMDQLAKELTAEKRLE 351 Rat 330 P-GGKGQELERKQQEEEEEEERLSREWED-KRWSRMDQLAKELTAEKRLE 377 Mouse 327 PQGGKGRELEHKQEEEEEEEERLSREWED-KRWSRMDQLAKELTAEKRLE 375

Human 360 GQEEEEDNRDSSMKLSFRARAYGFRGPGPQLRRGWRPSSREDSLEAGLPL 409 Cow 352 GEEEEEEDPDRSMRLSFRARGYGFRGPGLQLRRGWRPNSREDSVEAGLPL 401 Rat 378 G----EDDPDRSMKLSFRARAYGFRDPGPQLRRGWRPSSREDSVEA---- 419 Mouse 376 G----EDDPDRSMKLSFRTRAYGFRDPGPQLRRGWRPSSREDSVEA---- 417

Human 410 QVRGYPEEKKEEEGSANRRPEDQELESLSAIEAELEKVAHQLQALRRG 457 Cow 402 QVRGYPEEKKEEEGSANRRPEDQELESLSAIEAELEKVAHQLEELRRG 449 Rat 420 --RGDFEEKKEEEGSANRRAEDQELESLSAIEAELEKVAHQLQALRRG 465 Mouse 418 --RSDFEEKKEEEGSANRRAEDQELESLSAIEAELEKVAHQLQALRRG 463 125

Figure 4.9: Predicted coiled-coil conformation of SHR and WKY Chga

Since the human Chga protein is predicted to exhibit a coiled-coil 3D-structure, the probability of the WKY (A) and SHR (B) proteins to also assume a coiled- coil structure was computationally predicted with the COILS algorithm. In brief, the algorithm searches for strongly amphipathic regions that display a pattern of alternating hydrophilic and hydrophobic residues repeated every 7 residues (heptad repeat residues ag, with a & d being hydrophobic). The minimum window was two such heptads, over 14 residues. The probability of coiled-coil structure is presented as a function of amino acid position in the Chga protein. The location of the only two polymorphisms that differentiate SHR Chga from WKY Chga, the glutamine and glutamate repeat regions, are denoted with black rectangles. The glutamine repeat length polymorphism is predicted to change the probability of (by ~0.25) and number of residues (8 residue change) involved in a coiled-coil within the repeat region. Polymorphism within the glutamate repeat is not predicted to alter coiled-coil conformation. 126

FIGURE 4.9

A. WKY WKY glutamate repeat WKY glutamine repeat (16 residues) (20 residues) 1

0.8

0.6

0.4

0.2 Probability of coiled-coil

0 1 100 200 300 400 Residue number

B. SHR SHR glutamate repeat SHR glutamine repeat (15 residues) (12 residues) 1

0.8

0.6

0.4

0.2 Probability of coiled-coil

0 1 100 200 300 400 Residue number 127

Figure 4.10: Chga cDNA/EAP reporter constructs

The effect of the SHR glutamine repeat polymorphism on Chga protein trafficking through and secretion from the regulated secretory pathway was investigated using Chga cDNA/EAP reporter constructs. The SHR Chga cDNA (containing only the glutamine polymorphism) (A) and the WKY Chga cDNA (B) were inserted into vectors containing a CMV promoter and the cDNA for embryonic alkaline phosphatase (EAP). Transcription/translation of the constructs results in chimeric proteins wherein EAP is fused to the carboxy terminus of Chga. 128

FIGURE 4.10

A MV C prom ot er S e 0 5 H s 00 R ra 0 1 C fe 0 0 s 0 0 h 6 0 n g a a 0 r 1 t 0 5 c 5 o 0 D 5 0 h

0 s

0 SHR construct

0 o

4 y 0

o 0 0

3 0 5 n A N cD P EA

B MV C prom ot er e 0 W s 500 K ra Y 0 1 fe 0 0 C s 0 0 h 6 0 n g a a 0 r 1 t 0 5 c 5 o 0 D 5 0 h

A 0 s

WKY construct 2 0

0 o

4 y 0

o 0 0

3 0 5 n A N cD P EA 129

Figure 4.11 Chga cDNA/EAP Reporter Assay

The luminescent activity of EAP was measured in the supernatant (A) or lysate (B) fractions of PC12 cells transfected with mock, SEAP, human Chga, WKY Chga, or SHR Chga EAP reporter constructs. Cells were stimulated with calcium (negative control; basal level) or barium (a potent stimulus of regulated secretion). The SEAP construct lacks a Chga cDNA and, therefore, a signal peptide to the regulated secretory pathway. The human Chga-EAP construct is a positive control. Final EAP intensity is expressed as the normalized ratio of (EAP activity)/(3H-NE counts per minute) or (RLU/cpm). Replicates: n = 3 in each group. 130

FIGURE 4.11 A. Supernatant

16 Legend SEAP 14 Human WKY 12 SHR

Normalized intensity (RLU/cpm) 2

0 Mock SEAP Mock SEAP Calcium Barium Secretory stimulus B. Lysate

Legend SEAP 80 Human WKY SHR 60

20 Normalized intensity (RLU/cpm)

0 Mock SEAP Mock SEAP Calcium Barium Secretory stimulus 131

Figure 4.12 EAP Assay Relative Secretion and Sorting Index

EAP assay raw data (Figure 4.11) was used to compute the relative secretion (A) and sorting index (B) of the human, WKY, and SHR Chga-EAP reporter constructs. Relative secretion (Rs) is defined as the fraction of total EAP activity that is found in the supernatant: (EAP activity in supernatant)/(EAP activity in supernatant + EAP activity in lysate). There was no statistical difference between the basal level (i.e. cells treated with calcium) of SHR and WKY Chga-EAP relative secretion (unpaired t-test, p = 0.0933) or between barium stimulated relative secretion of SHR and WKY Chga-EAP (unpaired t- test, p = 0.0664). Sorting index is defined as the difference between relative secretion of barium and calcium treated cells normalized by the relative secretion of calcium treated cells: (Rsbarium – Rscalcium)/Rscalcium. No statistical difference exists between the sorting index of SHR and WKY. 132

FIGURE 4.12

A. Relative Secretion

30 Legend Human 25 WKY SHR

10 Relative Secretion (%) 5

Calcium Barium Secretory stimulus B. Sorting Index

5 Legend Human WKY 4 SHR

2 Sorting index

0 Chga-EAP construct 133

FIGURE 4.13

T-404C T-529C C-351T

-900 -800 -700 -600 -500 -300 -200 -100 0

C-396T In/Del -457 A/-

Figure 4.13: Polymorphism within the Pnmt promoter Resequencing of the Pnmt locus in the SHR and BN rat strains revealed 5 polymorphic sites in the promoter region. The promoter region is depicted as a horizontal line and is numbered relative to the transcriptional 5’ cap site (position “0”). Nucleotides upstream of the 5’ cap site are numbered negatively in descending order from right to left (e.g., -5, -4, -3, -2, -1, 0) in terms of base pair position. Polymorphic sites are named in the following convention: BN nuclotide, nucleotide position of the polymorphism, SHR nucleotide. For example, “T-529C” denotes that at position -529 in the Pnmt promoter, a “T” is found in the BN strain and a “C” is found in the SHR strain. The term “In/Del -457 A/-” indicates an insertion/deletion mutation at position -457 in the Pnmt promoter, wherein an “A” is present in the BN but is absent in the SHR.

134

FIGURE 4.14: Pnmt promoter constructs

The effect of the SHR Pnmt promoter polymorphisms on mRNA transcription was elucidated using promoter/luciferase reporter constructs. The SHR Pnmt promoter (A) and the BN Pnmt promoter (B) were inserted into promoter-less vectors containing the cDNA for luciferase. Transcription of luciferase mRNA within the SHR and BN constructs is dependent upon the nucleotide sequence of the inserted promoters. Differential effects of the Pnmt promoter polymorphisms on transcription can be measured through quantification of luciferase expression. 135

FIGURE 4.14

SHR Pn A mt p ro 0 m ) 5 o r 00 t p e 0 0 r m 0 A 5 1 ( 0 0 e 0

s 0

a 0

1 m 4

a 0

t SHR Pnmt Construct

0 l

0 -

a e

2 4

t s 0

0 a

b r

0 e

0 f

5 i

0 c

0 0 0 3 lu

SV40 late poly(A) signal

BN Pn mt B p ro 0 m ) 5 o r 00 te p 0 r 0 m 0 A 5 1 ( 0 0 e 0

s 0

a 0

1 m 4

a 0

t BN Pnmt Construct

0 l

0 -

a e

2 4

t s 0

0 a

b r

0 e

0 f

5 i

0 c

0 0 0 3 lu

SV40 late poly(A) signal 136

Figure 4.15: The SHR and BN Pnmt promoters lack differential response to PACAP and nicotine

Luciferase assays were performed on PC12 cells transfected with SHR or BN Pnmt promoter/luciferase reporter constructs. Cells were stimulated with 100 nM pituitary adenylate cyclase-activating peptide (PACAP) or 1 mM nicotine. The bioluminescent activity of luciferase is presented as a normalized intensity (mean ± SD), wherein the fluorescent intensity (in Relative Light Units or RLU) is normalized by total protein (μg of protein). The control group is the pGL3- Basic luciferase reporter vector without a Pnmt promoter insert. No difference exists in the activity of SHR and BN promoters in any of the treatment groups. Replicates: n = 3 control, n = 4 SHR, and n = 4 BN in each treatment group. Legend Normalized Intensity (RLU/μg protein) SHR BN Control (nopromoter) 100 120 140 160 180 20 40 60 80 Pnmt 0 Pnmt Mock no stimulation promoter construct promoter construct FIGURE 4.15 Mock (100 nM) PACAP Mock Nicotine (1 mM) 137 138

Figure 4.16: The SHR Pnmt promoter shows a blunted dose-dependent response to dexamethasone

Luciferase assays were performed on PC12 cells transfected with SHR and BN Pnmt promoter/luciferase reporter constructs. Cells were stimulated with 1 nM, 10 nM, or 100 nM dexamethasone. The bioluminescent activity of luciferase is presented as a normalized intensity (mean ± SD), wherein the fluorescent intensity (in Relative Light Units or RLU) is normalized by total protein (μg of protein). The control group is the pGL3-Basic luciferase reporter vector without a Pnmt promoter insert. The SHR promoter shows significantly less expression than the BN promoter at 10 nM (0.746-fold less in SHR, p = 0.016) and 100 nM (0.769-fold less in SHR, p = 0.004) doses. Replicates: n = 5 control, n = 5 SHR, and n = 5 BN for each dexamethasone dose. 139

FIGURE 4.16

120 p=0.004

100 g protein) μ 80 p=0.016

20 Normalized intensity (RLU/

0 Mock Mock Mock Mock

0 nM 1 nM 10 nM 100 nM Dexamethasone concentration (nM)

Legend Control (no promoter) BN Pnmt promoter construct SHR Pnmt promoter construct 140

FIGURE 4.17

T-529C

SHR -548 GGCCAGAACAGAGTGTCCTCTCTGAAGGAGGATAGAGACGGGGTAGAGGT -499 BN -548 GGCCAGAACAGAGTGTCCTTTCTGAAGGAGGATAGAGACGGGGTAGAGGT -499 GRE

Figure 4.17: The T-529C Pnmt promoter SNP is adjacent to a GRE The region of the Pnmt promoter in SHR/BN that harbors the T-529C SNP is shown above. The SNP is adjacent to a glucocorticoid response element (GRE). The GRE element in the SHR/BN Pnmt promoters differs from the consesus sequence for a GRE (5’-AGAACANNNTGTTCT-3’) by one nucleotide. 141

Figure 4.18: Pnmt promoter SNP variant luciferase assays

Luciferase assays were performed on PC12 cells transfected with Pnmt promoter SNP variant/luciferase reporter constructs. All cells were treated with 100 nM dexamethasone. (A) Constructs SNP1-SNP5 were formed by systematically mutating each of the 5 SNPs within the BN Pnmt promter to the SHR variant. (B) Constructs SNP1b-SNP5b were formed by systematically mutating each of the 5 SNPs within the SHR Pnmt promter to the BN variant. Constructs “SNP1” and “SNP1b” vary at the T-529C SNP, which is adjacent to a glucocorticoid response element (see Figure 4.17). The exact combination of all the SNPs within the constructs is presented in Table 4.4. (A) The BN construct is significantly higher than the SNP1 construct (p < 0.0001), and the SHR construct is significantly higher than the SNP1 construct (p < 0.0001). (B) The BN construct is significantly higher than the SNP1b construct (p = 0.0015), but the SHR construct is not significantly higher than the SNP1b construct. Statistical significance was determined with the Tukey test for multiple pairwise comparisons. The bioluminescent activity of luciferase is presented as a normalized intensity (mean ± SD), wherein the fluorescent intensity (in Relative Light Units or RLU) is normalized by total protein (μg of protein). The control group is the pGL3-Basic luciferase reporter vector without a Pnmt promoter insert. Replicates: n = 5 control, n = 5 SHR, n = 5 BN, and n = 5 for each SNP variant construct in (A); n = 4 control, n = 6 SHR, n = 6 BN, and n = 6 for each SNPb variant construct in (B). 142

FIGURE 4.18

Pnmt promoter SNP variants on a BN background p < 0.0001 A 250

p < 0.0001 200 g protein) μμ 150

100

50 Normalized Intensity (RLU/ 0 Mock Control BN SHR SNP1 SNP2 SNP3 SNP4 SNP5 Dexamethasone (100 nM)

Pnmt promoter SNP variants on a SHR background p < 0.0015 B 400

350

μg protein) 300 μ

250

200

150

100

50 Normalized Intensity (RLU/ 0 Mock Control BN SHR SNP1b SNP2b SNP3b SNP4b SNP5b Dexamethasone (100 nM) Table 4.1: SHR and WKY Chga promoters respond differentially to transcriptional stimuli

The response of the SHR and WKY Chga promoters to different transcriptional stimuli is listed. PC12 cells transfected with SHR or WKY Chga promoter/luciferase reporter constructs were incubated with the various stimuli for 16 hours, and then subjected to luciferase assays. Results of the luciferase assays (in terms of relative light units, RLU) are presented as “Transcriptional activity” (mean ± SD). Statistical significance between SHR and WKY “transcriptional activity” was determined using an unpaired t-test. Significant p-values (<0.05) and fold-changes are shown in bold. The “Fold change (SHR/WKY)” is computed as the quotient of SHR “transcriptional activity” and WKY “transcriptional activity.” Replicates: n = 4 SHR and n = 4 WKY for no stimulation, dexamethasone, and PACAP treated cells; n = 6 SHR and n = 6 WKY for nicotine treated cells.

Chga Promoter Stimulus Transcriptional activity (RLU) p-value Fold change (SHR/WKY) SHR None 1956 ± 110 0.002 0.73 WKY (basal level) 2697 ± 263

SHR Dexamethasone 2154 ± 88 0.003 0.72 WKY (100 nM) 3002 ± 342

SHR PACAP 16767 ± 996 0.102 0.84 WKY (100 nM) 20024 ± 3232

SHR Nicotine 3533 ± 433 0.001 1.35 WKY (1mM) 2615 ± 241 143 Table 4.2: SHR and BN Pnmt promoters lack differential response to PACAP and nicotine

The response of the SHR and BN Pnmt promoter/luciferase reporter constructs to PACAP and nicotine is shown. Luciferase assays were performed on PC12 cells transfected with SHR or WKY Pnmt promoter constructs and stimulated with PACAP or nicotine. Results of the luciferase assays (in terms of RLU) are presented as “Transcriptional activity” (mean ± SD). Statistical significance between SHR and BN “transcriptional activity” was determined using an unpaired t-test. Significant p-values (<0.05) and fold-changes are shown in bold. The “Fold change (SHR/ BN)” is computed as the quotient of SHR “transcriptional activity” and BN “transcriptional activity.” Replicates: n = 3 control, n = 4 SHR, and n = 4 BN for each stimulus.

Pnmt Promoter Stimulus Transcriptional activity (RLU) p-value Fold change (SHR/BN) SHR None 38 ± 3 0.773 1.00 BN (basal level) 38 ± 1

SHR PACAP 150 ± 14 0.888 0.99 BN (100 nM) 152 ± 18

SHR Nicotine 57 ± 5 0.274 1.10 BN (1mM) 52 ± 6 144 Table 4.3: The SHR Pnmt promoter shows a blunted dose-dependent response to dexamethasone

The response of the SHR and BN Pnmt promoter/luciferase reporter constructs to dexamethasone is presented. Transfected PC12 cells were incubated with various does of dexamethasone and subjected to luciferase assays. Results of the luciferase assays (in terms of RLU) are presented as “Transcriptional activity” (mean ± SD). Statistical significance between SHR and BN “transcriptional activity” was determined using an unpaired t-test. Significant p-values (<0.05) and fold-changes are shown in bold. The “Fold change (SHR/ BN)” is computed as the quotient of SHR “transcriptional activity” and BN “transcriptional activity.” Replicates: n = 5 control, n = 5 SHR, and n = 5 BN for each dexamethasone dose.

Pnmt Promoter Stimulus Transcriptional activity (RLU) p-value Fold change (SHR/BN) SHR None 58± 5 0.258 1.12 BN (basal level) 52 ± 10

SHR Dexamethasone 30 ± 4 0.525 0.938 BN (1 nM) 32 ± 7

SHR Dexamethasone 44 ± 6 0.016 0.746 BN (10 nM) 59 ± 10

SHR Dexamethasone 80 ± 9 0.004 0.769 BN (100 nM) 104 ± 9 145 146

Table 4.4: Pnmt promoter SNP constructs

SNP variants of the SHR/BN Pnmt promoters were constructed to reflect all possible combinations of individual SNPs found in the SHR and BN promoters. Individual SNPs within the SHR and BN promoters were systematically mutated to make a total of 10 possible promoter/reporter construct variants. Constructs named SNP1-SNP5 were developed from the BN promoter and, therefore, contain 4 SNPs matching the BN sequence and 1 SNP matching the SHR sequence. Constructs named SNP1b-SNP5b were developed from the SHR promoter and, therefore, contain 4 SNPs matching the SHR sequence and 1 SNP matching the BN sequence.

Construct T-529C -457 T-404C C-396T C-351T In/Del A/-- BN T A T C C SHR C -- C T T SNP1 C a (BN) t (BN) c (BN) c (BN) SNP2 t (BN) -- t (BN) c (BN) c (BN) SNP3 t (BN) a (BN) C c (BN) c (BN) SNP4 t (BN) a (BN) t (BN) T c (BN) SNP5 t (BN) a (BN) t (BN) c (BN) T SNP1b T -- (SHR) c (SHR) t (SHR) t (SHR) SNP2b c (SHR) A c (SHR) t (SHR) t (SHR) SNP3b c (SHR) -- (SHR) T t (SHR) t (SHR) SNP4b c (SHR) -- (SHR) c (SHR) C t (SHR) SNP5b c (SHR) -- (SHR) c (SHR) t (SHR) C

147

Table 4.5: Luciferase assay results from the Pnmt SNP variant constructs created from the BN promoter

The response of the BN Pnmt promoter SNP variant/luciferase reporter constructs to 100 nM dexamethasone is presented. Statistically significant differences (p<0.05) in mean luciferase assay signal intensity (determined by the Tukey test for multiple pairwise comparisons) between constructs are indicated in bold.

Difference between Comparison means (RLU) p-value BN vs SHR 52.15 < 0.0001 BN vs SNP1 104.89 < 0.0001 BN vs SNP2 76.75 < 0.0001 BN vs SNP3 45.23 < 0.0001 BN vs SNP4 38.07 0.0006 BN vs SNP5 23.36 0.0697

SHR vs SNP1 52.73 < 0.0001 SHR vs SNP2 24.60 0.0489 SHR vs SNP3 6.92 0.9703 SHR vs SNP4 14.09 0.5451 SHR vs SNP5 28.80 0.0135

SNP1 vs SNP2 28.14 0.0167 SNP1 vs SNP3 59.66 < 0.0001 SNP1 vs SNP4 66.82 < 0.0001 SNP1 vs SNP5 81.53 < 0.0001

SNP2 vs SNP3 31.52 0.0056 SNP2 vs SNP4 38.68 0.0005 SNP2 vs SNP5 53.39 < 0.0001

SNP3 vs SNP4 7.16 0.9649 SNP3 vs SNP5 21.87 0.1045

SNP4 vs SNP5 14.71 0.4951

148

Table 4.6: Luciferase assay results from the Pnmt SNP variant constructs created from the SHR promoter

The response of the SHR Pnmt promoter SNP variant/luciferase reporter constructs to 100 nM dexamethasone is presented. Statistically significant differences (p<0.05) in mean luciferase assay signal intensity (determined by the Tukey test for multiple pairwise comparisons) between constructs are indicated in bold.

Difference between Comparison means (RLU) p-value BN vs SHR 86.11 0.0051 BN vs SNP1b 95.47 0.0015 BN vs SNP2b 122.97 < 0.0001 BN vs SNP3b 133.78 < 0.0001 BN vs SNP4b 109.82 0.0002 BN vs SNP5b 29.06 0.8219

SHR vs SNP1b 9.36 0.9994 SHR vs SNP2b 36.86 0.6101 SHR vs SNP3b 47.67 0.3112 SHR vs SNP4b 23.70 0.9227 SHR vs SNP5b 115.18 0.0001

SNP1b vs SNP2b 27.50 0.856 SNP1b vs SNP3b 38.31 0.567 SNP1b vs SNP4b 14.34 0.9936 SNP1b vs SNP5b 124.54 < 0.0001

SNP2b vs SNP3b 10.81 0.9986 SNP2b vs SNP4b 13.16 0.996 SNP2b vs SNP5b 152.04 < 0.0001

SNP3b vs SNP4b 23.97 0.9189 SNP3b vs SNP5b 162.85 < 0.0001

SNP4b vs SNP5b 138.88 < 0.0001

149

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Chapter 5: Conclusion

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Genetic studies of human essential hypertension, a complex, polygenic, and age-dependent disorder, have not been able to completely elucidate the genes responsible for development of the trait. The series of experiments conducted in Chapters 2 through 4 formulate a novel approach to dissect the genetic basis of essential hypertension.

In Chapter 2, a comparison of gene expression in the adrenal gland of two independent rodent models of human essential hypertension (the spontaneously hypertensive rat, SHR, and the blood pressure high mouse,

BPH) was performed with the goal of uncovering shared, common genetic mechanisms of hypertension across mammalian species that might, therefore, be pertinent to human hypertension. The gene expression analysis revealed a diverse set of differentially expressed genes and biochemical systems within and between the SHR and BPH strains, reinforcing the multifactorial and complex nature of essential hypertension.

In Chapter 3, candidate genes for hypertension in the SHR were identified using a novel method that integrated the adrenal gland microarray data from Chapter 2 with QTL data. The underlying hypothesis of the method was that the nucleotide mutations in the genes responsible for hypertension in the SHR manifest as changes in mRNA transcript abundance in the adrenal gland, even before penetrance of hypertensive disease phenotypes.

Approximately 80 SHR blood pressure Quantitative Trait Loci (QTLs) have

157 been published, yet discussion of candidate genes for these QTLs has seldom exceeded conjecture. Therefore, the identification of 7 candidate genes was followed by resequencing to identify polymorphisms that could contribute to the mRNA differential expression of the genes in the SHR adrenal gland.

Chromogranin A (Chga) and phenylethanolamine-N-methyltransferase (Pnmt) emerged as the strongest candidate genes. Creating the link between gene polymorphisms and changes in mRNA transcription will help formulate mechanisms whereby abnormal mRNA transcription can lead to altered protein function and, ultimately, hypertension.

In Chapter 4, the effect of polymorphisms within the chromogranin A

(Chga) and phenylethanolamine-N-methyltransferase (Pnmt) candidate genes was studied using luciferase and embryonic alkaline phosphatase (EAP) reporter plasmid constructs. Luciferase and EAP assays demonstrated that the polymorphisms in the promoter regions of Chga and Pnmt are likely to contribute to the differential adrenal mRNA expression of these genes observed in vivo, and could also explain blood pressure, Chga, and Pnmt

QTLs in the SHR and in the SHR RI strains.

The crucial role of Chga and Pnmt in the biosynthesis and exocytosis of catecholamines makes them strong candidate genes for hypertension in the

SHR and worthwhile for additional study. Quantitative Real-Time PCR and immunohistochemistry experiments could be used to determine if Chga and

158

Pnmt mRNA and protein are differentially expressed in organs other than the adrenal gland. Measuring the abundance of the bioactive peptides derived from Chga protein could provide new insight into abnormal inflammatory and glucose homeostatic mechanisms in the SHR. In vivo siRNA (i.e., RNAi or

RNA interference) experiments could be used to knock-down adrenal Chga and determine if Chga overexpression directly contributes to hypertensive disease phenotypes such as inflammation and elevated blood pressure.

Conversely, increasing the in vivo adrenal expression of Pnmt with a cDNA could elucidate the effect of Pnmt underexpression on catecholamine biosynthesis and blood pressure. Since Chga and Pnmt abnormalities are also associated with human essential hypertension, any treatments that target

Chga and Pnmt in the SHR could potentially be adapted to help a subset of human patients with essential hypertension.

Appendix A: PCR and Sequencing Primers

159 160

The oligonucleotide primers used in PCR amplification and sequencing of the candidate genes are listed below.

Catechol-O-methyltransferase (Comt)

Primer Sequence (5’  3’) Target region of gene ComtPaF GTTTCCATGTCTGCTCAGCTC Proximal promoter ComtPaR GCTGGGTGAGCTCATGTGTA ComtPbF CCCAGTTAGATCCTGGGTTG Proximal promoter ComtPbR GAGATCTCTGTGTCCTTTCTCCT ComtPcF CAGGTTGATAATAGAGGTTAGTGGT Proximal promoter ComtPcR TGAGGAGGTCCAAGGTTCAG ComtE1F AAGTGACACCACCATCACGA Exon 1 ComtE1R TCCTACAAGGACTGCCATACC ComtE2F GGTCAGGGACATGAGAGGAG Exon 2 ComtE2R CAGGTGCTTAGTGGCTGACA ComtE3F CTCCAGAGCCCCAAAGAGAT Exon 3 ComtE3R TAAGAGGCCCAAGCTCAGTG ComtE4aF GGTTTGCCAAGCCTTCCT Exon 4 ComtE4aR CACTGAAACCCCGTGAAGAT ComtE4bF GAGCCCACTATGCAAAATCA Exon 4 ComtE4bR TGCAGAGTAACAGCAGTGTGG

161

Chromogranin A (Chga)

Primer Sequence (5’  3’) Target region of gene Chga_F_1 CGGACTCTGAAACTTGTGGTG Proximal promoter Chga_R_1 TGTACACTATGCTGGGTCATGG Chga_F_2 CGAGATGGTATTTTGGAGACAG Proximal promoter Chga_R_2 AGCTGGATATTTTGGGTGTGAG Chga_F_3 AGTTTCTCATTTAGGGGCATGA Proximal promoter Chga_R_3 TTCTCTTGATTTCACTCGGTTG Chga_F_4 GCACACATTGAACTTGTGTGAA Proximal promoter Chga_R_4 ACAGCAGAAGCGCCAAAG Chga_F_5 ATGACGTAATTTCCTGGGTGTG Exon 1 Chga_R_5 GAGTGCAGAGCTGAAATCAAGTT Chga_F_6 AACTATAGAGCCTGACCCAACC Exon 2 Chga_R_6 CTTTCTGCAGTTGCCTAAGGAC Chga_F_7 ATTCGATTGGCCCACAGTAAC Exon 3 Chga_R_7 TTGGAAAGGTGTGGTCTTTCTT Chga_F_8 GGGACCCTGAGGTTTGTAGACT Intron Chga_R_8 AAGTTCCTTCAGCAAATTCTGG Chga_F_9 GCAGTAGGAAGGTGATGGACAC Exon 4 Chga_R_9 TTAATCTCTTGGGGGCAAGTTA Chga_F_10 CCTCTGGTGTCTTGGACAGATA Exon 5 Chga_R_10 GACTGTTGGGAACTGGTCTTTC Chga_F_11 TGAGTGGGTAACTTCAATCCTT Intron Chga_R_11 CCACTCATCTTTCACGGTCAT Chga_F_12 AGAAGGCTGGGCCTAAAGAAGT Exon 6 Chga_R_12 ACCGGTCAGGTCATCTTCC Chga_F_13 GTGTGCTTGGCCTTAGAGGTAG Exon 7 Chga_R_13 CCTAAGAGGCAAGTCCTGCTAA Chga_F_14 TACAGCGTCCTAGCATTACTGG Intron Chga_R_14 ACCCAGCCCAGTGTAGAAATC Chga_F_15 AGATTCTTTCTCGGAACACAGG Exon 8 Chga_R_15 TTTCCAAATTGGGCCTAAGAC Chga_F_16 CTCCTGGACTGTCCCCTAGTTA Exon 8 Chga_R_16 ACGTTTAGCATCACCATCTCCT Chga_F_17 CACCACCCAACTTTCCTTTTTA 3’ downstream Chga_R_17 GACGTCATACAGGTGTCTCCAC Chga_F_18 AAGAGTCCTCGTCTCCAATGTG 3’ downstream Chga_R_18 TGCAGGACATAGGAGATGTTTC

162

Dopamine-beta-hydroxylase (Dbh)

Data not available: unpublished data from a collaborator.

163

Electron-transferring-flavoprotein dehydrogenase (Etfdh)

Primer Sequence (5’  3’) Target region of gene Etfdh_PaF CCTGACCTACACGCAGAACC Proximal promoter Etfdh_PaR GGGAGAGCGTTGTGGAATAA Etfdh_PbF TGAGCTGAGCTGACAGAACAT Proximal promoter Etfdh_PbR AGGGCTAGCTGGTGATGCTA Etfdh_PcF TGTTGGGAGTCAGGAAGTGA Proximal promoter Etfdh_PcR TCTTCATCGTCCCTCAGCAT Etfdh_PdF GCCCTCAACTCCAAGAACTG Proximal promoter Etfdh_PdR CGTGCGCACTAGAAGCATAG Etfdh_PeF GCCCCATCTTCTCGTTTGT Proximal promoter Etfdh_PeR TCTGTCAGCCTCTGGGATCT Etfdh_E1F TCCCCGCTATGCTTCTAGTG Exon 1 Etfdh_E1R GTGTTTGCAGTACCCCAGGT Etfdh_E2F AACATTCCATTTAGATTTGTGTCAA Exon 2 Etfdh_E2R TCTGGGATATATATTGGATGCTTT Etfdh_E3F TGGTCCTATTAATCCCAGAGTTG Exon 3 Etfdh_E3R GAGACAGCATAGATTAGACCTTGTG Etfdh_E4F TCAGAGGCATATTCACCCAAC Exon 4 Etfdh_E4R AAATAAAGTCAATATTAAAGCCTGAAA Etfdh_E5F TGCAGCAGTACACACTGGTT Exon 5 Etfdh_E5R TTCAATTCCTCTTTGGATTAGCA Etfdh_E6F CCCACCCTTCTTGCTTCA Exon 6 Etfdh_E6R TGTGCAGTTTGTAGGAAGACCT Etfdh_E7F AGTTTTCCTTCCAGTACATAGGTC Exon 7 Etfdh_E7R GGCAATCATTTGACCTGTTTTAG Etfdh_E8F AATTTTGTGCTGCTTTCATGT Exon 8 Etfdh_E8R CATGCTGGTAAGTTCAAAAGTCA Etfdh_E9F TGCCAATTATCCTTTTGCTT Exon 9 Etfdh_E9R TGTTAAGCTCCTTAAAGTTTTGTCC Etfdh_E10F CCCTTTTCCAGGCGTTTACT Exon 10 Etfdh_E10R ATGTGCTGAAAGGGGACATC Etfdh_E11F ATGATTCTGCATGGGTCCAC Exon 11 Etfdh_E11R GGGGAACAACCCTACAACAA Etfdh_E12F ATGAGCTGCCTGTATCACCA Exon 12 Etfdh_E12R TGAACTGGGAAATTGTTAAATGT Etfdh_E13F AAGGAAGGGCTGGAGTCAAT Exon 13 Etfdh_E13R GGAGCTTAGTAGCACAAGTTTCTGT

164

Endothelin receptor, type B (Ednrb)

Primer Sequence (5’  3’) Target region of gene Ednrb_PaF AACCTTGAGCACCCGTAATG Proximal promoter Ednrb_PaR CCCTGTGCCCAATATAGAAC Ednrb_PbF GTCATTGGCCCTTCTGACAA Proximal promoter Ednrb_PbR TGTTAGGTGATGTTTTCCCTTTC Ednrb_PcF CAGTAGAAAAGAACACAGGAAAAGTG Proximal promoter Ednrb_PcR TGGGAAGAAAGAAATGTTTATGA Ednrb_E1F AATTTTGCTCAGCTGCCTACA Exon 1 Ednrb_E1R CAGCAGAAGGCAATGATTCTC Ednrb_E2F CATGATCCCTAGCGATTTTAGG Exon 2 Ednrb_E2R GGGAGTCTTAATTGGCCTCTG Ednrb_E3F GGTGCTTTACAGGCAAATCG Exon 3 Ednrb_E3R TGATCAAGTCTAATTGTATCGGTGA Ednrb_E4aF AGAGGGGGACATGGAAAGAG Exon 4 Ednrb_E4aR GCTTTCCCGAGGCTTCAT Ednrb_E4bF TCCTCATCGTGGACAGATAGC Exon 4 Ednrb_E4bR TCAAACATCCAGGCTGTGC

Neuropeptide Y (Npy)

Primer Sequence (5’  3’) Target region of gene Npy_PaF AGACCGGTGCTTTGAATGAC Proximal promoter Npy_PaR TGAACACCAATATCCCATCC Npy_PbF TGACCGATGTTACTCCCTGA Proximal promoter Npy_PbR TTAAAAGACCAACGCCACTG Npy_PcF CATCCCTATTTAAACAATGCACA Proximal promoter Npy_PcR GCAGTCGAGCAAGGTTTTTC Npy_PdF AGTGTTCATTCGGGCGTTAG Proximal promoter Npy_PdR GTCTGGAGCCACCCACAC Npy_E1F GCTCCCCAAGTACAGTGTCTG Exon 1 Npy_E1R GGGTCGAACCAGAGTCCA Npy_E2F GCCCTCTGCTTCTCACTAGG Exon 2 Npy_E2R TATCCAGTTTGTGGCGTGTG Npy_E3F TGAGAATACTTATTAGCTCATGAACAG Exon 3 Npy_E3R CCTTGAAAGTTGAGATTTGCTG Npy_E4F GGCAAAAGCTGATGAACTGG Exon 4 Npy_E4R TCATCCACTCATGCCTGCTA

165

Phenylethanolamine-N-methyltransferase (Pnmt)

Data not available: unpublished data from a collaborator.

Appendix B: Candidate Gene Nucleotide Sequence

166 167

The nucleotide sequence (from 5’ to 3’) for each candidate gene resequenced in genomic DNA of the SHR/WKY/BN rat strains is listed below.

The 5’-cap site for the mRNA encoded by each gene is designated as position zero (“0”). The first nucleotide of the proximal promoter, therefore, is denoted as

“-1” while the first nucleotide of exon 1 is denoted as “+1”. Polymorphic regions are shown in bold text and enclosed in a box (e.g., ACTACTACT). Exons are denoted with bold, underlined, and capitalized letters (e.g., ATCGTCGGGCA).

Untranslated regions (5’ and 3’ UTR), if present, are shown in bold, underlined, and lowercase text (e.g., atcggct). Intronic regions that were not resequenced are presented as strings of “NNNNNNNN….”. The “RefSeq” sequence corresponds to the publicly available Rattus norvegicus genomic DNA sequence that is derived from the Brown Norway (BN) strain. In this case, the “RefSeq” sequence was downloaded from the University of California, Santa Cruz, rat genome browser (http://genome.ucsc.edu/).

168

Catechol-O-methyltransferase (Comt)

SHR -1530 TCAGTAAGAGGTGGAGTCCAGGACCTTCCGAGAGCAGATCATGAGGACCA -1481 WKY -1530 TCAGTAAGAGGTGGAGTCCAGGACCTTCCGAGAGCAGATCATGAGGACCA -1481 RefSeq -1530 TCAGTAAGAGGTGGAGTCCAGGACCTTCCGAGAGCAGATCATGAGGACCA -1481

SHR -1480 GGGAGAACTGGATGTGAGATCACTTGGCTGATGGAAGCCTGTGGGTGTGT -1431 WKY -1480 GGGAGAACTGGATGTGAGATCACTTGGCTGATGGAAGCCTGTGGGTGTGT -1431 RefSeq -1480 GGGAGAACTGGATGTGAGATCACTTGGCTGATGGAAGCCTGTGGGTGTGT -1431

SHR -1430 GAAGGCTCAGGCCCAGGGTGTGTGCGAGAAACCACGTGAGCACTCTGCCT -1381 WKY -1430 GAAGGCTCAGGCCCAGGGTGTGTGCGAGAAACCACGTGAGCACTCTGCCT -1381 RefSeq -1430 GAAGGCTCAGGCCCAGGGTGTGTGCGAGAAACCACGTGAGCACTCTGCCT -1381

SHR -1380 TGTGGAAATAGGCTACATGAGTTTGTGTGGGCCGGGAAGCCTCAGCTTCG -1331 WKY -1380 TGTGGAAATAGGCTACATGAGTTTGTGTGGGCCGGGAAGCCTCAGCTTCG -1331 RefSeq -1380 TGTGGAAATAGGCTACATGAGTTTGTGTGGGCCGGGAAGCCTCAGCTTCG -1331

SHR -1330 AGGCCTCTCTTCTCTCCTGAGACTGTTCTGAAGCACCCATCAGATCTAGG -1281 WKY -1330 AGGCCTCTCTTCTCTCCTGAGACTGTTCTGAAGCACCCATCAGATCTAGG -1281 RefSeq -1330 AGGCCTCTCTTCTCTCCTGAGACTGTTCTGAAGCACCCATCAGATCTAGG -1281

SHR -1280 GCTTAGGCCTGCCTCCTCTTAGGACTCTTCCCAGCCTAGATCAGGTTGTG -1231 WKY -1280 GCTTAGGCCTGCCTCCTCTTAGGACTCTTCCCAGCCTAGATCAGGTTGTG -1231 RefSeq -1280 GCTTAGGCCTGCCTCCTCTTAGGACTCTTCCCAGCCTAGATCAGGTTGTG -1231

SHR -1230 TGTGGTAGCCACACCCTACCCTGGAAGGAACTGTAAATGGCCAATCTGTA -1181 WKY -1230 TGTGGTAGCCACACCCTACCCTGGAAGGAACTGTAAATGGCCAATCTGTA -1181 RefSeq -1230 TGTGGTAGCCACACCCTACCCTGGAAGGAACTGTAAATGGCCAATCTGTA -1181

SHR -1180 GTGGACATTTCCTCTTTCTTGTGTCTTTTTAGGTTCTTACAAGGTTGTGC -1131 WKY -1180 GTGGACATTTCCTCTTTCTTGTGTCTTTTTAGGTTCTTACAAGGTTGTGC -1131 RefSeq -1180 GTGGACATTTCCTCTTTCTTGTGTCTTTTTAGGTTCTTACAAGGTTGTGC -1131

SHR -1130 TGAGAGCTTACTGAACCCAGTATCAGGGACGCTGGTAAACCCAGTTAGAT -1081 WKY -1130 TGAGAGCTTACTGAACCCAGTATCAGGGACGCTGGTAAACCCAGTTAGAT -1081 RefSeq -1130 TGAGAGCTTACTGAACCCAGTATCAGGGACGCTGGTAAACCCAGTTAGAT -1081

SHR -1080 CCTGGGTTGTGCTCCCCAGCTCTTGCTTACCAGAAGTGAGTGTGTGTGAG -1031 WKY -1080 CCTGGGTTGTGCTCCCCAGCTCTTGCTTACCAGAAGTGAGTGTGTGTGAG -1031 RefSeq -1080 CCTGGGTTGTGCTCCCCAGCTCTTGCTTACCAGAAGTGAGTGTGTGTGAG -1031

SHR -1030 CAGAGTCACTCGCACTCACCTTGTACTGGGGACAGAACTCAGGGCCTTAT -981 WKY -1030 CAGAGTCACTCGCACTCACCTTGTACTGGGGACAGAACTCAGGGCCTTAT -981 RefSeq -1030 CAGAGTCACTCGCACTCACCTTGTACTGGGGACAGAACTCAGGGCCTTAT -981

SHR -980 GTGTGCTACACTACACATGAGCTCACCCAGCCCTGTGGTTTTAAAAGATC -931 WKY -980 GTGTGCTACACTACACATGAGCTCACCCAGCCCTGTGGTTTTAAAAGATC -931 RefSeq -980 GTGTGCTACACTACACATGAGCTCACCCAGCCCTGTGGTTTTAAAAGATC -931

SHR -930 TAAAATCTTCTAAAGAAGCTGTTCCCTACTTCAGTGGGGAGAGGTCCCAG -881 WKY -930 TAAAATCTTCTAAAGAAGCTGTTCCCTACTTCAGTGGGGAGAGGTCCCAG -881 RefSeq -930 TAAAATCTTCTAAAGAAGCTGTTCCCTACTTCAGTGGGGAGAGGTCCCAG -881

169

SHR -880 TATCTTACTTAGTTCCTCTGTGAGTCTTCAGCCGAGTCTAGAAAGTCAGT -831 WKY -880 TATCTTACTTAGTTCCTCTGTGAGTCTTCAGCCGAGTCTAGAAAGTCAGT -831 RefSeq -880 TATCTTACTTAGTTCCTCTGTGAGTCTTCAGCCGAGTCTAGAAAGTCAGT -831

SHR -830 GCAAGGCCTAACGGCAAGAGGAAGAGTTCACGTTTTATTAATTTTGCACA -781 WKY -830 GCAAGGCCTAACGGCAAGAGGAAGAGTTCACGTTTTATTAATTTTGCACA -781 RefSeq -830 GCAAGGCCTAACGGCAAGAGGAAGAGTTCACGTTTTATTAATTTTGCACA -781

SHR -780 GCCCTGGAGATTTACACAAGGAGATGGGCCCGGACCAAAACACTGGACAC -731 WKY -780 GCCCTGGAGATTTACACAAGGAGATGGGCCCGGACCAAAACACTGGACAC -731 RefSeq -780 GCCCTGGAGATTTACACAAGGAGATGGGCCCGGACCAAAACACTGGACAC -731

SHR -730 GGTACATGGTACTGTACATGCCTATAATCCCAGCCCTCAGAAGGACTGGG -681 WKY -730 GGTACATGGTACTGTACATGCCTATAATCCCAGCCCTCAGAAGGACTGGG -681 RefSeq -730 GGTACATGGTACTGTACATGCCTATAATCCCAGCCCTCAGAAGGACTGGG -681

SHR -680 CACTTAAGACCAGCCTGGGCTGCACAGCCTGGCTGAAGAAACAGGTTGAT -631 WKY -680 CACTTAAGACCAGCCTGGGCTGCACAGCCTGGCTGAAGAAACAGGTTGAT -631 RefSeq -680 CACTTAAGACCAGCCTGGGCTGCACAGCCTGGCTGAAGAAACAGGTTGAT -631

SHR -630 AATAGAGGTTAGTGGTGGGGTGTTTACCTTGTACACGTAAAGTTCCACCC -581 WKY -630 AATAGAGGTTAGTGGTGGGGTGTTTACCTTGTACACGTAAAGTTCCACCC -581 RefSeq -630 AATAGAGGTTAGTGGTGGGGTGTTTACCTTGTACACGTAAAGTTCCACCC -581

SHR -580 AGGAGAAAGTAAAGTGCACAGAACCACAGCTTTCCTACTTTCTGAGGCCA -531 WKY -580 AGGAGAAAGTAAAGTGCACAGAACCACAGCTTTCCTACTTTCTGAGGCCA -531 RefSeq -580 AGGAGAAAGTAAAGTGCACAGAACCACAGCTTTCCTACTTTCTGAGGCCA -531

SHR -530 AGTGATCATTTTATAGGAGAAAGGACACAGAGATCTCTGGACTAGGGATG -481 WKY -530 AGTGATCATTTTATAGGAGAAAGGACACAGAGATCTCTGGACTAGGGATG -481 RefSeq -530 AGTGATCATTTTATAGGAGAAAGGACACAGAGATCTCTGGACTAGGGATG -481

SHR -480 GTACTTGGTAATCTATGGGGGCTGGGAAGCCAGCTGGAAAACGAGGGCCA -431 WKY -480 GTACTTGGTAATCTATGGGGGCTGGGAAGCCAGCTGGAAAACGAGGGCCA -431 RefSeq -480 GTACTTGGTAATCTATGGGGGCTGGGAAGCCAGCTGGAAAACGAGGGCCA -431

SHR -430 CTCCAACGAGTTTATTCAGGTCCCCAGCCCTGACAAAGGCTATTTTTCTG -381 WKY -430 CTCCAACGAGTTTATTCAGGTCCCCAGCCCTGACAAAGGCTATTTTTCTG -381 RefSeq -430 CTCCAACGAGTTTATTCAGGTCCCCAGCCCTGACAAAGGCTATTTTTCTG -381

SHR -380 TCCTGGTACTAGAGAATTCATCCTCACATTCTGCCACAAGCAGGAACAAG -331 WKY -380 TCCTGGTACTAGAGAATTCATCCTCACATTCTGCCACAAGCAGGAACAAG -331 RefSeq -380 TCCTGGTACTAGAGAATTCATCCTCACATTCTGCCACAAGCAGGAACAAG -331

SHR -330 CAGGTCATGTGGCCCATCCGGCAGCACCCACTGTTCCTCTTTCTGCCAGA -281 WKY -330 CAGGTCATGTGGCCCATCCGGCAGCACCCACTGTTCCTCTTTCTGCCAGA -281 RefSeq -330 CAGGTCATGTGGCCCATCCGGCAGCACCCACTGTTCCTCTTTCTGCCAGA -281

SHR -280 AACAGTCAGAATGCTACTTCAGTGCAAAAGTGACACCACCATCACGAGAA -231 WKY -280 AACAGTCAGAATGCTACTTCAGTGCAAAAGTGACACCACCATCACGAGAA -231 RefSeq -280 AACAGTCAGAATGCTACTTCAGTGCAAAAGTGACACCACCATCACGAGAA -231

170

SHR -230 AGCACTCAGGGTTTGGGACCATGCCTCACAGTCCTCTACACAGGACTCCG -181 WKY -230 AGCACTCAGGGTTTGGGACCATGCCTCACAGTCCTCTACACAGGACTCCG -181 RefSeq -230 AGCACTCAGGGTTTGGGACCATGCCTCACAGTCCTCTACACAGGACTCCG -181

SHR -180 GGAGGCACGCTTGTACCTGCCTCACCTGGCTCTGGCCCAGTGAGGCACTG -131 WKY -180 GGAGGCACGCTTGTACCTGCCTCACCTGGCTCTGGCCCAGTGAGGCACTG -131 RefSeq -180 GGAGGCACGCTTGTACCTGCCTCACCTGGCTCTGGCCCAGTGAGGCACTG -131

SHR -130 AGTCATGTGGATGCATTCTCTGAACCTTGGACCTCCTCAGCGCAGGAAAC -81 WKY -130 AGTCATGTGGATGCATTCTCTGAACCTTGGACCTCCTCAGCGCAGGAAAC -81 RefSeq -130 AGTCATGTGGATGCATTCTCTGAACCTTGGACCTCCTCAGCGCAGGAAAC -81

SHR -80 CAATTAAGCCAAGAACAGGGTGGGGTGAGGCAGGAAGGGCACAAGACACA -31 WKY -80 CAATTAAGCCAAGAACAGGGTGGGGTGAGGCAGGAAGGGCACAAGACACA -31 RefSeq -80 CAATTAAGCCAAGAACAGGGTGGGGTGAGGCAGGAAGGGCACAAGACACA -31 ++1|-1 SHR -30 CAGCCTGTCTGCTCTGCCCCCGCCCTGCAGATGCCGTTGGCTGCAGTCTC +20 WKY -30 CAGCCTGTCTGCTCTGCCCCCGCCCTGCAGATGCCGTTGGCTGCAGTCTC +20 RefSeq -30 CAGCCTGTCTGCTCTGCCCCCGCCCTGCAGATGCCGTTGGCTGCAGTCTC +20

SHR +21 ATTGGGTCTCCTGTTGTTGGCCCTCCTCCTGCTCTTGCGACACCTGGGCT +70 WKY +21 ATTGGGTCTCCTGTTGTTGGCCCTCCTCCTGCTCTTGCGACACCTGGGCT +70 RefSeq +21 ATTGGGTCTCCTGTTGTTGGCCCTCCTCCTGCTCTTGCGACACCTGGGCT +70

SHR +71 GGGGCTTGGTGACTATTTTCTGGTTTGAGTACGTGCTGCAGCCAGTCCAC +120 WKY +71 GGGGCTTGGTGACTATTTTCTGGTTTGAGTACGTGCTGCAGCCAGTCCAC +120 RefSeq +71 GGGGCTTGGTGACTATTTTCTGGTTTGAGTACGTGCTGCAGCCAGTCCAC +120

SHR +121 AACCTGATCATGGGTGACACAAAGGAGCAGCGCATCCTGCGCTACGTGCA +170 WKY +121 AACCTGATCATGGGTGACACAAAGGAGCAGCGCATCCTGCGCTACGTGCA +170 RefSeq +121 AACCTGATCATGGGTGACACAAAGGAGCAGCGCATCCTGCGCTACGTGCA +170

SHR +171 GCAGAATGCAAAGCCTGGAGACCCTCAGAGCGTCCTGGAGGCCATCGACA +220 WKY +171 GCAGAATGCAAAGCCTGGAGACCCTCAGAGCGTCCTGGAGGCCATCGACA +220 RefSeq +171 GCAGAATGCAAAGCCTGGAGACCCTCAGAGCGTCCTGGAGGCCATCGACA +220  EXON 1| SHR +221 CCTACTGCACACAGAAGGAATGGGCCATGAATGTGGGTGACGCGAAAGGT +270 WKY +221 CCTACTGCACACAGAAGGAATGGGCCATGAATGTGGGTGACGCGAAAGGT +270 RefSeq +221 CCTACTGCACACAGAAGGAATGGGCCATGAATGTGGGTGACGCGAAAGGT +270

SHR +271 ATGTGGCCAGCATGCAGAGGACAGCCTAGAAATGGGGAGCCTGNNNNNNN +320 WKY +271 ATGTGGCCAGCATGCAGAGGACAGCCTAGAAATGGGGAGCCTGNNNNNNN +320 RefSeq +271 ATGTGGCCAGCATGCAGAGGACAGCCTAGAAATGGGGAGCCTGAAGTGGG +320

SHR +321 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +370 WKY +321 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +370 RefSeq +321 TATGGCAGTCCTTGTAGGAGAGCCTGTTAGAAGGAGAGAAAATCGGGGTC +370

SHR +371 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +420 WKY +371 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +420 RefSeq +371 ATGCTCCTGTCTAGCCCCTGTGGGGACTGAATCATTTACACTTCTAAGAG +420

SHR +421 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +470 WKY +421 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +470 RefSeq +421 CCAGGGGGCTGGGTGAGGGGGCTCCCTGTGTGTGGATTCTCCCATGGTGT +470

171

SHR +471 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +520 WKY +471 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +520 RefSeq +471 GTACAGTGGGGTCCTCTGGGCTATTGGGAGGCAAAGGGCCTTTCTCAGCG +520

SHR +521 NNNNNNNNNNNNNNGAGGAGTGTGATGCTACTGGTCTGGATAGGGGCAAA +570 WKY +521 NNNNNNNNNNNNNNGAGGAGTGTGATGCTACTGGTCTGGATAGGGGCAAA +570 RefSeq +521 GGTCAGGGACATGAGAGGAGTGTGATGCTACTGGTCTGGATAGGGGCAAA +570

SHR +571 GCCAGAGTTGGGGACGGGTTCCACCCTCTACCTGTACTTAGAGCCTCCTC +620 WKY +571 GCCAGAGTTGGGGACGGGTTCCACCCTCTACCTGTACTTAGAGCCTCCTC +620 RefSeq +571 GCCAGAGTTGGGGACGGGTTCCACCCTCTACCTGTACTTAGAGCCTCCTC +620 |EXON 2  SHR +621 ACCACTTTTCAGGCCAAATCATGGATGCAGTGATTCGGGAGTACAGCCCC +670 WKY +621 ACCACTTTTCAGGCCAAATCATGGATGCAGTGATTCGGGAGTACAGCCCC +670 RefSeq +621 ACCACTTTTCAGGCCAAATCATGGATGCAGTGATTCGGGAGTACAGCCCC +670

SHR +671 TCCCTGGTGCTGGAGCTGGGAGCTTACTGTGGCTACTCAGCAGTGCGAAT +720 WKY +671 TCCCTGGTGCTGGAGCTGGGAGCTTACTGTGGCTACTCAGCAGTGCGAAT +720 RefSeq +671 TCCCTGGTGCTGGAGCTGGGAGCTTACTGTGGCTACTCAGCAGTGCGAAT +720

SHR +721 GGCTCGCCTGCTGCAGCCTGGAGCCAGGCTTCTCACCATGGAGATGAACC +770 WKY +721 GGCTCGCCTGCTGCAGCCTGGAGCCAGGCTTCTCACCATGGAGATGAACC +770 RefSeq +721 GGCTCGCCTGCTGCAGCCTGGAGCCAGGCTTCTCACCATGGAGATGAACC +770

SHR +771 CTGACTACGCTGCCATCACCCAGCAAATGCTGAACTTTGCAGGCCTACAG +820 WKY +771 CTGACTACGCTGCCATCACCCAGCAAATGCTGAACTTTGCAGGCCTACAG +820 RefSeq +771 CTGACTACGCTGCCATCACCCAGCAAATGCTGAACTTTGCAGGCCTACAG +820  EXON 2| SHR +821 GACAAAGTATGGCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +870 WKY +821 GACAAAGTATGGCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +870 RefSeq +821 GACAAAGTATGGCCCAGTGTGGCGGCTGTGGGGTGTAGGGGGCGGGGGGG +870

SHR +871 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +920 WKY +871 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +920 RefSeq +871 CTGGCAGGCAATGGGGGAGGATGAGGTCCTTGTCAGCCACTAAGCACCTG +920

SHR +921 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +970 WKY +921 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +970 RefSeq +921 CCACATCTGTAACACTGGAGTCAAGGAAGGAACCCCAGCAAGTCAGGGAC +970

SHR +971 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1020 WKY +971 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1020 RefSeq +971 ATGGTCTTACTGATCTATCCGGTAGAGCAAGCAACAACCAGGGCTGCCAG +1020

SHR +1021 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAGATGTAGACACG +1070 WKY +1021 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGAGATGTAGACACG +1070 RefSeq +1021 GGAGACTCAGTATGAACTCCTCTCCAGAGCCCCAAAGAGATGTAGACACG +1070

SHR +1071 GGTGGGGGTCCAGGCAAGCTCCCGCATATCACTGTGGGCACAGTACTGAC +1120 WKY +1071 GGTGGGGGTCCAGGCAAGCTCCCGCATATCACTGTGGGCACAGTACTGAC +1120 RefSeq +1071 GGTGGGGGTCCAGGCAAGCTCCCGCATATCACTGTGGGCACAGTACTGAC +1120

172

|EXON 3  SHR +1121 AGAACACTGTTCCAGGTCACCATCCTCAATGGGGCATCCCAGGATCTTAT +1170 WKY +1121 AGAACACTGTTCCAGGTCACCATCCTCAATGGGGCATCCCAGGATCTTAT +1170 RefSeq +1121 AGAACACTGTTCCAGGTCACCATCCTCAATGGGGCATCCCAGGATCTTAT +1170

SHR +1171 CCCCCAGCTGAAGAAGAAGTACGACGTGGACACACTAGACATGGTCTTTC +1220 WKY +1171 CCCCCAGCTGAAGAAGAAGTACGACGTGGACACACTAGACATGGTCTTTC +1220 RefSeq +1171 CCCCCAGCTGAAGAAGAAGTACGACGTGGACACACTAGACATGGTCTTTC +1220  EXON 3| SHR +1221 TTGACCACTGGAAAGACCGCTACCTTCCAGACACACTTCTCCTGGAGGTG +1270 WKY +1221 TTGACCACTGGAAAGACCGCTACCTTCCAGACACACTTCTCCTGGAGGTG +1270 RefSeq +1221 TTGACCACTGGAAAGACCGCTACCTTCCAGACACACTTCTCCTGGAGGTG +1270

SHR +1271 AGCTGAACTCCCACTGCTCTGCAGGATGGCCTCTGCGGTCACTGCTGGGG +1320 WKY +1271 AGCTGAACTCCCACTGCTCTGCAGGATGGCCTCTGCGGTCACTGCTGGGG +1320 RefSeq +1271 AGCTGAACTCCCACTGCTCTGCAGGATGGCCTCTGCGGTCACTGCTGGGG +1320

SHR +1321 TCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1370 WKY +1321 TCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1370 RefSeq +1321 TCACTGAGCTTGGGCCTCTTAAGAGCCCTGCAGATGTGGGCCCAGGGACA +1370

SHR +1371 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +4020 WKY +1371 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +4020 RefSeq +1371 GTCTGTGAGTTCTGT...... CCCATGGATGGCACT +4020 |EXON 4  SHR +4021 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGTCCCCTGCAGAAATGT +4070 WKY +4021 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTGTCCCCTGCAGAAATGT +4070 RefSeq +4021 GGGTTTGCCAAGCCTTCCTGACACTCCCTGTCTGTCCCCTGCAGAAATGT +4070

SHR +4071 GGCCTGCTGCGCAAGGGGACAGTGCTCCTAGCTGACAACGTCATCGTCCC +4120 WKY +4071 GGCCTGCTGCGCAAGGGGACAGTGCTCCTAGCTGACAACGTCATCGTCCC +4120 RefSeq +4071 GGCCTGCTGCGCAAGGGGACAGTGCTCCTAGCTGACAACGTCATCGTCCC +4120

SHR +4121 GGGAACCCCTGACTTCCTGGCGTATGTGAGAGGGAGCAGCAGCTTCGAGT +4170 WKY +4121 GGGAACCCCTGACTTCCTGGCGTATGTGAGAGGGAGCAGCAGCTTCGAGT +4170 RefSeq +4121 GGGAACCCCTGACTTCCTGGCGTATGTGAGAGGGAGCAGCAGCTTCGAGT +4170

SHR +4171 GCACACACTACAGCTCATACCTGGAGTACATGAAAGTTGTAGACGGCTTG +4220 WKY +4171 GCACACACTACAGCTCATACCTGGAGTACATGAAAGTTGTAGACGGCTTG +4220 RefSeq +4171 GCACACACTACAGCTCATACCTGGAGTACATGAAAGTTGTAGACGGCTTG +4220 |3’UTR  SHR +4221 GAGAAGGCAATCTACCAGGGTCCAAGTAGCCCTGACAAGTCTTGAtccct +4270 WKY +4221 GAGAAGGCAATCTACCAGGGTCCAAGTAGCCCTGACAAGTCTTGAtccct +4270 RefSeq +4221 GAGAAGGCAATCTACCAGGGTCCAAGTAGCCCTGACAAGTCTTGAtccct +4270

SHR +4271 cagcctgcccccctgctccccttccagcttctctctgcgagatgacacac +4320 WKY +4271 cagcctgcccccctgctccccttccagcttctctctgcgagatgacacac +4320 RefSeq +4271 cagcctgcccccctgctccccttccagcttctctctgcgagatgacacac +4320

SHR +4321 attcaatctgaccctttctatgctcctggggcttgtcctcgggggctgtg +4370 WKY +4321 attcaatctgaccctttctatgctcctggggcttgtcctcgggggctgtg +4370 RefSeq +4321 attcaatctgaccctttctatgctcctggggcttgtcctcgggggctgtg +4370

173

SHR +4371 gttccagactgtcatacactggcacattttaaaggtagtgagcccactat +4420 WKY +4371 gttccagactgtcatacactggcacattttaaaggtagtgagcccactat +4420 RefSeq +4371 gttccagactgtcatacactggcacattttaaaggtagtgagcccactat +4420

SHR +4421 gcaaaatcacttcaataaccctgaaaagcacctatggatgaaaggctgaa +4470 WKY +4421 gcaaaatcacttcaataaccctgaaaagcacctatggatgaaaggctgaa +4470 RefSeq +4421 gcaaaatcacttcaataaccctgaaaagcacctatggatgaaaggctgaa +4470

SHR +4471 ttgaggccaaaggtacaatagatcacagcctgcccagcatgcaaaaggcc +4520 WKY +4471 ttgaggccaaaggtacaatagatcacagcctgcccagcatgcaaaaggcc +4520 RefSeq +4471 ttgaggccaaaggtacaatagatcacagcctgcccagcatgcaaaaggcc +4520

SHR +4521 tcaaggttaatcctctgcacccgagcctcatggtctcgctaactagaaga +4570 WKY +4521 tcaaggttaatcctctgcacccgagcctcatggtctcgctaactagaaga +4570 RefSeq +4521 tcaaggttaatcctctgcacccgagcctcatggtctcgctaactagaaga +4570

SHR +4571 ggagatcttcacggggtttcagtgtgggatccctaggcactgcatcacca +4620 WKY +4571 ggagatcttcacggggtttcagtgtgggatccctaggcactgcatcacca +4620 RefSeq +4571 ggagatcttcacggggtttcagtgtgggatccctaggcactgcatcacca +4620

SHR +4621 gttaggcccagcaggaagtcagcaaactgtaaatgtccagacgccaaata +4670 WKY +4621 gttaggcccagcaggaagtcagcaaactgtaaatgtccagacgccaaata +4670 RefSeq +4621 gttaggcccagcaggaagtcagcaaactgtaaatgtccagacgccaaata +4670

SHR +4671 acagcagaggctcagagacagtactgtcactcactgtccccagcagctca +4720 WKY +4671 acagcagaggctcagagacagtactgtcactcactgtccccagcagctca +4720 RefSeq +4671 acagcagaggctcagagacagtactgtcactcactgtccccagcagctca +4720

SHR +4721 caccacatctcatctgaagaatgagtcacaagctttccaggtatggtggt +4770 WKY +4721 caccacatctcatctgaagaatgagtcacaagctttccaggtatggtggt +4770 RefSeq +4721 caccacatctcatctgaagaatgagtcacaagctttccaggtatggtggt +4770

SHR +4771 gcacgcctgtcatccccagtatccaggaggcagaggcatcggagttcagc +4820 WKY +4771 gcacgcctgtcatccccagtatccaggaggcagaggcatcggagttcagc +4820 RefSeq +4771 gcacgcctgtcatccccagtatccaggaggcagaggcatcggagttcagc +4820

SHR +4821 gccagcctggtctacataagtgagttccaagacagccagggctacgtaga +4870 WKY +4821 gccagcctggtctacataagtgagttccaagacagccagggctacgtaga +4870 RefSeq +4821 gccagcctggtctacataagtgagttccaagacagccagggctacgtaga +4870  EXON 4| SHR +4871 aatactttttaaaaaaagttataacctCTTTTATATTATAAAAGAAATTA +4920 WKY +4871 aatactttttaaaaaaagttataacctCTTTTATATTATAAAAGAAATTA +4920 RefSeq +4871 aatactttttaaaaaaagttataacctCTTTTATATTATAAAAGAAATTA +4920

SHR +4921 AAGA +4924 WKY +4921 AAGA +4924 RefSeq +4921 AAGA +4924

174

Chromogranin A (Chga)

SHR -1767 ATATGATATGAAAATAAATATAAAAATAAAAAAGCCAGCACACCTAAACT -1718 WKY -1767 ATATGATATGAAAATAAATATAAAAATAAAAAAGCCAGCACACCTAAACT -1718 RefSeq -1767 ATATGATATGAAAATAAATATAAAAATAAAAAAGCCAGCACACCTAAACT -1718 -1694 In/Del -/G SHR -1717 GTGACCTACAAAAGGGTGCCTGGGTTGATGATGTCCAGGGATCAGGTAGT -1668 WKY -1717 GTGACCTACAAAAGGGTGCCTGG-TTGATGATGTCCAGGGATCAGGTAGT -1668 RefSeq -1717 GTGACCTACAAAAGGGTGCCTGG-TTGATGATGTCCAGGGATCAGGTAGT -1668

SHR -1667 ATTTATTTATTTATTTATTTATTTATTTTATTTATTTAGAGACAGGGTCT -1618 WKY -1667 ATTTATTTATTTATTTATTTATTTATTTTATTTATTTAGAGACAGGGTCT -1618 RefSeq -1667 ATTTATTTATTTATTTATTTATTTATTTTATTTATTTAGAGACAGGGTCT -1618 A-1616T SHR -1617 CTCTGTGTAGCTCTGGCTATCCTGGAACTTATCATGTGGACCAGGCTGGC -1568 WKY -1617 CACTGTGTAGCTCTGGCTATCCTGGAACTTATCATGTGGACCAGGCTGGC -1568 RefSeq -1617 CACTGTGTAGCTCTGGCTATCCTGGAACTTATCATGTGGACCAGGCTGGC -1568

SHR -1567 ATCGAACTCACAGAGATCTGCTTGATTCTGCCTCCCACGCTCGGGGGTTA -1518 WKY -1567 ATCGAACTCACAGAGATCTGCTTGATTCTGCCTCCCACGCTCGGGGGTTA -1518 RefSeq -1567 ATCGAACTCACAGAGATCTGCTTGATTCTGCCTCCCACGCTCGGGGGTTA -1518

SHR -1517 GAGGTTTGTGCCACCATACTTTGCTGCAGCTAGTTTTTACGAGATGGTAT -1468 WKY -1517 GAGGTTTGTGCCACCATACTTTGCTGCAGCTAGTTTTTACGAGATGGTAT -1468 RefSeq -1517 GAGGTTTGTGCCACCATACTTTGCTGCAGCTAGTTTTTACGAGATGGTAT -1468

SHR -1467 TTTGGAGACAGCATGCCGGGAGCTTGCGTGTGATCACAGTGACTTCATGT -1418 WKY -1467 TTTGGAGACAGCATGCCGGGAGCTTGCGTGTGATCACAGTGACTTCATGT -1418 RefSeq -1467 TTTGGAGACAGCATGCCGGGAGCTTGCGTGTGATCACAGTGACTTCATGT -1418

SHR -1417 CCTAGGACCTGGAACACTCTGGAATTCTCCCAGTGCTGAGCTGGAGTCCT -1368 WKY -1417 CCTAGGACCTGGAACACTCTGGAATTCTCCCAGTGCTGAGCTGGAGTCCT -1368 RefSeq -1417 CCTAGGACCTGGAACACTCTGGAATTCTCCCAGTGCTGAGCTGGAGTCCT -1368

SHR -1367 TTTCTAGGAACTAATATATATGAATGGAGACGCCTCAGTGCAGAATAAAT -1318 WKY -1367 TTTCTAGGAACTAATATATATGAATGGAGACGCCTCAGTGCAGAATAAAT -1318 RefSeq -1367 TTTCTAGGAACTAATATATATGAATGGAGACGCCTCAGTGCAGAATAAAT -1318

SHR -1317 GCTTTAAATTTGCTGGCCCTTGTCCCCGGGTAGAGCCAGCCTCTCCCATA -1268 WKY -1317 GCTTTAAATTTGCTGGCCCTTGTCCCCGGGTAGAGCCAGCCTCTCCCATA -1268 RefSeq -1317 GCTTTAAATTTGCTGGCCCTTGTCCCCGGGTAGAGCCAGCCTCTCCCATA -1268

SHR -1267 CATTCCTGTCCCTCACTAGAACTCTGCCGTCTTCTCCCCTTTATGCCTGT -1218 WKY -1267 CATTCCTGTCCCTCACTAGAACTCTGCCGTCTTCTCCCCTTTATGCCTGT -1218 RefSeq -1267 CATTCCTGTCCCTCACTAGAACTCTGCCGTCTTCTCCCCTTTATGCCTGT -1218

SHR -1217 AGCACGGCCATGACCCAGCATAGTGTACACCTTGCTTCTTTCTCTGGAAA -1168 WKY -1217 AGCACGGCCATGACCCAGCATAGTGTACACCTTGCTTCTTTCTCTGGAAA -1168 RefSeq -1217 AGCACGGCCATGACCCAGCATAGTGTACACCTTGCTTCTTTCTCTGGAAA -1168

SHR -1167 GGGAATTCTATAAGGGTTGGGTTTGCTGTTTTGTTTACTGCCGTGTCTTT -1118 WKY -1167 GGGAATTCTATAAGGGTTGGGTTTGCTGTTTTGTTTACTGCCGTGTCTTT -1118 RefSeq -1167 GGGAATTCTATAAGGGTTGGGTTTGCTGTTTTGTTTACTGCCGTGTCTTT -1118

175

SHR -1117 GGCATCTGGCACAGTCAAGTGGTGTTCTGAGGTGTTCTAAGCCCACGTTG -1068 WKY -1117 GGCATCTGGCACAGTCAAGTGGTGTTCTGAGGTGTTCTAAGCCCACGTTG -1068 RefSeq -1117 GGCATCTGGCACAGTCAAGTGGTGTTCTGAGGTGTTCTAAGCCCACGTTG -1068

SHR -1067 ATGCTTAACACATGATTGTTGAATGAATGCATGCAAAGCAGTTTCTCATT -1018 WKY -1067 ATGCTTAACACATGATTGTTGAATGAATGCATGCAAAGCAGTTTCTCATT -1018 RefSeq -1067 ATGCTTAACACATGATTGTTGAATGAATGCATGCAAAGCAGTTTCTCATT -1018

SHR -1017 TAGGGGCATGAGTGGGCAGAGGTGTGGGCAGGAAGCAGGGAAGAGCAGAA -968 WKY -1017 TAGGGGCATGAGTGGGCAGAGGTGTGGGCAGGAAGCAGGGAAGAGCAGAA -968 RefSeq -1017 TAGGGGCATGAGTGGGCAGAGGTGTGGGCAGGAAGCAGGGAAGAGCAGAA -968

SHR -967 GCAGGTGGGGACGGAAGGGGGCGGGGCTCTGAAGGATGCCAGTCAGTGCC -918 WKY -967 GCAGGTGGGGACGGAAGGGGGCGGGGCTCTGAAGGATGCCAGTCAGTGCC -918 RefSeq -967 GCAGGTGGGGACGGAAGGGGGCGGGGCTCTGAAGGATGCCAGTCAGTGCC -918

SHR -917 AAACTGTCATCCAGATACCAGGCTCATTATGGCACTGGGTGCAGGCTTCA -868 WKY -917 AAACTGTCATCCAGATACCAGGCTCATTATGGCACTGGGTGCAGGCTTCA -868 RefSeq -917 AAACTGTCATCCAGATACCAGGCTCATTATGGCACTGGGTGCAGGCTTCA -868

SHR -867 CAGGGCTTCCCATGTGGTCCACAGGGTGAGAGCAGAGCTGGGGATGGAGC -818 WKY -867 CAGGGCTTCCCATGTGGTCCACAGGGTGAGAGCAGAGCTGGGGATGGAGC -818 RefSeq -867 CAGGGCTTCCCATGTGGTCCACAGGGTGAGAGCAGAGCTGGGGATGGAGC -818

SHR -817 GGGGCAGAAGGAAACCAACCAGGAAGCAAGCTCACACCCAAAATATCCAG -768 WKY -817 GGGGCAGAAGGAAACCAACCAGGAAGCAAGCTCACACCCAAAATATCCAG -768 RefSeq -817 GGGGCAGAAGGAAACCAACCAGGAAGCAAGCTCACACCCAAAATATCCAG -768 “A” repeat polymorphism (-753 to -539) SHR -767 CTTTTAAGAGCATTAAAAAAAAAAA----GACAAGGCGTGGCTGTTGAAG -718 WKY -767 CTTTTAAGAGCATTAAAAAAAAAAAAAAAGACAAGGCGTGGCTGTTGAAG -718 RefSeq -767 CTTTTAAGAGCATTAAAAAAAAAAAAAAAGACAAGGCGTGGCTGTTGAAG -718

SHR -717 ACAGAGGTGTTCCTGGAGTGCTGGACTAGGACTGACTACTTTTGTTTTAG -668 WKY -717 ACAGAGGTGTTCCTGGAGTGCTGGACTAGGACTGACTACTTTTGTTTTAG -668 RefSeq -717 ACAGAGGTGTTCCTGGAGTGCTGGACTAGGACTGACTACTTTTGTTTTAG -668

SHR -667 CTTAATGGTGAGAACTGCCTCCCACTGCTACCTGCCTTACTTGCCACTTG -618 WKY -667 CTTAATGGTGAGAACTGCCTCCCACTGCTACCTGCCTTACTTGCCACTTG -618 RefSeq -667 CTTAATGGTGAGAACTGCCTCCCACTGCTACCTGCCTTACTTGCCACTTG -618

SHR -617 AAATACTAGGACACACTCATGTGTGGGCTGGATCTTCAATGCACACATTG -568 WKY -617 AAATACTAGGACACACTCATGTGTGGGCTGGATCTTCAATGCACACATTG -568 RefSeq -617 AAATACTAGGACACACTCATGTGTGGGCTGGATCTTCAATGCACACATTG -568

SHR -567 AACTTGTGTGAAGCCATTGGTTGTCAGTGAGGAGCTCTCAGCACTGAGAA -518 WKY -567 AACTTGTGTGAAGCCATTGGTTGTCAGTGAGGAGCTCTCAGCACTGAGAA -518 RefSeq -567 AACTTGTGTGAAGCCATTGGTTGTCAGTGAGGAGCTCTCAGCACTGAGAA -518

SHR -517 AGCAGTGACCACTATCCCCTATCAAATAACTATTAAATACACACAGAACG -468 WKY -517 AGCAGTGACCACTATCCCCTATCAAATAACTATTAAATACACACAGAACG -468 RefSeq -517 AGCAGTGACCACTATCCCCTATCAAATAACTATTAAATACACACAGAACG -468

176

SHR -467 AGGCACAAAACTGAGTTTCAGGAGACGCCTCACTCAGGTAGGGATCCAAG -418 WKY -467 AGGCACAAAACTGAGTTTCAGGAGACGCCTCACTCAGGTAGGGATCCAAG -418 RefSeq -467 AGGCACAAAACTGAGTTTCAGGAGACGCCTCACTCAGGTAGGGATCCAAG -418

SHR -417 AGCCTTCTGTGGGACCCGCTGTATGTTCCAGGGAGTTCTGAAAGACAAGC -368 WKY -417 AGCCTTCTGTGGGACCCGCTGTATGTTCCAGGGAGTTCTGAAAGACAAGC -368 RefSeq -417 AGCCTTCTGTGGGACCCGCTGTATGTTCCAGGGAGTTCTGAAAGACAAGC -368

SHR -367 GTGCCTCCAACCGAGTGAAATCAAGAGAAAAGTACGCTAAGTATAGGAAA -318 WKY -367 GTGCCTCCAACCGAGTGAAATCAAGAGAAAAGTACGCTAAGTATAGGAAA -318 RefSeq -367 GTGCCTCCAACCGAGTGAAATCAAGAGAAAAGTACGCTAAGTATAGGAAA -318

SHR -317 ATTCAGCAGCCTGGAGAGGAACCCTAAACAGGGAAGGGATGTGAGGCTCA -268 WKY -317 ATTCAGCAGCCTGGAGAGGAACCCTAAACAGGGAAGGGATGTGAGGCTCA -268 RefSeq -317 ATTCAGCAGCCTGGAGAGGAACCCTAAACAGGGAAGGGATGTGAGGCTCA -268

SHR -267 GAGACAGGAGGACTTGCCCAAGGACACACAGCAAATTGACAGGTGGAAGT -218 WKY -267 GAGACAGGAGGACTTGCCCAAGGACACACAGCAAATTGACAGGTGGAAGT -218 RefSeq -267 GAGACAGGAGGACTTGCCCAAGGACACACAGCAAATTGACAGGTGGAAGT -218 C-177T SHR -217 TCAGCTGTGCCACCTTCTGAAGCCGTGTATCCTTCACAGCTACCAAATAG -168 WKY -217 TCAGCTGTGCCACCTTCTGAAGCCGTGTATCCTTCACAGCCACCAAATAG -168 RefSeq -217 TCAGCTGTGCCACCTTCTGAAGCCGTGTATCCTTCACAGCCACCAAATAG -168

SHR -167 AAGCAGGATGGAGGCAGCTCACCGTGAAGCTGGAGGTAGGGGGCGGGACC -118 WKY -167 AAGCAGGATGGAGGCAGCTCACCGTGAAGCTGGAGGTAGGGGGCGGGACC -118 RefSeq -167 AAGCAGGATGGAGGCAGCTCACCGTGAAGCTGGAGGTAGGGGGCGGGACC -118

SHR -117 CCGAAGGTGGGGAAAGGGCGCAGGGGGCGGTCCTATGACGTAATTTCCTG -68 WKY -117 CCGAAGGTGGGGAAAGGGCGCAGGGGGCGGTCCTATGACGTAATTTCCTG -68 RefSeq -117 CCGAAGGTGGGGAAAGGGCGCAGGGGGCGGTCCTATGACGTAATTTCCTG -68 C-59T SHR -67 GGTGTGTGTGTGTGCGTGCGTGTGTATAAAAGAGGGCATAGCATTGCTTC -18 WKY -67 GGTGTGTGCGTGTGCGTGCGTGTGTATAAAAGAGGGCATAGCATTGCTTC -18 RefSeq -67 GGTGTGTGCGTGTGCGTGCGTGTGTATAAAAGAGGGCATAGCATTGCTTC -18 -1|+1 SHR -17 GGGGCTGCTGCTACCGCcaccaccatcaccgccactgccaccaccaccgc +33 WKY -17 GGGGCTGCTGCTACCGCcaccaccatcaccgccactgccaccaccaccgc +33 RefSeq -17 GGGGCTGCTGCTACCGCcaccaccatcaccgccactgccaccaccaccgc +33

SHR +34 taccgcagtgctcccactggtgcagagctccagtggtcacagacccactt +83 WKY +34 taccgcagtgctcccactggtgcagagctccagtggtcacagacccactt +83 RefSeq +34 taccgcagtgctcccactggtgcagagctccagtggtcacagacccactt +83

SHR +84 ccgccatcctcctgcagcagctcgccctctctttccgcaccgtccggctc +133 WKY +84 ccgccatcctcctgcagcagctcgccctctctttccgcaccgtccggctc +133 RefSeq +84 ccgccatcctcctgcagcagctcgccctctctttccgcaccgtccggctc +133  5’UTR|  EXON 1| SHR +134 gctATGCGCTCCTCCGCGGCTTTGGCGCTTCTGCTGTGCGCCGGGCAAGG +183 WKY +134 gctATGCGCTCCTCCGCGGCTTTGGCGCTTCTGCTGTGCGCCGGGCAAGG +183 RefSeq +134 gctATGCGCTCCTCCGCGGCTTTGGCGCTTCTGCTGTGCGCCGGGCAAGG +183

SHR +184 TGAGAGGCTCGGCAGCGCGGTCCCCTCTCTTGCCCCAGGTCCCTTGCAGC +233 WKY +184 TGAGAGGCTCGGCAGCGCGGTCCCCTCTCTTGCCCCAGGTCCCTTGCAGC +233 RefSeq +184 TGAGAGGCTCGGCAGCGCGGTCCCCTCTCTTGCCCCAGGTCCCTTGCAGC +233

177

SHR +234 GCCCGAGGATTCAGCACCTCGGACAGCACCAGGTCTCCTGCTTGAGTCTG +283 WKY +234 GCCCGAGGATTCAGCACCTCGGACAGCACCAGGTCTCCTGCTTGAGTCTG +283 RefSeq +234 GCCCGAGGATTCAGCACCTCGGACAGCACCAGGTCTCCTGCTTGAGTCTG +283

SHR +284 GTCGCAAGACCTGTAATGTGGCCTATGGTCTCCCACCACACCCGGAGGAA +333 WKY +284 GTCGCAAGACCTGTAATGTGGCCTATGGTCTCCCACCACACCCGGAGGAA +333 RefSeq +284 GTCGCAAGACCTGTAATGTGGCCTATGGTCTCCCACCACACCCGGAGGAA +333

SHR +334 CTATAGAGCCTGACCCAACCCCACGGGAAAGCCCATCCCCATTCACACCC +383 WKY +334 CTATAGAGCCTGACCCAACCCCACGGGAAAGCCCATCCCCATTCACACCC +383 RefSeq +334 CTATAGAGCCTGACCCAACCCCACGGGAAAGCCCATCCCCATTCACACCC +383 T+413C SHR +384 GCACATCACCCACGGGTGCCCTGGCTCCACGCCGACCAAGGTCACTGGGG +433 WKY +384 GCACATCACCCACGGGTGCCCTGGCTCCATGCCGACCAAGGTCACTGGGG +433 RefSeq +384 GCACATCACCCACGGGTGCCCTGGCTCCATGCCGACCAAGGTCACTGGGG +433

SHR +434 GGAGATAGGGGACTGGCACTGGCTCCCCATCCCTTTTCCTGTCCTTCCAC +483 WKY +434 GGAGATAGGGGACTGGCACTGGCTCCCCATCCCTTTTCCTGTCCTTCCAC +483 RefSeq +434 GGAGATAGGGGACTGGCACTGGCTCCCCATCCCTTTTCCTGTCCTTCCAC +483

SHR +484 CCTCCAGTCTGTCCTTGATCTCTTTCCTCACCCCTGCTCTTTCCCGCCAC +533 WKY +484 CCTCCAGTCTGTCCTTGATCTCTTTCCTCACCCCTGCTCTTTCCCGCCAC +533 RefSeq +484 CCTCCAGTCTGTCCTTGATCTCTTTCCTCACCCCTGCTCTTTCCCGCCAC +533

SHR +534 GTCTGGGGTTTCTCCCCTTGCCTGCCTCTGTCCCTACTTCAGTCCCACTT +583 WKY +534 GTCTGGGGTTTCTCCCCTTGCCTGCCTCTGTCCCTACTTCAGTCCCACTT +583 RefSeq +534 GTCTGGGGTTTCTCCCCTTGCCTGCCTCTGTCCCTACTTCAGTCCCACTT +583

SHR +584 CATGGCTGGCCTTGTTTTCACTGAACTTGATTTCAGCTCTGCACTCCCAT +633 WKY +584 CATGGCTGGCCTTGTTTTCACTGAACTTGATTTCAGCTCTGCACTCCCAT +633 RefSeq +584 CATGGCTGGCCTTGTTTTCACTGAACTTGATTTCAGCTCTGCACTCCCAT +633

SHR +634 GGGGAAGGGTTGAGGCTCTTCAGAGCAGCCTCTGAGCCGACCCTCTACCC +683 WKY +634 GGGGAAGGGTTGAGGCTCTTCAGAGCAGCCTCTGAGCCGACCCTCTACCC +683 RefSeq +634 GGGGAAGGGTTGAGGCTCTTCAGAGCAGCCTCTGAGCCGACCCTCTACCC +683

SHR +684 TGGGGGTGGGGCGCAGCTTGGAGGAGTAGCAGAATCAATTCAGCCAAGTT +733 WKY +684 TGGGGGTGGGGCGCAGCTTGGAGGAGTAGCAGAATCAATTCAGCCAAGTT +733 RefSeq +684 TGGGGGTGGGGCGCAGCTTGGAGGAGTAGCAGAATCAATTCAGCCAAGTT +733

SHR +734 ATTTGGGTTTGGGAGGGGATCAATGGGGAGGGTGTTGGTTGAGTGGGTCC +783 WKY +734 ATTTGGGTTTGGGAGGGGATCAATGGGGAGGGTGTTGGTTGAGTGGGTCC +783 RefSeq +734 ATTTGGGTTTGGGAGGGGATCAATGGGGAGGGTGTTGGTTGAGTGGGTCC +783

SHR +784 CTTGAGCCTCCCAAAGGGAAGGAATGGGCTTCCCTGGATGCAGAGATAGC +833 WKY +784 CTTGAGCCTCCCAAAGGGAAGGAATGGGCTTCCCTGGATGCAGAGATAGC +833 RefSeq +784 CTTGAGCCTCCCAAAGGGAAGGAATGGGCTTCCCTGGATGCAGAGATAGC +833

SHR +834 CCCAGTCAGCAATCTCAGACACACTCACTCACTCCGTGTGTGTGTGTGTG +883 WKY +834 CCCAGTCAGCAATCTCAGACACACTCACTCACTCCGTGTGTGTGTGTGTG +883 RefSeq +834 CCCAGTCAGCAATCTCAGACACACTCACTCACTCCGTGTGTGTGTGTGTG +883

178

C+885T SHR +884 TGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC +933 WKY +884 TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC +933 RefSeq +884 TGTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC +933 |EXON 2  TC repeat polymorphism (+886 to +943) SHR +934 ------CAGTCTTTGCCCTTCCTGTGAACAGCCCTATGACAAAAGG +983 WKY +934 ------CAGTCTTTGCCCTTCCTGTGAACAGCCCTATGACAAAAGG +983 RefSeq +934 TCTCTCTCTCCAGTCTTTGCCCTTCCTGTGAACAGCCCTATGACAAAAGG +983  EXON 2| SHR +984 GGACACTAAGGTAAGAAGGAATGTTGGGGTATCCTGGGAGAGGGAGGGTG +1033 WKY +984 GGACACTAAGGTAAGAAGGAATGTTGGGGTATCCTGGGAGAGGGAGGGTG +1033 RefSeq +984 GGACACTAAGGTAAGAAGGAATGTTGGGGTATCCTGGGAGAGGGAGGGTG +1033

SHR +1034 CCCTGTGGACTCCTCATGGCCAGTCCTTAGGCAACTGCAGAAAGGAGCCT +1083 WKY +1034 CCCTGTGGACTCCTCATGGCCAGTCCTTAGGCAACTGCAGAAAGGAGCCT +1083 RefSeq +1034 CCCTGTGGACTCCTCATGGCCAGTCCTTAGGCAACTGCAGAAAGGAGCCT +1083 A+1113G SHR +1084 GGCATGGAGCCAAAAGGCAGCACTGCAGCGGCTGAGCCTGCACAGGGGAC +1133 WKY +1084 GGCATGGAGCCAAAAGGCAGCACTGCAGCAGCTGAGCCTGCACAGGGGAC +1133 RefSeq +1084 GGCATGGAGCCAAAAGGCAGCACTGCAGCAGCTGAGCCTGCACAGGGGAC +1133

SHR +1134 AGCTCGCACAAGAAGGCATCAAAGATCTCCTGCTGTTCAGCTACAGTGAC +1183 WKY +1134 AGCTCGCACAAGAAGGCATCAAAGATCTCCTGCTGTTCAGCTACAGTGAC +1183 RefSeq +1134 AGCTCGCACAAGAAGGCATCAAAGATCTCCTGCTGTTCAGCTACAGTGAC +1183 A+1196T SHR +1184 TTCCCAAGGCCCTTGTCTCACCTGCTGGGGCCTGGGACCAGGGACACTGG +1233 WKY +1184 TTCCCAAGGCCCATGTCTCACCTGCTGGGGCCTGGGACCAGGGACACTGG +1233 RefSeq +1184 TTCCCAAGGCCCATGTCTCACCTGCTGGGGCCTGGGACCAGGGACACTGG +1233

SHR +1234 TCTGAGCCAGGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1283 WKY +1234 TCTGAGCCAGGACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1283 RefSeq +1234 TCTGAGCCAGGACCATAGTTTCTCCTACCCTAGGTGCACAGACCTGCCTG +1283

SHR +1284 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +2983 WKY +1284 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +2983 RefSeq +1284 GTTGTGGCCTCTCTT...... TGATTCGATTGGCCC +2983 G+3033T SHR +2984 NNNNNNNNNNNNNNGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT +3033 WKY +2984 NNNNNNNNNNNNNNGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGG +3033 RefSeq +2984 ACAGTAACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGG +3033

SHR +3034 GTGGTTTTCTCTCTCTATGGCTCTATGATCTTGTTATGGAAGCAGATCTG +3083 WKY +3034 GTGGTTTTCTCTCTCTATGGCTCTATGATCTTGTTATGGAAGCAGATCTG +3083 RefSeq +3034 GTGGTTTTCTCTCTCTATGGCTCTATGATCTTGTTATGGAAGCAGATCTG +3083

SHR +3084 GGCCCCGTGGCCTCATGCAGACAACACAACTGCTCTACTGGGAAAGTTGC +3133 WKY +3084 GGCCCCGTGGCCTCATGCAGACAACACAACTGCTCTACTGGGAAAGTTGC +3133 RefSeq +3084 GGCCCCGTGGCCTCATGCAGACAACACAACTGCTCTACTGGGAAAGTTGC +3133 C+3168T SHR +3134 TCTGGCACCTCAGGGGCTGAACTCCATGTTCCTATCAGCCACACCCCGCC +3183 WKY +3134 TCTGGCACCTCAGGGGCTGAACTCCATGTTCCTACCAGCCACACCCCGCC +3183 RefSeq +3134 TCTGGCACCTCAGGGGCTGAACTCCATGTTCCTACCAGCCACACCCCGCC +3183

179

|EXON 3  SHR +3184 TTGCTGTCTCCCAGGTGATGAAGTGTGTCCTGGAGGTCATCTCCGACTCG +3233 WKY +3184 TTGCTGTCTCCCAGGTGATGAAGTGTGTCCTGGAGGTCATCTCCGACTCG +3233 RefSeq +3184 TTGCTGTCTCCCAGGTGATGAAGTGTGTCCTGGAGGTCATCTCCGACTCG +3233

SHR +3234 CTGTCCAAACCCAGCCCCATGCCTGTCAGCCCTGAGTGTCTGGAGACCCT +3283 WKY +3234 CTGTCCAAACCCAGCCCCATGCCTGTCAGCCCTGAGTGTCTGGAGACCCT +3283 RefSeq +3234 CTGTCCAAACCCAGCCCCATGCCTGTCAGCCCTGAGTGTCTGGAGACCCT +3283

 EXON 3| SHR +3284 CCAAGGAGGTAGGAGTCAGAGGCTGGAGGAGGAATTGGGAGGCCAGGGTG +3333 WKY +3284 CCAAGGAGGTAGGAGTCAGAGGCTGGAGGAGGAATTGGGAGGCCAGGGTG +3333 RefSeq +3284 CCAAGGAGGTAGGAGTCAGAGGCTGGAGGAGGAATTGGGAGGCCAGGGTG +3333

SHR +3334 TGGGTGGGTAGCTCTTGATAGGATTAATCATCTCATCCAGCAGTCATTGC +3383 WKY +3334 TGGGTGGGTAGCTCTTGATAGGATTAATCATCTCATCCAGCAGTCATTGC +3383 RefSeq +3334 TGGGTGGGTAGCTCTTGATAGGATTAATCATCTCATCCAGCAGTCATTGC +3383 +3386 In/Del C/- SHR +3384 CC-AGGGCCCTGCTGTGTGCTAGAATCTGGGGAGGACACATGGATCAGTG +3433 WKY +3384 CCCAGGGCCCTGCTGTGTGCTAGAATCTGGGGAGGACACATGGATCAGTG +3433 RefSeq +3384 CCCAGGGCCCTGCTGTGTGCTAGAATCTGGGGAGGACACATGGATCAGTG +3433

SHR +3434 ACAGCTCCTGGGACCCTGAGGTTTGTAGACTCTCAGAGGGGACATAGGGC +3483 WKY +3434 ACAGCTCCTGGGACCCTGAGGTTTGTAGACTCTCAGAGGGGACATAGGGC +3483 RefSeq +3434 ACAGCTCCTGGGACCCTGAGGTTTGTAGACTCTCAGAGGGGACATAGGGC +3483

SHR +3484 AGTTAACTAGAAGGGACTGGAACAGCAAGTTGCAGACAGGGCACCTTGAG +3533 WKY +3484 AGTTAACTAGAAGGGACTGGAACAGCAAGTTGCAGACAGGGCACCTTGAG +3533 RefSeq +3484 AGTTAACTAGAAGGGACTGGAACAGCAAGTTGCAGACAGGGCACCTTGAG +3533

SHR +3534 GACCGGGGTTCTGTGTGTTGTAGTCCCAGTGTTTGGAGTCTTGCTGACAC +3583 WKY +3534 GACCGGGGTTCTGTGTGTTGTAGTCCCAGTGTTTGGAGTCTTGCTGACAC +3583 RefSeq +3534 GACCGGGGTTCTGTGTGTTGTAGTCCCAGTGTTTGGAGTCTTGCTGACAC +3583

SHR +3584 CAGGGGCCATTCTATGCATCTGGGGTGAATACGGCGTTGAGTGGGCCTAA +3633 WKY +3584 CAGGGGCCATTCTATGCATCTGGGGTGAATACGGCGTTGAGTGGGCCTAA +3633 RefSeq +3584 CAGGGGCCATTCTATGCATCTGGGGTGAATACGGCGTTGAGTGGGCCTAA +3633

SHR +3634 GAAAGACCACACCTTTCCAAACTGACAGTCCAGTTGGTCTAGCGCAGGAT +3683 WKY +3634 GAAAGACCACACCTTTCCAAACTGACAGTCCAGTTGGTCTAGCGCAGGAT +3683 RefSeq +3634 GAAAGACCACACCTTTCCAAACTGACAGTCCAGTTGGTCTAGCGCAGGAT +3683

SHR +3684 AGCGCTGGGACAGGTAGACGAAGAAAGATGTCAGGGGGTGCATACAGAGC +3733 WKY +3684 AGCGCTGGGACAGGTAGACGAAGAAAGATGTCAGGGGGTGCATACAGAGC +3733 RefSeq +3684 AGCGCTGGGACAGGTAGACGAAGAAAGATGTCAGGGGGTGCATACAGAGC +3733

SHR +3734 AGGCAGCAGGGAAATGGGAAAGGGAGTATTTACACATTCACTGGAGCCCG +3783 WKY +3734 AGGCAGCAGGGAAATGGGAAAGGGAGTATTTACACATTCACTGGAGCCCG +3783 RefSeq +3734 AGGCAGCAGGGAAATGGGAAAGGGAGTATTTACACATTCACTGGAGCCCG +3783

SHR +3784 AGGCCCGAGGCCCTTTCCTTGGAGGCTGGGACTAGAGTCAGCAGGTGACC +3833 WKY +3784 AGGCCCGAGGCCCTTTCCTTGGAGGCTGGGACTAGAGTCAGCAGGTGACC +3833 RefSeq +3784 AGGCCCGAGGCCCTTTCCTTGGAGGCTGGGACTAGAGTCAGCAGGTGACC +3833

180

C+3863T SHR +3834 TTTCTGCTGCAGTAGGAAGGTGATGGACATCTTGGAGATGTCAAAGCGTT +3883 WKY +3834 TTTCTGCTGCAGTAGGAAGGTGATGGACACCTTGGAGATGTCAAAGCGTT +3883 RefSeq +3834 TTTCTGCTGCAGTAGGAAGGTGATGGACACCTTGGAGATGTCAAAGCGTT +3883

SHR +3884 CTACCAAGATCCAGGCTGTCGTTGCCTCCTGGTGAGTTTCCAGGATAAAT +3933 WKY +3884 CTACCAAGATCCAGGCTGTCGTTGCCTCCTGGTGAGTTTCCAGGATAAAT +3933 RefSeq +3884 CTACCAAGATCCAGGCTGTCGTTGCCTCCTGGTGAGTTTCCAGGATAAAT +3933 T+3961C SHR +3934 GCTTGAGCTCAGCTGTAAGTGTCCACACCAATCTCTCCCGGGGCTGTCCT +3983 WKY +3934 GCTTGAGCTCAGCTGTAAGTGTCCACATCAATCTCTCCCGGGGCTGTCCT +3983 RefSeq +3934 GCTTGAGCTCAGCTGTAAGTGTCCACATCAATCTCTCCCGGGGCTGTCCT +3983 |EXON 4  SHR +3984 CTGGAAGCCACCAGTAACTGTGCTCTCTCCCTGCAGATGAGAGGGTCCTC +4033 WKY +3984 CTGGAAGCCACCAGTAACTGTGCTCTCTCCCTGCAGATGAGAGGGTCCTC +4033 RefSeq +3984 CTGGAAGCCACCAGTAACTGTGCTCTCTCCCTGCAGATGAGAGGGTCCTC +4033

SHR +4034 TCCATCCTTCGACACCAGAATTTGCTGAAGGAACTTCAAGACCTGGCGCT +4083 WKY +4034 TCCATCCTTCGACACCAGAATTTGCTGAAGGAACTTCAAGACCTGGCGCT +4083 RefSeq +4034 TCCATCCTTCGACACCAGAATTTGCTGAAGGAACTTCAAGACCTGGCGCT +4083  EXON 4| SHR +4084 TCAAGGTATTTCAGCACCATACACAGTTCCACACAGAAAAGCCAGCCACA +4133 WKY +4084 TCAAGGTATTTCAGCACCATACACAGTTCCACACAGAAAAGCCAGCCACA +4133 RefSeq +4084 TCAAGGTATTTCAGCACCATACACAGTTCCACACAGAAAAGCCAGCCACA +4133

SHR +4134 GAGCCTGAAGGGACCTGGGTGGAGGTCGAGTGTACTCTTTCAGGGGCTCA +4183 WKY +4134 GAGCCTGAAGGGACCTGGGTGGAGGTCGAGTGTACTCTTTCAGGGGCTCA +4183 RefSeq +4134 GAGCCTGAAGGGACCTGGGTGGAGGTCGAGTGTACTCTTTCAGGGGCTCA +4183

SHR +4184 GTTCCGGAGCAGAAAGGTGGGGCCCTCTATGTCACCTGGCTCCATTGTCT +4233 WKY +4184 GTTCCGGAGCAGAAAGGTGGGGCCCTCTATGTCACCTGGCTCCATTGTCT +4233 RefSeq +4184 GTTCCGGAGCAGAAAGGTGGGGCCCTCTATGTCACCTGGCTCCATTGTCT +4233

SHR +4234 GTACTACTAAAGAGCTCAAGGCTCAGTCCCCCAAGGTCACAGTGAGTAGA +4283 WKY +4234 GTACTACTAAAGAGCTCAAGGCTCAGTCCCCCAAGGTCACAGTGAGTAGA +4283 RefSeq +4234 GTACTACTAAAGAGCTCAAGGCTCAGTCCCCCAAGGTCACAGTGAGTAGA +4283

SHR +4284 CCTTGTTAAGAGCAGACACAGCTCTTCGGACATTCCTCCGCCTCAGGTAG +4333 WKY +4284 CCTTGTTAAGAGCAGACACAGCTCTTCGGACATTCCTCCGCCTCAGGTAG +4333 RefSeq +4284 CCTTGTTAAGAGCAGACACAGCTCTTCGGACATTCCTCCGCCTCAGGTAG +4333

SHR +4334 TCATCCAGGAAGGCCCAGAGAGTTCTCTGCAGACCCAGAAGTGAATGAAC +4383 WKY +4334 TCATCCAGGAAGGCCCAGAGAGTTCTCTGCAGACCCAGAAGTGAATGAAC +4383 RefSeq +4334 TCATCCAGGAAGGCCCAGAGAGTTCTCTGCAGACCCAGAAGTGAATGAAC +4383

SHR +4384 CCACCCAGAGAGAAGGCTTTCGTTTCCTTACAGACTCTTTGTTCTCTGGG +4433 WKY +4384 CCACCCAGAGAGAAGGCTTTCGTTTCCTTACAGACTCTTTGTTCTCTGGG +4433 RefSeq +4384 CCACCCAGAGAGAAGGCTTTCGTTTCCTTACAGACTCTTTGTTCTCTGGG +4433

SHR +4434 TAATGGGGAGGCTAAACANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +4483 WKY +4434 TAATGGGGAGGCTAAACANNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +4483 RefSeq +4434 TAATGGGGAGGCTAAACACAGACCACATTATATTGTGTTTCAGACTGAGA +4483

181

SHR +4484 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +6083 WKY +4484 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +6083 RefSeq +4484 TGTAACTTGCCCCCA...... ATGAGGAGTCCAGGT +6083

SHR +6084 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGCTGCCTTGGAGACC +6133 WKY +6084 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTGCTGCCTTGGAGACC +6133 RefSeq +6084 GTACTATACGGTCTTAACGATTCCTTATTGTTCCTGCTGCCTTGGAGACC +6133

SHR +6134 TGTCTGTTCCATTTGGGCCTTTATTGCTCTCTAAGGTATAAGTAGAACAT +6183 WKY +6134 TGTCTGTTCCATTTGGGCCTTTATTGCTCTCTAAGGTATAAGTAGAACAT +6183 RefSeq +6134 TGTCTGTTCCATTTGGGCCTTTATTGCTCTCTAAGGTATACTCAGAACAT +6183

SHR +6184 TCTCTCATTGCCCACTGGGGACACTG--AGTCAGAGGGGTGGTGTGGCCT +6233 WKY +6184 TCTCTCATTGCCCACTGGGGACACTG--AGTCAGAGGGGTGGTGTGGCCT +6233 RefSeq +6184 TCTCTCATTGCCCACTGACTATTTTTTTATTCTATCGACTGGCGCGGCCT +6233

SHR +6234 GATGGGTAGAACTGAGAGCCTGCACCCTCCGGGGAGGCCTCTGGTGTCTT +6283 WKY +6234 GATGGGTAGAACTGAGAGCCTGCACCCTCCGGGGAGGCCTCTGGTGTCTT +6283 RefSeq +6234 GATGTATACAGCTTATAGCCCGCACCCTCCGGGGAGGCCTCTGGTGTCTT +6283

SHR +6284 GGACAGATATGTCAGGGCTTGGTCACATGGGACCTTCCC-TTGTCCCTTC +6333 WKY +6284 GGACAGATATGTCAGGGCTTGGTCACATGGGACCTTCCC-TTGTCCCTTC +6333 RefSeq +6284 GGACAGATATGTCAGGGCTTGGTCACATGGGACCTTCCCCTTGTCCCTTC +6333 |EXON 5  SHR +6334 CTGCTCCCAGGTGCCAAGGAGCGGGCCCAGCAGCAGCAGCAGCAGCAGCA +6383 WKY +6334 CTGCTCCCAGGTGCCAAGGAGCGGGCCCAGCAGCAGCAGCAGCAGCAGCA +6383 RefSeq +6334 CTGCTCCCAGGTGCCAAGGAGCGGGCCCAGCAGCAGCAGCAGCAGCAGCA +6383 GLN (codon = CAG, CAA) repeat polymorphism (+6361 to +6420) SHR +6384 GCA------ACAACAACAACACAGCAGCTTTG +6433 WKY +6384 GCAGCAGCAGCAGCAGCAGCAGCAGCAACAACAACAACACAGCAGCTTTG +6433 RefSeq +6384 GCAGCAGCAGCAGCAGCAGCAGCA---ACAACAGCAACACAGCAGCTTTG +6433

SHR +6434 AGGATGAACTCTCGGAAGTATTTGAGAACCAGAGCCCTGCAGCCAAGCAT +6483 WKY +6434 AGGATGAACTCTCGGAAGTATTTGAGAACCAGAGCCCTGCAGCCAAGCAT +6483 RefSeq +6434 AGGATGAACTCTCGGAAGTATTTGAGAACCAGAGCCCTGCAGCCAAGCAT +6483  EXON 5| SHR +6484 GGAGGTTAGTGTGGTCAGCTAGGAGGGGAGGGGGCTACAAGGAGAACAGT +6533 WKY +6484 GGAGGTTAGTGTGGTCAGCTAGGAGGGGAGGGGGCTACAAGGAGAACAGT +6533 RefSeq +6484 GGAGGTTAGTGTGGTCAGCTAGGAGGGGAGGGGGCTACAAGGAGAACAGT +6533

SHR +6534 GGTTCCCAGAGGATGCTGCTTTCAGAGCAAGAAGGTGGCTACCATTCCCT +6583 WKY +6534 GGTTCCCAGAGGATGCTGCTTTCAGAGCAAGAAGGTGGCTACCATTCCCT +6583 RefSeq +6534 GGTTCCCAGAGGATGCTGCTTTCAGAGCAAGAAGGTGGCTACCATTCCCT +6583 C+6587T SHR +6584 TCCTTACACAAGGGAAACTGAGGCCAAGAAAATTAAGTCTCCTGGGAATT +6633 WKY +6584 TCCCTACACAAGGGAAACTGAGGCCAAGAAAATTAAGTCTCCTGGGAATT +6633 RefSeq +6584 TCCCTACACAAGGGAAACTGAGGCCAAGAAAATTAAGTCTCCTGGGAATT +6633

SHR +6634 TGAATCTGGCTTTTATCCAAACCAAAGCCAGTGGCTTGTCTTGTACCAGC +6683 WKY +6634 TGAATCTGGCTTTTATCCAAACCAAAGCCAGTGGCTTGTCTTGTACCAGC +6683 RefSeq +6634 TGAATCTGGCTTTTATCCAAACCAAAGCCAGTGGCTTGTCTTGTACCAGC +6683

182

SHR +6684 GTCTCACTCCAGCAGCTGCAGTAATTCTGGACTAACCTCATGGCTCCTGC +6733 WKY +6684 GTCTCACTCCAGCAGCTGCAGTAATTCTGGACTAACCTCATGGCTCCTGC +6733 RefSeq +6684 GTCTCACTCCAGCAGCTGCAGTAATTCTGGACTAACCTCATGGCTCCTGC +6733

SHR +6734 CAACACAGACCCTGTTTCCCACTCCTGTCCACCTGGGACTCGATGCCATC +6783 WKY +6734 CAACACAGACCCTGTTTCCCACTCCTGTCCACCTGGGACTCGATGCCATC +6783 RefSeq +6734 CAACACAGACCCTGTTTCCCACTCCTGTCCACCTGGGACTCGATGCCATC +6783

SHR +6784 TTAGTTGAAGGTGTTGAGGGAGGGGTCAGAGGAGCGGGTTCTAAATACAA +6833 WKY +6784 TTAGTTGAAGGTGTTGAGGGAGGGGTCAGAGGAGCGGGTTCTAAATACAA +6833 RefSeq +6784 TTAGTTGAAGGTGTTGAGGGAGGGGTCAGAGGAGCGGGTTCTAAATACAA +6833

SHR +6834 AATGACCTGGGTCATCCACCATGAAGCTAAAGGGGTCATCCTTGGACAGC +6883 WKY +6834 AATGACCTGGGTCATCCACCATGAAGCTAAAGGGGTCATCCTTGGACAGC +6883 RefSeq +6834 AATGACCTGGGTCATCCACCATGAAGCTAAAGGGGTCATCCTTGGACAGC +6883

SHR +6884 TTCCTATTCTAGAAACACAACAAACTGCNNNNNNNNNNNNNNNNNNNNNN +6933 WKY +6884 TTCCTATTCTAGAAACACAACAAACTGCNNNNNNNNNNNNNNNNNNNNNN +6933 RefSeq +6884 TTCCTATTCTAGAAACACAACAAACTGCAAACTGGGATTTATTTTTCCTG +6933

SHR +6934 NNNNNNNNNNNNNNN...... NNNNTCAGGAGTCGC +7733 WKY +6934 NNNNNNNNNNNNNNN...... NNNNTCAGGAGTCGC +7733 RefSeq +6934 TCGAAAGCGGACAGA...... GGCCTCAGGAGTCGC +7733 |EXON 6  SHR +7734 CAAAGGCATTTGGGTCCCCTGCTTGACTCTATCCATTTGTGTTTGTAGAC +7783 WKY +7734 CAAAGGCATTTGGGTCCCCTGCTTGACTCTATCCATTTGTGTTTGTAGAC +7783 RefSeq +7734 CAAAGGCATTTGGGTCCCCTGCTTGACTCTATCCATTTGTGTTTGTAGAC +7783

SHR +7784 GCAGCATCAGAAGCCCCCTCCAAGGACACTGTAGAGAAGAGAGAGGATTC +7833 WKY +7784 GCAGCATCAGAAGCCCCCTCCAAGGACACTGTAGAGAAGAGAGAGGATTC +7833 RefSeq +7784 GCAGCATCAGAAGCCCCCTCCAAGGACACTGTAGAGAAGAGAGAGGATTC +7833

SHR +7834 TGACAAGGGGCAACAGGATGCCTTTGAGGGAACCACAGAAGGACCCAGAC +7883 WKY +7834 TGACAAGGGGCAACAGGATGCCTTTGAGGGAACCACAGAAGGACCCAGAC +7883 RefSeq +7834 TGACAAGGGGCAACAGGATGCCTTTGAGGGAACCACAGAAGGACCCAGAC +7883

SHR +7884 CTCAGGCCTTTCCAGAGCCCAAGCAGGAGTCCTCTATGATGGGAAACAGT +7933 WKY +7884 CTCAGGCCTTTCCAGAGCCCAAGCAGGAGTCCTCTATGATGGGAAACAGT +7933 RefSeq +7884 CTCAGGCCTTTCCAGAGCCCAAGCAGGAGTCCTCTATGATGGGAAACAGT +7933

SHR +7934 CAATCTCCAGGGGAGGACACAGCCAACAATACCCAATCACCAACCAGCCT +7983 WKY +7934 CAATCTCCAGGGGAGGACACAGCCAACAATACCCAATCACCAACCAGCCT +7983 RefSeq +7934 CAATCTCCAGGGGAGGACACAGCCAACAATACCCAATCACCAACCAGCCT +7983

SHR +7984 CCCCAGCCAGGAGCATGGGATTCCACAGACCACAGAAGGCAGTGAGAGAG +8033 WKY +7984 CCCCAGCCAGGAGCATGGGATTCCACAGACCACAGAAGGCAGTGAGAGAG +8033 RefSeq +7984 CCCCAGCCAGGAGCATGGGATTCCACAGACCACAGAAGGCAGTGAGAGAG +8033

SHR +8034 GTCCCAGTGCCCAGCAGCAAGCCAGAAAAGCCAAGCAAGAGGAGAAAGAG +8083 WKY +8034 GTCCCAGTGCCCAGCAGCAAGCCAGAAAAGCCAAGCAAGAGGAGAAAGAG +8083 RefSeq +8034 GTCCCAGTGCCCAGCAGCAAGCCAGAAAAGCCAAGCAAGAGGAGAAAGAG +8083 GLU (codon = GAG, CAA) repeat polymorphism (+8072 to +8128) SHR +8084 GAAGAGGAGGAG---AAAGAGGAAGAGGAGGAGGAGAAAGAGGAGAAGGC +8133 WKY +8084 GAAGAGGAGGAGGAGAAAGAGGAAGAGGAGGAGGAGAAAGAGGAGAAGGC +8133 RefSeq +8084 GAAGAGGAGGAGGAGAAAGAGGAAGAGGAGGAGGAGAAAGAGGAGAAGGC +8133

183

SHR +8134 GATCGCCAGAGAGAAGGCTGGGCCTAAAGAAGTCCCCACGGCAGCATCCA +8183 WKY +8134 GATCGCCAGAGAGAAGGCTGGGCCTAAAGAAGTCCCCACGGCAGCATCCA +8183 RefSeq +8134 GATCGCCAGAGAGAAGGCTGGGCCTAAAGAAGTCCCCACGGCAGCATCCA +8183  EXON 6| SHR +8184 GTTCTCACTTCTATTCAGGCTACAAGAAGATCCAGAAAGATGATGATGGT +8233 WKY +8184 GTTCTCACTTCTATTCAGGCTACAAGAAGATCCAGAAAGATGATGATGGT +8233 RefSeq +8184 GTTCTCACTTCTATTCAGGCTACAAGAAGATCCAGAAAGATGATGATGGT +8233

SHR +8234 ATGTATGGGGACAGGGACCTCAACTAATGTGTCTGGGAGATGGGTGGGTA +8283 WKY +8234 ATGTATGGGGACAGGGACCTCAACTAATGTGTCTGGGAGATGGGTGGGTA +8283 RefSeq +8234 ATGTATGGGGACAGGGACCTCAACTAATGTGTCTGGGAGATGGGTGGGTA +8283

SHR +8284 GGGACCTCATGCCTGCCACCTTCATAATGACCGTGAAAGATGAGTGGGGG +8333 WKY +8284 GGGACCTCATGCCTGCCACCTTCATAATGACCGTGAAAGATGAGTGGGGG +8333 RefSeq +8284 GGGACCTCATGCCTGCCACCTTCATAATGACCGTGAAAGATGAGTGGGGG +8333

SHR +8334 AACTGTCAGGGAGTGGGGGAACAAGAGGTTATATGATTTTCCCAAGGCTA +8383 WKY +8334 AACTGTCAGGGAGTGGGGGAACAAGAGGTTATATGATTTTCCCAAGGCTA +8383 RefSeq +8334 AACTGTCAGGGAGTGGGGGAACAAGAGGTTATATGATTTTCCCAAGGCTA +8383 A+8388G SHR +8384 CACAGCTAATTTAAGACAAGGTGCGGTTCCAAGCCCAGTTTATCTGACTC +8433 WKY +8384 CACAACTAATTTAAGACAAGGTGCGGTTCCAAGCCCAGTTTATCTGACTC +8433 RefSeq +8384 CACAACTAATTTAAGACAAGGTGCGGTTCCAAGCCCAGTTTATCTGACTC +8433

SHR +8434 CTGAACCACGTGTCAATCACTCTTCTACACCTGGGAGTAAGAGGAGCTTA +8483 WKY +8434 CTGAACCACGTGTCAATCACTCTTCTACACCTGGGAGTAAGAGGAGCTTA +8483 RefSeq +8434 CTGAACCACGTGTCAATCACTCTTCTACACCTGGGAGTAAGAGGAGCTTA +8483

SHR +8484 GGGGGAGGTGCTTGGAGAATGGAGAGATGACAGGGTGAGCTTCAGAGAGG +8533 WKY +8484 GGGGGAGGTGCTTGGAGAATGGAGAGATGACAGGGTGAGCTTCAGAGAGG +8533 RefSeq +8484 GGGGGAGGTGCTTGGAGAATGGAGAGATGACAGGGTGAGCTTCAGAGAGG +8533

SHR +8534 GGCTCTGCCAATTCCTTTGAGCTTGGGAGGGTCTTGCTCCATGCTCCTGG +8583 WKY +8534 GGCTCTGCCAATTCCTTTGAGCTTGGGAGGGTCTTGCTCCATGCTCCTGG +8583 RefSeq +8534 GGCTCTGCCAATTCCTTTGAGCTTGGGAGGGTCTTGCTCCATGCTCCTGG +8583

SHR +8584 CTGGACTCTGAATGGTGTGCTTGGCCTTAGAGGTAGCTGCACAAAGGACC +8633 WKY +8584 CTGGACTCTGAATGGTGTGCTTGGCCTTAGAGGTAGCTGCACAAAGGACC +8633 RefSeq +8584 CTGGACTCTGAATGGTGTGCTTGGCCTTAGAGGTAGCTGCACAAAGGACC +8633

SHR +8634 TTGAGGAGTGGCAGGGCCTGAGGGAATGGCAGTCCTTCCTCATTTCCTGT +8683 WKY +8634 TTGAGGAGTGGCAGGGCCTGAGGGAATGGCAGTCCTTCCTCATTTCCTGT +8683 RefSeq +8634 TTGAGGAGTGGCAGGGCCTGAGGGAATGGCAGTCCTTCCTCATTTCCTGT +8683 |EXON 7  SHR +8684 TCCCGCCACCACCTGCTCCAGGTCAGTCGGAGTCTCAGGCAGTGAATGGA +8733 WKY +8684 TCCCGCCACCACCTGCTCCAGGTCAGTCGGAGTCTCAGGCAGTGAATGGA +8733 RefSeq +8684 TCCCGCCACCACCTGCTCCAGGTCAGTCGGAGTCTCAGGCAGTGAATGGA +8733

SHR +8734 AAGACAGGGGCTTCTGAAGCTGTGCCATCTGAAGGGAAGGGGGAGCTGGA +8783 WKY +8734 AAGACAGGGGCTTCTGAAGCTGTGCCATCTGAAGGGAAGGGGGAGCTGGA +8783 RefSeq +8734 AAGACAGGGGCTTCTGAAGCTGTGNNNNNNNNNNNNNNNNNNNNNNNNNN +8783

184

SHR +8784 GCACTCTCAGCAGGAGGAAGATGGGGAAGAGGCCATGGCAGGGCCCCCCC +8833 WKY +8784 GCACTCTCAGCAGGAGGAAGATGGGGAAGAGGCCATGGCAGGGCCCCCCC +8833 RefSeq +8784 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +8833

SHR +8834 AAGGTCTCTTCCCAGGTGGGAAGGGCCAGGAGCTGGAACGTAAGCAGCAG +8883 WKY +8834 AAGGTCTCTTCCCAGGTGGGAAGGGCCAGGAGCTGGAACGTAAGCAGCAG +8883 RefSeq +8834 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +8883

SHR +8884 GAGGAGGAAGAGGAAGAGGAGCGTCTGTCCAGAGAATGGGAGGACAAGCG +8933 WKY +8884 GAGGAGGAAGAGGAAGAGGAGCGTCTGTCCAGAGAATGGGAGGACAAGCG +8933 RefSeq +8884 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +8933

SHR +8934 ATGGAGCAGGATGGACCAGCTGGCCAAGGAGCTGACAGCAGAGAAGCGGC +8983 WKY +8934 ATGGAGCAGGATGGACCAGCTGGCCAAGGAGCTGACAGCAGAGAAGCGGC +8983 RefSeq +8934 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +8983

SHR +8984 TGGAGGGGGAAGATGACCCTGACCGGTCCATGAAGCTCTCCTTCCGGGCC +9033 WKY +8984 TGGAGGGGGAAGATGACCCTGACCGGTCCATGAAGCTCTCCTTCCGGGCC +9033 RefSeq +8984 NNNNNNNGGAAGATGACC-TGACCGGTCCATGAAGCTCTCCTTCCGGGCC +9033

SHR +9034 CGGGCCTATGGCTTCAGGGACCCTGGGCCT-CAGCTACGCCG-GGGCTGG +9083 WKY +9034 CGGGCCTATGGCTTCAGGGACCCTGGGCCT-CAGCTACGCCG-GGGCTGG +9083 RefSeq +9034 CGGGC-TATGGCTTCAGGGACCCTGGGCCTACAGCTACGCCGAGGGCTGG +9083

SHR +9084 AGGCCATCTTCCAGAGAAGACAGTGTGGAGGCCCGAGGCGACTTTGAGGA +9133 WKY +9084 AGGCCATCTTCCAGAGAAGACAGTGTGGAGGCCCGAGGCGACTTTGAGGA +9133 RefSeq +9084 AGGCCATCTTCCAGAGAAGACAGTGTGGAGGCCCGAGGCGACTTTGAGGA +9133  EXON 7| SHR +9134 AAAGAAGGAAGAGGAGGGCAGCGCCAACCGCAGAGCAGAGGTTGGTATAG +9183 WKY +9134 AAAGAAGGAAGAGGAGGGCAGCGCCAACCGCAGAGCAGAGGTTGGTATAG +9183 RefSeq +9134 AAAGAAGGAAGAGGAGGGCAGCGCCAACCGCAGAGCAGAGGTTGGTATAG +9183

SHR +9184 GCAAGGGTAGCTGCGCCCAGCAGCCCCTCCCCCACTGAGCTGACATGGTT +9233 WKY +9184 GCAAGGGTAGCTGCGCCCAGCAGCCCCTCCCCCACTGAGCTGACATGGTT +9233 RefSeq +9184 GCAAGGGTAGCTGCGCCCAGCAGCCCCTCCCCCACTGAGCTGACATGGTT +9233

SHR +9234 CCTTAGATGGGTCCTGGAGAGGTGCTCAGATGGGCTTGACATAGGGGAAC +9283 WKY +9234 CCTTAGATGGGTCCTGGAGAGGTGCTCAGATGGGCTTGACATAGGGGAAC +9283 RefSeq +9234 CCTTAGATGGGTCCTGGAGAGGTGCTCAGATGGGCTTGACATAGGGGAAC +9283

SHR +9284 AGCCGGCTTATAGCAGGGGCTCTACAGAGTAACTGGGTCAGGTGGACAGA +9333 WKY +9284 AGCCGGCTTATAGCAGGGGCTCTACAGAGTAACTGGGTCAGGTGGACAGA +9333 RefSeq +9284 AGCCGGCTTATAGCAGGGGCTCTACAGAGTAACTGGGTCAGGTGGACAGA +9333

SHR +9334 CCTGCCTGAGGGGCATGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +9383 WKY +9334 CCTGCCTGAGGGGCATGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +9383 RefSeq +9334 CCTGCCTGAGGGGCATGGAGTGAGGACATTGCCTCTCCTGGTATGTAAGC +9383

SHR +9384 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +10783 WKY +9384 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +10783 RefSeq +9384 AGGACATGGGGTGAT...... CTGTGATGTGGCCAA +10783

185

SHR +10784 NNNNNNNNNNNNNNNNNGGCAAAGCTATTCTTCTGAGGCTAACAGCTAAA +10833 WKY +10784 NNNNNNNNNNNNNNNNNGGCAAAGCTATTCTTCTGAGGCTAACAGCTAAA +10833 RefSeq +10784 CTCCCCTGGGGCTCAGAGGCAAAGCTATTCTTCTGAGGCTAACAGCTAAA +10833 G+10882A SHR +10834 AAACATCAGGGCCAGCCTGGCACACTGTACCATCGACGCTTTTAACATAG +10883 WKY +10834 AAACATCAGGGCCAGCCTGGCACACTGTACCATCGACGCTTTTAACATGG +10883 RefSeq +10834 AAACATCAGGGCCAGCCTGGCACACTGTACCATCGACGCTTTTAACATGG +10883 |EXON 8  SHR +10884 GCCAGCCCCCAAGTGACCCTGTCTGTTCCCTCCTAGGACCAGGAGCTAGA +10933 WKY +10884 GCCAGCCCCCAAGTGACCCTGTCTGTTCCCTCCTAGGACCAGGAGCTAGA +10933 RefSeq +10884 GCCAGCCCCCAAGTGACCCTGTCTGTTCCCTCCTAGGACCAGGAGCTAGA +10933

SHR +10934 GAGCCTGTCAGCCATCGAGGCAGAGCTGGAGAAGGTGGCCCACCAGCTGC +10983 WKY +10934 GAGCCTGTCAGCCATCGAGGCAGAGCTGGAGAAGGTGGCCCACCAGCTGC +10983 RefSeq +10934 GAGCCTGTCAGCCATCGAGGCAGAGCTGGAGAAGGTGGCCCACCAGCTGC +10983 |3’UTR  SHR +10984 AGGCTTTGCGACGGGGATGAggcactggctggtggggtccggccatggct +11033 WKY +10984 AGGCTTTGCGACGGGGATGAggcactggctggtggggtccggccatggct +11033 RefSeq +10984 AGGCTTTGCGACGGGGATGAggcactggctggtggggtccggccatggct +11033

SHR +11034 tcaaggcccaactgctgctcaagtagggaggcttccagcctcagagccca +11083 WKY +11034 tcaaggcccaactgctgctcaagtagggaggcttccagcctcagagccca +11083 RefSeq +11034 tcaaggcccaactgctgctcaagtagggaggcttccagcctcagagccca +11083

SHR +11084 ggttaccccttgccccttcccttctcttgctctcagcccctgccctggac +11133 WKY +11084 ggttaccccttgccccttcccttctcttgctctcagcccctgccctggac +11133 RefSeq +11084 ggttaccccttgccccttcccttctcttgctctcagcccctgccctggac +11133 G+11177T SHR +11134 acttctgcagggcagccctgaaagtcaacacagattctttctctgaacac +11183 WKY +11134 acttctgcagggcagccctgaaagtcaacacagattctttctcggaacac +11183 RefSeq +11134 acttctgcagggcagccctgaaagtcaacacagattctttctcggaacac +11183

SHR +11184 aggcagctttctagaagtttctcttccaccttctaatccatggggcacaa +11233 WKY +11184 aggcagctttctagaagtttctcttccaccttctaatccatggggcacaa +11233 RefSeq +11184 aggcagctttctagaagtttctcttccaccttctaatccatggggcacaa +11233

SHR +11234 ctgcagtagttctgatctttgggcgaaagccgagaactcctgactattaa +11283 WKY +11234 ctgcagtagttctgatctttgggcgaaagccgagaactcctgactattaa +11283 RefSeq +11234 ctgcagtagttctgatctttgggcgaaagccgagaactcctgactattaa +11283

SHR +11284 gatattccagataaaatttattgaaggaagaataaacctgctttgggctc +11333 WKY +11284 gatattccagataaaatttattgaaggaagaataaacctgctttgggctc +11333 RefSeq +11284 gatattccagataaaatttattgaaggaagaataaacctgctttgggctc +11333  EXON 8| SHR +11334 ttcACTTTTCTTTGATTATTATTTTTCATTCTTTTTTACTCTGAGTTGGG +11383 WKY +11334 ttcACTTTTCTTTGATTATTATTTTTCATTCTTTTTTACTCTGAGTTGGG +11383 RefSeq +11334 ttcACTTTTCTTTGATTATTATTTTTCATTCTTTTTTACTCTGAGTTGGG +11383

SHR +11384 TCAGCCTGGGTCTGAGGGGCAGGTTGATTTCTACACTGGGCTGGGTGTGT +11433 WKY +11384 TCAGCCTGGGTCTGAGGGGCAGGTTGATTTCTACACTGGGCTGGGTGTGT +11433 RefSeq +11384 TCAGCCTGGGTCTGAGGGGCAGGTTGATTTCTACACTGGGCTGGGTGTGT +11433

SHR +11434 GGTTTGGGAGACTCCGTTCTGGGCCATTTCTGGGGCAAGCGATTTTGCAT +11483 WKY +11434 GGTTTGGGAGACTCCGTTCTGGGCCATTTCTGGGGCAAGCGATTTTGCAT +11483 RefSeq +11434 GGTTTGGGAGACTCCGTTCTGGGCCATTTCTGGGGCAAGCGATTTTGCAT +11483

186

SHR +11484 TGTGGAACCTCAGTTTCATCCGTAGATGGAGGCACTGAGCTGCCCAGAGT +11533 WKY +11484 TGTGGAACCTCAGTTTCATCCGTAGATGGAGGCACTGAGCTGCCCAGAGT +11533 RefSeq +11484 TGTGGAACCTCAGTTTCATCCGTAGATGGAGGCACTGAGCTGCCCAGAGT +11533

SHR +11534 GGGGGTGGAGCTGAACACCACCACAGGGGACTTCACCCCCTGCTTGCTTA +11583 WKY +11534 GGGGGTGGAGCTGAACACCACCACAGGGGACTTCACCCCCTGCTTGCTTA +11583 RefSeq +11534 GGGGGTGGAGCTGAACACCACCACAGGGGACTTCACCCCCTGCTTGCTTA +11583

SHR +11584 TCTCCCAGCCATGCCTGAGTCAGGTACTTTCTGCTGAGAATGTCCTCCTG +11633 WKY +11584 TCTCCCAGCCATGCCTGAGTCAGGTACTTTCTGCTGAGAATGTCCTCCTG +11633 RefSeq +11584 TCTCCCAGCCATGCCTGAGTCAGGTACTTTCTGCTGAGAATGTCCTCCTG +11633

SHR +11634 GACTGTCCCCTAGTTACCTCCATGCTGGGTAGAGGCGGACAGGCTCTAGG +11683 WKY +11634 GACTGTCCCCTAGTTACCTCCATGCTGGGTAGAGGCGGACAGGCTCTAGG +11683 RefSeq +11634 GACTGTCCCCTAGTTACCTCCATGCTGGGTAGAGGCGGACAGGCTCTAGG +11683

SHR +11684 ATAATACCTCCCATTGGCCATAGGCTGGCCTTGCTGGACTCCCTAGCATA +11733 WKY +11684 ATAATACCTCCCATTGGCCATAGGCTGGCCTTGCTGGACTCCCTAGCATA +11733 RefSeq +11684 ATAATACCTCCCATTGGCCATAGGCTGGCCTTGCTGGACTCCCTAGCATA +11733

SHR +11734 GCTCCTTGCTGCTTAAGTCCACAGTGCCCATGACCACAGGCTTGTGATGT +11783 WKY +11734 GCTCCTTGCTGCTTAAGTCCACAGTGCCCATGACCACAGGCTTGTGATGT +11783 RefSeq +11734 GCTCCTTGCTGCTTAAGTCCACAGTGCCCATGACCACAGGCTTGTGATGT +11783

SHR +11784 GCGTGGGACCGCAGGGTATCTGAGGGGTGGGGCAGGAACGATTTATTGAA +11833 WKY +11784 GCGTGGGACCGCAGGGTATCTGAGGGGTGGGGCAGGAACGATTTATTGAA +11833 RefSeq +11784 GCGTGGGACCGCAGGGTATCTGAGGGGTGGGGCAGGAACGATTTATTGAA +11833

SHR +11834 AACAGCAGGTCTTAGGCCCAATTTGGAAAAGAGGGAAACAGGCAGAGAAG +11883 WKY +11834 AACAGCAGGTCTTAGGCCCAATTTGGAAAAGAGGGAAACAGGCAGAGAAG +11883 RefSeq +11834 AACAGCAGGTCTTAGGCCCAATTTGGAAAAGAGGGAAACAGGCAGAGAAG +11883

SHR +11884 ATAAAAAGGCGCATTGAAGTCACAGGTAGAGGGTACATTCCCTTCCCATC +11933 WKY +11884 ATAAAAAGGCGCATTGAAGTCACAGGTAGAGGGTACATTCCCTTCCCATC +11933 RefSeq +11884 ATAAAAAGGCGCATTGAAGTCACAGGTAGAGGGTACATTCCCTTCCCATC +11933

SHR +11934 CCATTCCCCTTTTTGGTTTTAAGACAGGGCTTTTTTGTCTAGACTTGGAT +11983 WKY +11934 CCATTCCCCTTTTTGGTTTTAAGACAGGGCTTTTTTGTCTAGACTTGGAT +11983 RefSeq +11934 CCATTCCCCTTTTTGGTTTTAAGACAGGGCTTTTTTGTCTAGACTTGGAT +11983

SHR +11984 GTCCTGGAACTAGCTGGTCTACAACCCAGAGATCCATCCACCTACCCGCC +12033 WKY +11984 GTCCTGGAACTAGCTGGTCTACAACCCAGAGATCCATCCACCTACCCGCC +12033 RefSeq +11984 GTCCTGGAACTAGCTGGTCTACAACCCAGAGATCCATCCACCTACCCGCC +12033

SHR +12034 TGAGCTGAGGCACACACCACCATAGCCCTACTGACACACCACCCAACTTT +12083 WKY +12034 TGAGCTGAGGCACACACCACCATAGCCCTACTGACACACCACCCAACTTT +12083 RefSeq +12034 TGAGCTGAGGCACACACCACCATAGCCCTACTGACACACCACCCAACTTT +12083

SHR +12084 CCTTTTTAGATGTATTCATTTTATTTTGTGTGTTTTGCCTGCATGCATTT +12133 WKY +12084 CCTTTTTAGATGTATTCATTTTATTTTGTGTGTTTTGCCTGCATGCATTT +12133 RefSeq +12084 CCTTTTTAGATGTATTCATTTTATTTTGTGTGTTTTGCCTGCATGCATTT +12133

187

SHR +12134 CTGTGCACCATATGCTGTCTGGTGCCCTTGGATCCCCTGGAACAGGAGTT +12183 WKY +12134 CTGTGCACCATATGCTGTCTGGTGCCCTTGGATCCCCTGGAACAGGAGTT +12183 RefSeq +12134 CTGTGCACCATATGCTGTCTGGTGCCCTTGGATCCCCTGGAACAGGAGTT +12183

SHR +12184 AGGTTTATGTGTAGTTCTGCTGGGTTCAGTTTTAGGCAAACTACCTCATC +12233 WKY +12184 AGGTTTATGTGTAGTTCTGCTGGGTTCAGTTTTAGGCAAACTACCTCATC +12233 RefSeq +12184 AGGTTTATGTGTAGTTCTGCTGGGTTCAGTTTTAGGCAAACTACCTCATC +12233

SHR +12234 CTGTTCTCCGCCTGTGCACCTCAAACTAAAAGCCAGGAGATGGTGATGCT +12283 WKY +12234 CTGTTCTCCGCCTGTGCACCTCAAACTAAAAGCCAGGAGATGGTGATGCT +12283 RefSeq +12234 CTGTTCTCCGCCTGTGCACCTCAAACTAAAAGCCAGGAGATGGTGATGCT +12283

SHR +12284 AAACGTGTGGGGATGGCCTTAGTCTCAGCAGTCAGCCTGTGCAAGGCTCT +12333 WKY +12284 AAACGTGTGGGGATGGCCTTAGTCTCAGCAGTCAGCCTGTGCAAGGCTCT +12333 RefSeq +12284 AAACGTGTGGGGATGGCCTTAGTCTCAGCAGTCAGCCTGTGCAAGGCTCT +12333 T+12339C SHR +12334 CCTGCCGGGAAAAGCATGCACACTGACTTGAGAGACAGACATTGCTGCTT +12383 WKY +12334 CCTGCTGGGAAAAGCATGCACACTGACTTGAGAGACAGACATTGCTGCTT +12383 RefSeq +12334 CCTGCCGGGAAAAGCATGCACACTGACTTGAGAGACAGACATTGCTGCTT +12383

SHR +12384 CCCCAGGCAGAACCAGCTCTATAGAGTTCCAGCTGTTTTGAGGGCTGGGT +12433 WKY +12384 CCCCAGGCAGAACCAGCTCTATAGAGTTCCAGCTGTTTTGAGGGCTGGGT +12433 RefSeq +12384 CCCCAGGCAGAACCAGCTCTATAGAGTTCCAGCTGTTTTGAGGGCTGGGT +12433

SHR +12434 CCCAAGAGTCCTCGTCTCCAATGTGCCTCAGACCCCCCATGGAGCACTAC +12483 WKY +12434 CCCAAGAGTCCTCGTCTCCAATGTGCCTCAGACCCCCCATGGAGCACTAC +12483 RefSeq +12434 CCCAAGAGTCCTCGTCTCCAATGTGCCTCAGACCCCCCATGGAGCACTAC +12483

SHR +12484 CTGGAGTGCTTGGCCAGCTTTTGCTCTCCCAGAGCCTCAGTTTCCCAATC +12533 WKY +12484 CTGGAGTGCTTGGCCAGCTTTTGCTCTCCCAGAGCCTCAGTTTCCCAATC +12533 RefSeq +12484 CTGGAGTGCTTGGCCAGCTTTTGCTCTCCCAGAGCCTCAGTTTCCCAATC +12533

SHR +12534 TGGAATACTTACCTCTCTACTGAGGTAACTGTAAGGTTTGACAGAACCAG +12583 WKY +12534 TGGAATACTTACCTCTCTACTGAGGTAACTGTAAGGTTTGACAGAACCAG +12583 RefSeq +12534 TGGAATACTTACCTCTCTACTGAGGTAACTGTAAGGTTTGACAGAACCAG +12583 G+12611T SHR +12584 ACAGCACAGACTAAGAGCTACAGCAGTTCGAGACATGGTAGGATAGTGGT +12633 WKY +12584 ACAGCACAGACTAAGAGCTACAGCAGTGCGAGACATGGTAGGATAGTGGT +12633 RefSeq +12584 ACAGCACAGACTAAGAGCTACAGCAGTGCGAGACATGGTAGGATAGTGGT +12633

SHR +12634 GGGGTGGGAGGTCTTAGCCTTAACTTGTGAGACCTGGCTTCCTGCTGCAG +12683 WKY +12634 GGGGTGGGAGGTCTTAGCCTTAACTTGTGAGACCTGGCTTCCTGCTGCAG +12683 RefSeq +12634 GGGGTGGGAGGTCTTAGCCTTAACTTGTGAGACCTGGCTTCCTGCTGCAG +12683

SHR +12684 TGCAGCCTGCACTGAAGTGGAGACACCTGTATGACGTCACCGCCGAGGCG +12733 WKY +12684 TGCAGCCTGCACTGAAGTGGAGACACCTGTATGACGTCACCGCCGAGGCG +12733 RefSeq +12684 TGCAGCCTGCACTGAAGTGGAGACACCTGTATGACGTCACCGCCGAGGCG +12733

SHR +12734 TGGTTCTTCACGTTATACCTCCTGATAGTTATATAATTATGCAAACTCCC +12783 WKY +12734 TGGTTCTTCACGTTATACCTCCTGATAGTTATATAATTATGCAAACTCCC +12783 RefSeq +12734 TGGTTCTTCACGTTATACCTCCTGATAGTTATATAATTATGCAAACTCCC +12783

SHR +12784 ATCAGGCCTCCAGAGACCAAGAACACCCACCAACCAAGACTAAGCAGCAA +12833 WKY +12784 ATCAGGCCTCCAGAGACCAAGAACACCCACCAACCAAGACTAAGCAGCAA +12833 RefSeq +12784 ATCAGGCCTCCAGAGACCAAGAACACCCACCAACCAAGACTAAGCAGCAA +12833

188

SHR +12834 CAGACCACAGGGGCACTGTGTCATCAAAGGACCCCATCCCAGACACAAGC +12883 WKY +12834 CAGACCACAGGGGCACTGTGTCATCAAAGGACCCCATCCCAGACACAAGC +12883 RefSeq +12834 CAGACCACAGGGGCACTGTGTCATCAAAGGACCCCATCCCAGACACAAGC +12883

SHR +12884 CACCTGCATTGTATCATGGGAAGGAAAGATGCAGCCCAGGTGGGCGCCGA +12933 WKY +12884 CACCTGCATTGTATCATGGGAAGGAAAGATGCAGCCCAGGTGGGCGCCGA +12933 RefSeq +12884 CACCTGCATTGTATCATGGGAAGGAAAGATGCAGCCCAGGTGGGCGCCGA +12933

SHR +12934 CAGACGGCTTCGTAGGCAGTAGAGGGCCAAGGACACTTGGTGTACAGATG +12983 WKY +12934 CAGACGGCTTCGTAGGCAGTAGAGGGCCAAGGACACTTGGTGTACAGATG +12983 RefSeq +12934 CAGACGGCTTCGTAGGCAGTAGAGGGCCAAGGACACTTGGTGTACAGATG +12983

SHR +12984 TGCACAAGATACATGGGGAGGCAAGCAGCAGCCATCGC +13033 WKY +12984 TGCACAAGATACATGGGGAGGCAAGCAGCAGCCATCGC +13033 RefSeq +12984 TGCACAAGATACATGGGGAGGCAAGCAGCAGCCATCGC +13033

189

Dopamine beta-hydroxylase (Dbh)

Data not available: unpublished data from a collaborator.

190

Electron-transferring-flavoprotein dehydrogenase (Etfdh)

SHR -1459 ATCAGAGAACTGATATTCTGAGCTGAGCTGACAGAACATCTAACATCACA -1410 WKY -1459 ATCAGAGAACTGATATTCTGAGCTGAGCTGACAGAACATCTAACATCACA -1410 RefSeq -1459 ATCAGAGAACTGATATTCTGAGCTGAGCTGACAGAACATCTAACATCACA -1410

SHR -1409 CACAGCTATGAAAAACTCACTTTTAAATCAAATGTTTTCCTCACCAAAAA -1360 WKY -1409 CACAGCTATGAAAAACTCACTTTTAAATCAAATGTTTTCCTCACCAAAAA -1360 RefSeq -1409 CACAGCTATGAAAAACTCACTTTTAAATCAAATGTTTTCCTCACCAAAAA -1360

SHR -1359 TATATAAAGTAATACTTTTTTATATCACATCAGCATTTATTCTACACATT -1310 WKY -1359 TATATAAAGTAATACTTTTTTATATCACATCAGCATTTATTCTACACATT -1310 RefSeq -1359 TATATAAAGTAACACTTTTTTATATCACATCAGCATTTATTCTACACATT -1310

SHR -1309 TATCTGCAACCAGTATTGTTTTGGGAAAAAAAATTAAAAACCTGGACCAG -1260 WKY -1309 TATCTGCAACCAGTATTGTTTTGGGAAAAAAAATTAAAAACCTGGACCAG -1260 RefSeq -1309 TATCTGCAACCAGTATTGTTTTGGGAAAAAAAATTAAAAACCTGGACCAG -1260

SHR -1259 CACTTTAGCCATCTCTTTTGATGAAACAGTAAAAACCTGTCAACCAGTTC -1210 WKY -1259 CACTTTAGCCATCTCTTTTGATGAAACAGTAAAAACCTGTCAACCAGTTC -1210 RefSeq -1259 CACTTTAGCCATCTCTTTTGATGAAACAGTAAAAACCTGTCAACCAGTTC -1210

SHR -1209 TAGTTTCCAATCTTTAGTGTGGTTATTCCACAACGCTCTCCCAGAAAGTT -1160 WKY -1209 TAGTTTCCAATCTTTAGTGTGGTTATTCCACAACGCTCTCCCAGAAAGTT -1160 RefSeq -1209 TAGTTTCCAATCTTTAGTGTGGTTATTCCACAACGCTCTCCCAGAAAGTT -1160

SHR -1159 TGTCCGCTACATACTTCATTCCTCTCTCCCTCTCAAGAGTCCTTATAAAG -1110 WKY -1159 TGTCCGCTACATACTTCATTCCTCTCTCCCTCTCAAGAGTCCTTATAAAG -1110 RefSeq -1159 TGTCCGCTACATACTTCATTCCTCTCTCCCTCTCAAGAGTCCTTATAAAG -1110

SHR -1109 TGACGCTGTGACCCTCCAACCACCTCAGTGTCATATGTGTTGGGAGTCAG -1060 WKY -1109 TGACGCTGTGACCCTCCAACCACCTCAGTGTCATATGTGTTGGGAGTCAG -1060 RefSeq -1109 TGACGCTGTGACCCTCCAACCACCTCAGTGTCATATGTGTTGGGAGTCAG -1060

SHR -1059 GAAGTGATTTTTGGGAATTCGAGATTGAAAAAAAAA-TCCCGTAATTTTT -1010 WKY -1059 GAAGTGATTTTTGGGAATTCGAGATTGAAAAAAAAA-TCCCGTAATTTTT -1010 RefSeq -1059 GAAGTGATTTTTGGGAATTCGAGATTGAAAAAAAAAATCCCGTAATTTTT -1010

SHR -1009 GCATTACAGGGATGTCTAATCTGTTCTGTCTAACCATCAGTGAAACCAAT -960 WKY -1009 GCATTACAGGGATGTCTAATCTGTTCTGTCTAACCATCAGTGAAACCAAT -960 RefSeq -1009 GCATTACAGGGATGTCTAATCTGTTCTGTCTAACCATCAGTGAAACCAAT -960

SHR -959 GACCTCGGCTGCCGGCTGTGATTAGCCCTGGTCCACTCAAGTCCAACACA -910 WKY -959 GACCTCGGCTGCCGGCTGTGATTAGCCCTGGTCCACTCAAGTCCAACACA -910 RefSeq -959 GACCTCGGCTGCCGGCTGTGATTAGCCCTGGTCCACTCAAGTCCAACACA -910

SHR -909 GAGCAAGTATGCGACACACAATAAATAGCATCACCAGCTAGCCCTGGGCT -860 WKY -909 GAGCAAGTATGCGACACACAATAAATAGCATCACCAGCTAGCCCTGGGCT -860 RefSeq -909 GAGCAAGTATGCGACACACAATAAATAGCATCACCAGCTAGCCCTGGGCT -860

SHR -859 GATGACCAAGAAGCGCAGGCTCAAGTCCCTGCTCAAAATGTTCCGAAGGA -810 WKY -859 GATGACCAAGAAGCGCAGGCTCAAGTCCCTGCTCAAAATGTTCCGAAGGA -810 RefSeq -859 GATGACCAAGAAGCGCAGGCTCAAGTCCCTGCTCAAAATGTTCCGAAGGA -810

191

SHR -809 TGGAAGGAGGCGGAGAGAGGATCCCGGTAGCGGTGCTGTGGGTGACTGCA -760 WKY -809 TGGAAGGAGGCGGAGAGAGGATCCCGGTAGCGGTGCTGTGGGTGACTGCA -760 RefSeq -809 TGGAAGGAGGCGGAGAGAGGATCCCGGTAGCGGTGCTGTGGGTGACTGCA -760

SHR -759 AGCAGAAGAAGGAAAGAAAGATGCGGACGGAGAGAGCCGAGGCTAGCCCT -710 WKY -759 AGCAGAAGAAGGAAAGAAAGATGCGGACGGAGAGAGCCGAGGCTAGCCCT -710 RefSeq -759 AGCAGAAGAAGGAAAGAAAGATGCGGACGGAGAGAGCCGAGGCTAGCCCT -710

SHR -709 CAACTCCAAGAACTGCAGGCAGGAGCACCCTAGCTGCAAGCAGCTCCCGG -660 WKY -709 CAACTCCAAGAACTGCAGGCAGGAGCACCCTAGCTGCAAGCAGCTCCCGG -660 RefSeq -709 CAACTCCAAGAACTGCAGGCAGGAGCACCCTAGCTGCAAGCAGCTCCCGG -660

SHR -659 CCGCAGCGGCCACACTCACGTCTCCGATCTGCAGCTCCACTTGCTCCAGC -610 WKY -659 CCGCAGCGGCCACACTCACGTCTCCGATCTGCAGCTCCACTTGCTCCAGC -610 RefSeq -659 CCGCAGCGGCCACACTCACATCTCCGATCTGCAGCTCCACTTGCTCCAGC -610

SHR -609 GGGTCCACCGTTGGACCGTGGCTCCGGCTCGGACCCCGCCAGCCCAAACT -560 WKY -609 GGGTCCACCGTTGGACCGTGGCTCCGGCTCGGACCCCGCCAGCCCAAACT -560 RefSeq -609 GGGTCCACCGTTGGACCGTGGCTCCGGCTCGGACCCCGCCAGCCCAAACT -560

SHR -559 CACTGGCATGCTGAGGGACGACGAAGAGGCGCCTGACGACGGAGACGAGG -510 WKY -559 CACTGGCATGCTGAGGGACGACGAAGAGGCGCCTGACGACGGAGACGAGG -510 RefSeq -559 CACTGGCATGCTGAGGGACGATGAAGAGGCGCCTGACGACGGAGACGAGG -510

SHR -509 GAGGCGGCGGCGGTGGCGAAGAAGCCCGAAACTCCTCAGAATCAGCCATG -460 WKY -509 GAGGCGGCGGCGGTGGCGAAGAAGCCCGAAACTCCTCAGAATCAGCCATG -460 RefSeq -509 GAGGCGGCGGCGGTGGCGAAGAAGCCCGAAACTCCTCAGAATCAGCCATG -460

SHR -459 GGACTGAGCTGGTACCGCCGGACAGGACCGAGCAACCGAACCCTCACCAA -410 WKY -459 GGACTGAGCTGGTACCGCCGGACAGGACCGAGCAACCGAACCCTCACCAA -410 RefSeq -459 GGACTGAGCTGGTACCGCCGGACAGGACCGAGCAACCGAACCCTCACCAA -410

SHR -409 CTGCCTTTCGACCGCTCGCGCCCAAAGCCCACAGGCTCCGCCTCCTCCCA -360 WKY -409 CTGCCTTTCGACCGCTCGCGCCCAAAGCCCACAGGCTCCGCCTCCTCCCA -360 RefSeq -409 CTGCCTTTCGACCGCTCGCGCCCAAAGCCCACAGGCTCCGCCTCCTCCCA -360

SHR -359 CACGGAGTCCCGCCCCATCTTCTCGTTTGTTTCTGCCCTGGACCACAAGT -310 WKY -359 CACGGAGTCCCGCCCCATCTTCTCGTTTGTTTCTGCCCTGGACCACAAGT -310 RefSeq -359 CACGGAGTCCCGCCCCATCTTCTCGTTTGTTTCTGCCCTGGACCACAAGT -310

SHR -309 ACTTTTCATTTTCCCAGACAGCCAATCAGATATCAAGTCAGGCACTCCTT -260 WKY -309 ACTTTTCATTTTCCCAGACAGCCAATCAGATATCAAGTCAGGCACTCCTT -260 RefSeq -309 ACTTTTCATTTTCCCAGACAGCCAATCAGATATCAAGTCAGGCACTCCTT -260

SHR -259 GCGCACACCTCGGGTCCGGATCGGGAGACACTGGAGCTGAACACTACAAC -210 WKY -259 GCGCACACCTCGGGTCCGGATCGGGAGACACTGGAGCTGAACACTACAAC -210 RefSeq -259 GCGCACACCTCGGGTCCGGATCGGGAGACACTGGAGCTGAACACTACAAC -210

SHR -209 TCCCAGAATGCTGAGCGACTCATCCCCGCTATGCTTCTAGTGCGCACGCG -160 WKY -209 TCCCAGAATGCTGAGCGACTCATCCCCGCTATGCTTCTAGTGCGCACGCG -160 RefSeq -209 TCCCAGAATGCTGAGCGACTCATCCCCGCTATGCTTCTAGTGCGCACGCG -160

192

SHR -159 CACATGCGAGACCGTGTGGGCCTCTTCCGTACACTAGGTTCCTGGATGAG -110 WKY -159 CACATGCGAGACCGTGTGGGCCTCTTCCGTACACTAGGTTCCTGGATGAG -110 RefSeq -159 CACATGCGAGACCGTGTGGGCCTCTTCCGTACACTAGGTTCCTGGATGAG -110

SHR -109 GATGACTGGGAGATGGAGTCTTCTGCCTTCTCCTCGCTTGTACGTGACCC -60 WKY -109 GATGACTGGGAGATGGAGTCTTCTGCCTTCTCCTCGCTTGTACGTGACCC -60 RefSeq -109 GATGACTGGGAGATGGAGTCTTCTGCCTTCTCCTCGCTTGTACGTGACCC -60

SHR -59 GGAAACCTTACTGGGTATCGTGTCCTCTGTGGCGCCGGCGCAAAGCAGAA -10 WKY -59 GGAAACCTTACTGGGTATCGTGTCCTCTGTGGCGCCGGCGCAAAGCAGAA -10 RefSeq -59 GGAAACCTTACTGGGTATCGTGTCCTCTGTGGCGCCGGCGCAAAGCAGAA -10  -1|+1  SHR -9 GAGGGGCGGgtacgccgcgaagcaagggcgatcagcagatcccagaggct +41 WKY -9 GAGGGGCGGgtacgccgcgaagcaagggcgatcagcagatcccagaggct +41 RefSeq -9 GAGGGGCGGgtacgccgcgaagcaagggcgatcagcagatcccagaggct +41

SHR +42 gacagaagctgagcagtaggggcttcttggcaggtgatggcgcccctcgt +91 WKY +42 gacagaagctgagcagtaggggcttcttggcaggtgatggcgcccctcgt +91 RefSeq +42 gacagaagctgagcagtaggggcttcttggcaggtgatggcgcccctcgt +91

SHR +92 ggcctagaagtccagcgccgtcctcaagccacagacccgcctctctctgt +141 WKY +92 ggcctagaagtccagcgccgtcctcaagccacagacccgcctctctctgt +141 RefSeq +92 ggcctagaagtccagcgccgtcctcaagccacagacccgcctctctctgt +141  5’UTR| SHR +142 tgtatctgatcaagtctccgggtcactttgaacATGTTGGTGCGACTAAC +191 WKY +142 tgtatctgatcaagtctccgggtcactttgaacATGTTGGTGCGACTAAC +191 RefSeq +142 tgtatctgatcaagtctccgggtcactttgaacATGTTGGTGCGACTAAC +191  Exon 1| SHR +192 CAAGCTGTCCTGCCCTGGTGAGATGAAACTCCTGGTGGGGACAGATGGAG +241 WKY +192 CAAGCTGTCCTGCCCTGGTGAGATGAAACTCCTGGTGGGGACAGATGGAG +241 RefSeq +192 CAAGCTGTCCTGCCCTGGTGAGATGAAACTCCTGGTGGGGACAGATGGAG +241

SHR +242 CATGGTGGATAAGTTGGGACAAAAGAGACTGGGGCCACACCTGGGGTACT +291 WKY +242 CATGGTGGATAAGTTGGGACAAAAGAGACTGGGGCCACACCTGGGGTACT +291 RefSeq +242 CATGGTGGATAAGTTGGGACAAAAGAGACTGGGGCCACACCTGGGGTACT +291

SHR +292 GCNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +4091 WKY +292 GCNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +4091 RefSeq +292 GCAAACACCTCTCTC...... TAAGAAAAAACATTC +4091

SHR +4092 NNNNNAGATTTGTGTCAATTATTTAAAATATTCGTTCAGCTGAGAGACTT +4141 WKY +4092 NNNNNAGATTTGTGTCAATTATTTAAAATATTCGTTCAGCTGAGAGACTT +4141 RefSeq +4092 CATTTAGATTTGTGTCAATTATTTAAAATATTCGTTCAGCTGAGAGACTT +4141

SHR +4142 TTCAGTATACGACAGATGGAAACTAATTTTTAAAAAGATACTGTTTTCAT +4191 WKY +4142 TTCAGTATACGACAGATGGAAACTAATTTTTAAAAAGATACTGTTTTCAT +4191 RefSeq +4142 TTCAGTATACGACAGATGAAAACTAATTTTTAAAAAGATACTGTTTTCAT +4191 |Exon 2  SHR +4192 TTTTATGCAGCATATCAGTGGTTTCATGCCTTAAAAATTAAAAAGTGCCT +4241 WKY +4192 TTTTATGCAGCATATCAGTGGTTTCATGCCTTAAAAATTAAAAAGTGCCT +4241 RefSeq +4192 TTTTATGCAGCATATCAGTGGTTTCATGCCTTAAAAATTAAAAAGTGCCT +4241

SHR +4242 ACCTCTGTGTGCTCCAAGATGCTCTTCAACCTCTGCTGTACCTCAGATTA +4291 WKY +4242 ACCTCTGTGTGCTCCAAGATGCTCTTCAACCTCTGCTGTACCTCAGATTA +4291 RefSeq +4242 ACCTCTGTGTGCTCCAAGATGCTCTTCAACCTCTGCTGTACCTCAGATTA +4291

193

 Exon 2| SHR +4292 CCACTCACTACACTATTCATCCCCGGGAAAAAGACAAAAGATGGGAAGGT +4341 WKY +4292 CCACTCACTACACTATTCATCCCCGGGAAAAAGACAAAAGATGGGAAGGT +4341 RefSeq +4292 CCACTCACTACACTATTCATCCCCGGGAAAAAGACAAAAGATGGGAAGGT +4341

SHR +4342 AGGCCATAGTTTTCATAATATTCTTNNNNNNNNNNNNNNNNNNNNNNNNN +4391 WKY +4342 AGGCCATAGTTTTCATAATATTCTTNNNNNNNNNNNNNNNNNNNNNNNNN +4391 RefSeq +4342 AGGCCATAGTTTTCATAATATTCTTGAGACTTCCCTAGATATTTTATATT +4391

SHR +4392 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +4841 WKY +4392 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +4841 RefSeq +4392 ATCTTGTTGGTATTA...... AAAACACATCTTAAT +4841

SHR +4842 NNNNNNNNNNNNNNNNNNNNNNNNNNNNGGTCCTATTAATCCCAGAGTTG +4891 WKY +4842 NNNNNNNNNNNNNNNNNNNNNNNNNNNNGGTCCTATTAATCCCAGAGTTG +4891 RefSeq +4842 GTATCCTAAACCAGCATAAGACTTATGTGGTCCTATTAATCCCAGAGTTG +4891 |Exon 3  SHR +4892 TTCTTTATCTTTAACCTGTTTTCCTCCCAGGAGTGAATATGGAGAGATTT +4941 WKY +4892 TTCTTTATCTTTAACCTGTTTTCCTCCCAGGAGTGAATATGGAGAGATTT +4941 RefSeq +4892 TTCTTTATCTTTAACCTGTTTTCCTCCCAGGAGTGAATATGGAGAGATTT +4941

SHR +4942 GCAGAAGAAGCAGATGTGGTAATAGTTGGTGCCGGCCCAGCAGGGCTCTC +4991 WKY +4942 GCAGAAGAAGCAGATGTGGTAATAGTTGGTGCCGGCCCAGCAGGGCTCTC +4991 RefSeq +4942 GCAGAAGAAGCAGATGTGGTAATAGTTGGTGCCGGCCCAGCAGGGCTCTC +4991

SHR +4992 TGCAGCTATTCGGCTAAAGCAGCTGGCTGCTGAACAGGAAAAGGACATCC +5041 WKY +4992 TGCAGCTATTCGGCTAAAGCAGCTGGCTGCTGAACAGGAAAAGGACATCC +5041 RefSeq +4992 TGCAGCTATTCGGCTAAAGCAGCTGGCTGCTGAACAGGAAAAGGACATCC +5041

SHR +5042 GTGTGTGTCTGGTGGAGAAAGCTGCTCAGATAGGAGCTCATACGCTCTCA +5091 WKY +5042 GTGTGTGTCTGGTGGAGAAAGCTGCTCAGATAGGAGCTCATACGCTCTCA +5091 RefSeq +5042 GTGTGTGTCTGGTGGAGAAAGCTGCTCAGATAGGAGCTCATACGCTCTCA +5091

SHR +5092 GGGGCTTGCCTTGATCCAGCTGCTTTTAAAGAGCTCTTCCCAGACTGGAA +5141 WKY +5092 GGGGCTTGCCTTGATCCAGCTGCTTTTAAAGAGCTCTTCCCAGACTGGAA +5141 RefSeq +5092 GGGGCTTGCCTTGATCCAGCTGCTTTTAAAGAGCTCTTCCCAGACTGGAA +5141  Exon 3| SHR +5142 GGAGAAGGGGGTAGGACAATGATTCTTACACAAGGTCTAATCTATGCTGT +5191 WKY +5142 GGAGAAGGGGGTAGGACAATGATTCTTACACAAGGTCTAATCTATGCTGT +5191 RefSeq +5142 GGAGAAGGGGGTAGGACAATGATTCTTACACAAGGTCTAATCTATGCTGT +5191

SHR +5192 CTNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +7241 WKY +5192 CTNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +7241 RefSeq +5192 CTCTACTTTCAAATT...... TTCTCTTTCAGAGGC +7241

SHR +7242 NNNNNNNCCCAACATACAAGTAACATCATCTCCTTCCTGTTCTCATTTGT +7291 WKY +7242 NNNNNNNCCCAACATACAAGTAACATCATCTCCTTCCTGTTCTCATTTGT +7291 RefSeq +7242 ATATTCACCCAACATACAAGTAACATCATCTCCTTCCGGTTCTCATTTGT +7291 |Exon 4  SHR +7292 TCCCAGGCTCCACTTAACACTCCTGTAACAGAAGACAGGTTTGCGATTTT +7341 WKY +7292 TCCCAGGCTCCACTTAACACTCCTGTAACAGAAGACAGGTTTGCGATTTT +7341 RefSeq +7292 TCCCAGGCTCCACTTAACACTCCTGTAACAGAAGACAGGTTTGCGATTTT +7341

194

 Exon 4| SHR +7342 AACAGAGAAACACAGAATTCCTGTGCCAATTCTTCCAGGTAGAGTATGCA +7391 WKY +7342 AACAGAGAAACACAGAATTCCTGTGCCAATTCTTCCAGGTAGAGTATGCA +7391 RefSeq +7342 AACAGAGAAACACAGAATTCCTGTGCCAATTCTTCCAGGTAGAGTATGCA +7391

SHR +7392 CAGTCGCGAAAGCATGGGCTTGCACGGCTGGAGGGGCTGCATCTCGGTGA +7441 WKY +7392 CAGTCGCGAAAGCATGGGCTTGCACGGCTGGAGGGGCTGCATCTCGGTGA +7441 RefSeq +7392 CAGTCGCGAAAGCATGGGCTTGCACGGCTGGAGGGGCTGCATCTCAGTGA +7441

SHR +7442 GGAAAACCCTCAGATGAGAACACAGAAGCCGAAATGTGGAGGACTTGCCC +7491 WKY +7442 GGAAAACCCTCAGATGAGAACACAGAAGCCGAAATGTGGAGGACTTGCCC +7491 RefSeq +7442 GGAAAACCCTCAGATGAGAACACAGAAGCCGAAATGTGGAGGACTTGCCC +7491

SHR +7492 AGAGATGGCTAAGCAAGCTAATGAGAAGGCCAGACTANNNNNNNNNNNNN +7541 WKY +7492 AGAGATGGCTAAGCAAGCTAATGAGAAGGCCAGACTANNNNNNNNNNNNN +7541 RefSeq +7492 AGAGATGGCTAAGCAAGCTAATGAGAAGGCCAGACTAGGGTGCAGTTCTT +7541

SHR +7542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +7591 WKY +7542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +7591 RefSeq +7542 TTGTCTTCCTCTCTAAAAGAGCCTTGAAAGCCAGCGAGGAATTTTTTATT +7591

SHR +7592 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +7641 WKY +7592 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +7641 RefSeq +7592 TCAGGCTTTAATATTGACTTTATTTTAATAAGCTAGGACTATACCCAAAC +7641

SHR +7642 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +7691 WKY +7642 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +7691 RefSeq +7642 TTTTAAAAAATGTCATGATAGCCAAAAAGATAATGTGTATATTTATGCAG +7691

SHR +7692 NNNNNNNNNNTGGTTAGATTTTTTAAATTAAGTATATTAGATTATCAGAG +7741 WKY +7692 NNNNNNNNNNTGGTTAGATTTTTTAAATTAAGTATATTAGATTATCAGAG +7741 RefSeq +7692 CAGTACACACTGGTTAGATTTTTTAAATTAAGTACATTAGATTATCCGAG +7741 |Exon 5  SHR +7742 TGCCTTAGCAATAAAATGTCAGGTTTTAAATTGCATAAAAATTGCAGGTC +7791 WKY +7742 TGCCTTAGCAATAAAATGTCAGGTTTTAAATTGCATAAAAATTGCAGGTC +7791 RefSeq +7742 TGCCTTAGCAATAAAATGTCAGGTTTTAAATTGCATAAAAATTGCAGGTC +7791

SHR +7792 TTCCGATGAACAATCATGGCAATTACATCGTACGCCTCGGACACCTCGTG +7841 WKY +7792 TTCCGATGAACAATCATGGCAATTACATCGTACGCCTCGGACACCTCGTG +7841 RefSeq +7792 TTCCGATGAACAATCATGGCAATTACATCGTACGCCTCGGACACCTTGTG +7841

SHR +7842 AGCTGGATGGGAGAACAGGCAGAGGCTCTGGGAGTTGAAGTGTACCCTGG +7891 WKY +7842 AGCTGGATGGGAGAACAGGCAGAGGCTCTGGGAGTTGAAGTGTACCCTGG +7891 RefSeq +7842 AGCTGGATGGGAGAACAGGCAGAGGCTCTGGGAGTTGAAGTGTACCCTGG +7891  Exon 5| SHR +7892 ATATGCTGCTGCTGAGGTCTGTGTAGTTTTGTTTTTTTTAACATTTATAG +7941 WKY +7892 ATATGCTGCTGCTGAGGTCTGTGTAGTTTTGTTTTTTTTAACATTTATAG +7941 RefSeq +7892 ATATGCTGCTGCTGAGGTCTGTGTAGTTTTGTTTTTTTTAACATTTATAG +7941

SHR +7942 AAAACAATCTCTTTCTTACAAAAGTAAACCAATAATTTTACANNNNNNNN +7991 WKY +7942 AAAACAATCTCTTTCTTACAAAAGTAAACCAATAATTTTACANNNNNNNN +7991 RefSeq +7942 AAAACAATCTCTTTCTTACAAAAGTAAACCAATAATTTTACAGAGGTAAA +7991

195

SHR +7992 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +9891 WKY +7992 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +9891 RefSeq +7992 TTGTTTAGAAAAATC...... CTTCATGGTTATTTA +9891

SHR +9892 NNNNNNNNNNNNNNNNNNNNNNCAAAAATAAACTGTAATCATCATCATTT +9941 WKY +9892 NNNNNNNNNNNNNNNNNNNNNNCAAAAATAAACTGTAATCATCATCATTT +9941 RefSeq +9892 CGTTCCCCCACCCTTCTTGCTTCAAAAATAAACTGTAATCATCATCATTT +9941

SHR +9942 CAAATATGTCAAAGTTAATATGAATCTGAGGTAATACTAAGTTTGTTTTT +9991 WKY +9942 CAAATATGTCAAAGTTAATATGAATCTGAGGTAATACTAAGTTTGTTTTT +9991 RefSeq +9942 CAAATATGTCAAAGTTAATATGAATCTGAGGTAATACTAAGTTTGTTTTT +9991

|Exon 6  SHR +9992 GTCATTTTTAGGTCCTTTATCATGAAGATGGTAGTGTGAAAGGAATCGCC +10041 WKY +9992 GTCATTTTTAGGTCCTTTATCATGAAGATGGTAGTGTGAAAGGAATCGCC +10041 RefSeq +9992 ATCATTTTTAGGTCCTTTATCATGAAGATGGTAGTGTGAAAGGAATCGCC +10041  Exon 6| SHR +10042 ACTAACGATGTGGGAATACAGAAGGATGGTGCACCAAAGGTATGCCTACG +10091 WKY +10042 ACTAACGATGTGGGAATACAGAAGGATGGTGCACCAAAGGTATGCCTACG +10091 RefSeq +10042 ACTAACGATGTGGGAATACAGAAGGATGGCGCACCAAAGGTATGCCTACG +10091

SHR +10092 AACAACAGCTATGTGCTGAGGCAGGGCAGGGCATGGAGCACATCCGCAGG +10141 WKY +10092 AACAACAGCTATGTGCTGAGGCAGGGCAGGGCATGGAGCACATCCGCAGG +10141 RefSeq +10092 AACAACAGCTATGTGCTGAGGCAGGGCAGGGCATGGCGCACATCCGCAGG +10141

SHR +10142 CCCAGTACTTGGGAGGCCGAGGCTGAGGCTCAAGGCCANNNNNNNNNNNN +10191 WKY +10142 CCCAGTACTTGGGAGGCCGAGGCTGAGGCTCAAGGCCANNNNNNNNNNNN +10191 RefSeq +10142 CCCAGTACTTGGGAGGCCGAGGCTGAGGCTCAAGGCCAGCCTAAGCTAGC +10191

SHR +10192 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +12691 WKY +10192 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +12691 RefSeq +10192 TACATACCAGATCCT...... ATAGTCCTGATAGTT +12691

SHR +12692 NNNNNNNNNNNNNNNNNNNNTACTATGTTTTGTGTACCTAAATCGTTGTT +12741 WKY +12692 NNNNNNNNNNNNNNNNNNNNTACTATGTTTTGTGTACCTAAATCGTTGTT +12741 RefSeq +12692 TTCCTTCCAGTACATAGGTCTACTATGTTTTGTGTACCTAAATCGTTGTT +12741 |Exon 7  SHR +12742 AGCTTATTTTATCAAATACTGTTTTTTAACAAGTTTTTGCTTTTCAGACA +12791 WKY +12742 AGCTTATTTTATCAAATACTGTTTTTTAACAAGTTTTTGCTTTTCAGACA +12791 RefSeq +12742 AGCTTATTTTATCAAATACTGTTTTTTAACAAGTTTTTGCTTTTCAGACA +12791

SHR +12792 ACATTTGAGAGAGGCCTGGAGTTGCATGCCAAAGTCACAATCTTTGCAGA +12841 WKY +12792 ACATTTGAGAGAGGCCTGGAGTTGCATGCCAAAGTCACAATCTTTGCAGA +12841 RefSeq +12792 ACATTTGAGAGAGGCCTGGAGTTGCATGCCAAAGTCACAATCTTTGCAGA +12841

SHR +12842 AGGCTGCCATGGACACCTAGCCAAGCAGCTTTATAAAAAGTTTGATTTGA +12891 WKY +12842 AGGCTGCCATGGACACCTAGCCAAGCAGCTTTATAAAAAGTTTGATTTGA +12891 RefSeq +12842 AGGCTGCCATGGACACCTAGCCAAGCAGCTTTATAAAAAGTTTGATTTGA +12891  Exon 7| SHR +12892 GGGCCAGCTGTGATGCCCAGACTTACGGAATTGGTTTGAAGGAGGTATCC +12941 WKY +12892 GGGCCAGCTGTGATGCCCAGACTTACGGAATTGGTTTGAAGGAGGTATCC +12941 RefSeq +12892 GGGCCAGCTGTGATGCCCAGACTTACGGAATTGGTTTGAAGGAGGTATCC +12941

196

SHR +12942 TGGTTTGCTTGTGCAATTTTAACCTTAAGTGATGGAATTTAATTTGTATT +12991 WKY +12942 TGGTTTGCTTGTGCAATTTTAACCTTAAGTGATGGAATTTAATTTGTATT +12991 RefSeq +12942 TGGTTTGCTTGTGCAATTTTAACCTTAAGTGATGGAATTTAATTTGTATT +12991

SHR +12992 ATCATNNNNNNNNNN...... NNNNNNNNNNNNNNN +13991 WKY +12992 ATCATNNNNNNNNNN...... NNNNNNNNNNNNNNN +13991 RefSeq +12992 ATCATGTAGTTTATA...... AAGTGCTAATGGGTT +13991

SHR +13992 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTACAGTATTAATTACATTA +14041 WKY +13992 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNCTTACAGTATTAATTACATTA +14041 RefSeq +13992 CATCACTTTCATGAACCATTTCTTTATGTTGGCTTACAATCATTTAATTA +14041

SHR +14042 ATATTATGTAACATGTAATTTTGTGCTGCTTTCATGTAATTAATATTACA +14091 WKY +14042 ATATTATGTAACATGTAATTTTGTGCTGCTTTCATGTAATTAATATTACA +14091 RefSeq +14042 TATTTTTGTCCAAAATAATTTTGTGCTGCTTTCATGTAATTAATATTACA +14091

SHR +14092 CAATATTACATGTTACATAATATTAATGTAATTAATACTGTAAGACTTCA +14141 WKY +14092 CAATATTACATGTTACATAATATTAATGTAATTAATACTGTAAGACTTCA +14141 RefSeq +14092 CAATATTACATGTTACATAATATTAATGTAATTAATACTGTAAGACTTCA +14141 |Exon 8  SHR +14142 AGGACTGTGTTTTGTTTTTAGTTATGGGTTATTGATGAGAAGAAGTGGAA +14191 WKY +14142 AGGACTGTGTTTTGTTTTTAGTTATGGGTTATTGATGAGAAGAAGTGGAA +14191 RefSeq +14142 AGGACTGTGTTTTGTTTTTAGTTATGGGTTATTGATGAGAAGAAGTGGAA +14191

SHR +14192 ACCTGGGAGAGTAGATCACACTGTTGGCTGGCCCTTGGACAGACATACTT +14241 WKY +14192 ACCTGGGAGAGTAGATCACACTGTTGGCTGGCCCTTGGACAGACATACTT +14241 RefSeq +14192 ACCTGGGAGAGTAGATCACACTGTTGGCTGGCCCTTGGACAGACATACTT +14241

SHR +14242 ATGGAGGCTCTTTCCTCTATCACTTAAATGAAGGTGAACCTCTAGTAGCT +14291 WKY +14242 ATGGAGGCTCTTTCCTCTATCACTTAAATGAAGGTGAACCTCTAGTAGCT +14291 RefSeq +14242 ATGGAGGCTCTTTCCTCTATCACTTAAATGAAGGTGAACCTCTAGTAGCT +14291  Exon 8| SHR +14292 GTTGGTTTTGTGGTAAGTTTTCTCCCATTGCAAAAAGTTTTACTAGANNN +14341 WKY +14292 GTTGGTTTTGTGGTAAGTTTTCTCCCATTGCAAAAAGTTTTACTAGANNN +14341 RefSeq +14292 GTTGGTTTTGTGGTAAGTTTTCTCCCATTGCAAAAAGTTTTACTAGAGGT +14341

SHR +14342 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +15791 WKY +14342 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +15791 RefSeq +14342 ATAGGGTGATCCATG...... AATGCCAATTATCCT +15791

SHR +15792 NNNGCTTACTTTCTTACCCTCAGTGTAGTTTATCTCTGCTTTAATTTTTG +15841 WKY +15792 NNNGCTTACTTTCTTACCCTCAGTGTAGTTTATCTCTGCTTTAATTTTTG +15841 RefSeq +15792 TTTGCTTACTTTCTTACCCTCAGTGTAGTTT-TCTCTGCTTTAATTTTTG +15841 |Exon 9  SHR +15842 TTTTGCTTTAAGGTTGGCCTGGACTATCAGAATCCATACCTGAGTCCATT +15891 WKY +15842 TTTTGCTTTAAGGTTGGCCTGGACTATCAGAATCCATACCTGAGTCCATT +15891 RefSeq +15842 TTTTGCTTTAAGGTTGGCCTGGACTATCAGAATCCATACCTGAGTCCATT +15891

SHR +15892 CAGAGAGTTCCAGAGGTGGAAGCATCACCCCAGCATCCGACCAACCCTGG +15941 WKY +15892 CAGAGAGTTCCAGAGGTGGAAGCATCACCCCAGCATCCGACCAACCCTGG +15941 RefSeq +15892 CAGAGAGTTCCAGAGGTGGAAGCATCACCCCAGCATCCGACCAACCCTGG +15941

197

SHR +15942 AAGGTGGGAAAAGGATAGCCTATGGAGCCCGAGCTCTCAATGAAGGTGGC +15991 WKY +15942 AAGGTGGGAAAAGGATAGCCTATGGAGCCCGAGCTCTCAATGAAGGTGGC +15991 RefSeq +15942 AAGGTGGGAAAAGGATAGCCTATGGAGCCCGAGCTCTCAATGAAGGTGGC +15991  Exon 9| SHR +15992 TTGCAGGTAGCCTCCAGCACTTCTCAAATTAAGAACATAATTTTTATTTC +16041 WKY +15992 TTGCAGGTAGCCTCCAGCACTTCTCAAATTAAGAACATAATTTTTATTTC +16041 RefSeq +15992 TTGCAGGTAGCCTCCAGCACTTCTCAAATTAAGAACATAATTTTTATTTC +16041

SHR +16042 ATAGGACAAAACTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +16091 WKY +16042 ATAGGACAAAACTTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +16091 RefSeq +16042 ATAGGACAAAACTTTAAGGAGCTTAACAAATACTTATAAATTATGCATAA +16091

SHR +16092 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +17441 WKY +16092 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +17441 RefSeq +16092 ACACAGTGATTACAG...... TTCCTTGCGACACCC +17441

SHR +17442 NNNNNNNNNNNNNNNNNTCTTCTTTGCCCTACAACTTTAGGTTTGAAATA +17491 WKY +17442 NNNNNNNNNNNNNNNNNTCTTCTTTGCCCTACAACTTTAGGTTTGAAATA +17491 RefSeq +17442 CTTTTCCAGGCGTTTACTCTTCTTTGCCCTACAACTTTAGGTTTGAAATA +17491

SHR +17492 TTTGTGTTTGTTTTGTCTCATTTGAGCTGACGAATGTATTATTCTTCATC +17541 WKY +17492 TTTGTGTTTGTTTTGTCTCATTTGAGCTGACGAATGTATTATTCTTCATC +17541 RefSeq +17492 TTTGTGTTTGTTTTGTCTCATTTGAGCTGACGAATGTATTATTCTTCATC +17541 |Exon 10  SHR +17542 TTCTTTTCAGTCCATACCAAAACTCACTTTTCCTGGTGGCTTACTAATTG +17591 WKY +17542 TTCTTTTCAGTCCATACCAAAACTCACTTTTCCTGGTGGCTTACTAATTG +17591 RefSeq +17542 TTCTTTTCAGTCCATACCAAAACTCACTTTTCCTGGTGGCTTACTAATTG +17591

SHR +17592 GTTGCAGTCCTGGATTCATGAATGTTCCCAAGATCAAAGGTACCCACACA +17641 WKY +17592 GTTGCAGTCCTGGATTCATGAATGTTCCCAAGATCAAAGGTACCCACACA +17641 RefSeq +17592 GTTGCAGTCCTGGATTCATGAATGTTCCCAAGATCAAAGGTACCCACACA +17641

SHR +17642 GCAATGAAAAGTGGAAGCTTGGCAGCAGAAGCAATTTTTAAGCAACTAAC +17691 WKY +17642 GCAATGAAAAGTGGAAGCTTGGCAGCAGAAGCAATTTTTAAGCAACTAAC +17691 RefSeq +17642 GCAATGAAAAGTGGAAGCTTGGCAGCAGAAGCAATTTTTAAGCAACTAAC +17691  Exon 10| SHR +17692 TAGTGAAAATCTCCAATCAAAGACAGCAGGTGAGAAACTCCTATATGAAA +17741 WKY +17692 TAGTGAAAATCTCCAATCAAAGACAGCAGGTGAGAAACTCCTATATGAAA +17741 RefSeq +17692 TAGTGAAAATCTCCAATCAAAGACAGCAGGTGAGAAACTCCTATATGAAA +17741

SHR +17742 GAAATACTAAGACAGGCAGACATTTTCTTCTAAAGATTTATTTATTTATT +17791 WKY +17742 GAAATACTAAGACAGGCAGACATTTTCTTCTAAAGATTTATTTATTTATT +17791 RefSeq +17742 GAAATACTAAGACAGGCAGACATTTTCTTCTAAAGATTTATTTATTTATT +17791

SHR +17792 TAATCTATATAAGTACACTGTAGCTACCTTCACACACCAGAAGAGGGCAT +17841 WKY +17792 TAATCTATATAAGTACACTGTAGCTACCTTCACACACCAGAAGAGGGCAT +17841 RefSeq +17792 TAATCTATATAAGTACACTGTAGCTACCTTCACACACCAGAAGAGGGCAT +17841

SHR +17842 TGGATACCATTATAGATGGTTGTGAGCTCAGGACCTTTGGAAGAGCAGTC +17891 WKY +17842 TGGATACCATTATAGATGGTTGTGAGCTCAGGACCTTTGGAAGAGCAGTC +17891 RefSeq +17842 TGGATACCATTATAGATGGTTGTGAGCTCAGGACCTTTGGAAGAGCAGTC +17891

SHR +17892 AAGTGCTCTTAACCACTGAACCATCNNNNNNNNNNNNNNNNNNNNNNNNN +17941 WKY +17892 AAGTGCTCTTAACCACTGAACCATCNNNNNNNNNNNNNNNNNNNNNNNNN +17941 RefSeq +17892 AAGTGCTCTTAACCACTGAACCATCTCTACAGCCCCTACAGTCAGAAATT +17941

198

SHR +17942 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +19991 WKY +17942 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +19991 RefSeq +17942 TTAACTGTCATTTTC...... TTCTGTTATACTCAG +19991

SHR +19992 NNNNNNNNNNNNNNNNNNCCACATGACGTTTTTAAAAACTAAGCCTCACT +20041 WKY +19992 NNNNNNNNNNNNNNNNNNCCACATGACGTTTTTAAAAACTAAGCCTCACT +20041 RefSeq +19992 ACATGATTCTGCATGGGTCCACATGACGTTTTTAAAAACTAAGCCTCACT +20041 |Exon 11  SHR +20042 ATCTATCTTTCACTTAATATTTGTTTTTCATAAAAGGACTCCACGTAACT +20091 WKY +20042 ATCTATCTTTCACTTAATATTTGTTTTTCATAAAAGGACTCCACGTAACT +20091 RefSeq +20042 ATCTATCTTTCACTTAATATTTGTTTTTCATAAAAGGACTCCACGTAACT +20091

SHR +20092 GAGTATGAGGACAATTTGAAGCAATCCTGGGTGTGGAAAGAGCTACATGC +20141 WKY +20092 GAGTATGAGGACAATTTGAAGCAATCCTGGGTGTGGAAAGAGCTACATGC +20141 RefSeq +20092 GAGTATGAGGACAATTTGAAGCAATCCTGGGTGTGGAAAGAGCTACATGC +20141

SHR +20142 TGTCAGAAATATAAGGCCATCCTGCCACGGGATCCTGGGGGTATATGGAG +20191 WKY +20142 TGTCAGAAATATAAGGCCATCCTGCCACGGGATCCTGGGGGTATATGGAG +20191 RefSeq +20142 TGTCAGAAATATAAGGCCATCCTGCCACGGGATCCTGGGGGTATATGGAG +20191

SHR +20192 GGATGATTTACACTGGAATATTTTACTGGATATTGAGAGGAATGGAGCCA +20241 WKY +20192 GGATGATTTACACTGGAATATTTTACTGGATATTGAGAGGAATGGAGCCA +20241 RefSeq +20192 GGATGATTTACACTGGAATATTTTACTGGATATTGAGAGGAATGGAGCCA +20241  Exon 11| SHR +20242 TGGACTCTAAAACATAAAGGTAATTCAAATATACTTAATGCTAGTATTNN +20291 WKY +20242 TGGACTCTAAAACATAAAGGTAATTCAAATATACTTAATGCTAGTATTNN +20291 RefSeq +20242 TGGACTCTAAAACATAAAGGTAATTCAAATATACTTAATGCTAGTATTGA +20291

SHR +20292 NNNNNNNNNNNNNNN...... NNNNNNNNNNACACA +20691 WKY +20292 NNNNNNNNNNNNNNN...... NNNNNNNNNNACACA +20691 RefSeq +20292 TATTAAGTACCTATA...... ACCAACACACACACA +20691

SHR +20692 CACACACACACACACACACACACACACACACACACACAGAGTGAGTCCTG +20741 WKY +20692 CACACACACACACACACACACACACACACACACACACAGAGTGAGTCCTG +20741 RefSeq +20692 CACACACACACACACACACACACACACACACACACACAGAGTGAGTCCTG +20741 |Exon 12  SHR +20742 GAAAATGATGTTACTTTATTAAACATCTTCCTCAAAATTGCTTAAGGCTC +20791 WKY +20742 GAAAATGATGTTACTTTATTAAACATCTTCCTCAAAATTGCTTAAGGCTC +20791 RefSeq +20742 GAAAATGATGTTACTTTATTAAACATCTTCCTCAAAATTGCTTAAGGCTC +20791

SHR +20792 AGACTCTGAACAGCTCAAACCAGCCAAGGACTGTACACCCATTGAGTATC +20841 WKY +20792 AGACTCTGAACAGCTCAAACCAGCCAAGGACTGTACACCCATTGAGTATC +20841 RefSeq +20792 AGACTCTGAACAGCTCAAACCAGCCAAGGACTGTACACCCATTGAGTATC +20841

SHR +20842 CAAAACCTGACGGACAGATCAGTTTTGACCTCTTATCCTCTGTGGCTCTG +20891 WKY +20842 CAAAACCTGACGGACAGATCAGTTTTGACCTCTTATCCTCTGTGGCTCTG +20891 RefSeq +20842 CAAAACCTGACGGACAGATCAGTTTTGACCTCTTATCCTCTGTGGCTCTG +20891

SHR +20892 AGTGGTACTAATCATGAACATGACCAGCCAGCACATTTAACCTTGAAGGA +20941 WKY +20892 AGTGGTACTAATCATGAACATGACCAGCCAGCACATTTAACCTTGAAGGA +20941 RefSeq +20892 AGTGGTACTAATCATGAACATGACCAGCCAGCACATTTAACCTTGAAGGA +20941

199

SHR +20942 TGACAGCATACCTGTTAATAGAAATCTGTCAATATATGATGGGCCTGAGC +20991 WKY +20942 TGACAGCATACCTGTTAATAGAAATCTGTCAATATATGATGGGCCTGAGC +20991 RefSeq +20942 TGACAGCATACCTGTTAATAGAAATCTGTCAATATATGATGGGCCTGAGC +20991  Exon 12| SHR +20992 AGCGATTCTGCCCTGCAGGTGAGTAACAATTTCTTTCTGTTCCTAAACAT +21041 WKY +20992 AGCGATTCTGCCCTGCAGGTGAGTAACAATTTCTTTCTGTTCCTAAACAT +21041 RefSeq +20992 AGCGATTCTGCCCTGCAGGTGAGTAACAATTTCTTTCTGTTCCTAAACAT +21041

SHR +21042 TTAACAATTTCCCAGTTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +21091 WKY +21042 TTAACAATTTCCCAGTTCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +21091 RefSeq +21042 TTAACAATTTCCCAGTTCAAGATGCTCCCTTCCTATTGCAAAGAGACCAT +21091

SHR +21092 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +21541 WKY +21092 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +21541 RefSeq +21092 TAAAACACACACACA...... TATCTTTGACTTGTC +21541

SHR +21542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGCTTGTTAACTAACCCA +21591 WKY +21542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGCTTGTTAACTAACCCA +21591 RefSeq +21542 TGTCAGAAGGAAGGGCTGGAGTCAATTTCTTTAGCTTGTTAACTAACCCA +21591

SHR +21592 GAGAATACTGCTGCACTTCTTTTATGTGATTAGTTTATACCCACTCACTT +21641 WKY +21592 GAGAATACTGCTGCACTTCTTTTATGTGATTAGTTTATACCCACTCACTT +21641 RefSeq +21592 GAGAATACTGCTGCACTTCTTTTATGTGATTAGTTTATACCCACTCACTT +21641

SHR +21642 TTTCATTATTTGCCTTTTGTTGCATAGCTAAATATATATATAATTTTTTT +21691 WKY +21642 TTTCATTATTTGCCTTTTGTTGCATAGCTAAATATATATATAATTTTTTT +21691 RefSeq +21642 TTTCATTATTTGCCTTTTGTTGCATAGCTAAATATATATATAATTTTTTT +21691 |Exon 13  SHR +21692 TACAGGAGTTTATGAATTTGTTCCTCTGGAACAAGGTGATGGATTTCGGT +21741 WKY +21692 TACAGGAGTTTATGAATTTGTTCCTCTGGAACAAGGTGATGGATTTCGGT +21741 RefSeq +21692 -ACAGGAGTTTATGAATTTGTTCCTCTGGAACAAGGTGATGGATTTCGGT +21741

SHR +21742 TACAGATAAATGCTCAGAACTGTGTGCATTGTAAAACATGTGATATCAAA +21791 WKY +21742 TACAGATAAATGCTCAGAACTGTGTGCATTGTAAAACATGTGATATCAAA +21791 RefSeq +21742 TACAGATAAATGCTCAGAACTGTGTGCATTGTAAAACATGTGATATCAAA +21791

SHR +21792 GACCCAAGTCAAAATATTAACTGGGTGGTCCCAGAAGGTGGAGGAGGACC +21841 WKY +21792 GACCCAAGTCAAAATATTAACTGGGTGGTCCCAGAAGGTGGAGGAGGACC +21841 RefSeq +21792 GACCCAAGTCAAAATATTAACTGGGTGGTCCCAGAAGGTGGAGGAGGACC +21841 |3’UTR  SHR +21842 TGCTTACAATGGCATGTAAagcccaagtgcctccacttactggcacattt +21891 WKY +21842 TGCTTACAATGGCATGTAAagcccaagtgcctccacttactggcacattt +21891 RefSeq +21842 TGCTTACAATGGCATGTAAagcccaagtgcctccacttactggcacactt +21891

SHR +21892 gacagccagtttctagaatactgtaaatgtatgccaaactaacctcccat +21941 WKY +21892 gacagccagtttctagaatactgtaaatgtatgccaaactaacctcccat +21941 RefSeq +21892 gacagccagtttctagaatactgtaaatgtatgccaaactaacctcccat +21941

SHR +21942 atgtttggataacttctgaacaagtgtccttcaaacactgaagtaaaaaa +21991 WKY +21942 atgtttggataacttctgaacaagtgtccttcaaacactgaagtaaaaaa +21991 RefSeq +21942 atgtttggataacttctgaacaagtgtccttcaaacactgaagtaaaaaa +21991

SHR +21992 ctttgtatctaacgtcccataaaatcatgaaatatttgtcattaataaaa +22041 WKY +21992 ctttgtatctaacgtcccataaaatcatgaaatatttgtcattaataaaa +22041 RefSeq +21992 ctttgtatctaacgtcccataaaatcatgaaatatttgtcattaataaaa +22041

200

 Exon 13| SHR +22042 ctttataaatAAATAATAGAGCATCTACTTACTTTTCAAGTCCTTCCAGT +22091 WKY +22042 ctttataaatAAATAATAGAGCATCTACTTACTTTTCAAGTCCTTCCAGT +22091 RefSeq +22042 ctttataaatAAATAATAGAGCATCTACTTACTTTTCAAGTCCTTCCAGT +22091

SHR +22092 ACCAACAGAAACTTG +22106 WKY +22092 ACCAACAGAAACTTG +22106 RefSeq +22092 ACCAACAGAAACTTG +22106

201

Endothelin receptor, type B (Ednrb)

SHR -2309 TATTTAACCTTTTGTTTAATAAAACTTATATTCTAACATGGTCATTACCT -2260 WKY -2309 TATTTAACCTTTTGTTTAATAAAACTTATATTCTAACATGGTCATTACCT -2260 RefSeq -2309 TATTTAACCTTTTGTTTAATAAAACTTATATTCTAACATGGTCATTACCT -2260

SHR -2259 GAGCTCTGAAGCTTTGTGAAATATCCAGTTTCCATTTTCTAAACCAGTGG -2210 WKY -2259 GAGCTCTGAAGCTTTGTGAAATATCCAGTTTCCATTTTCTAAACCAGTGG -2210 RefSeq -2259 GAGCTCTGAAGCTTTGTGAAATATCCAGTTTCCATTTTCTAAACCAGTGG -2210

SHR -2209 CCCTCAACCTTCCTAAAGCTTTCACTCTTTAATACATCCACCACACATGT -2160 WKY -2209 CCCTCAACCTTCCTAAAGCTTTCACTCTTTAATACATCCACCACACATGT -2160 RefSeq -2209 CCCTCAACCTTCCTAAAGCTTTCACTCTTTAATACATCCACCACACATGT -2160

SHR -2159 TGTGGTGACCACCACACATCCCTCCAACCATAAACTTATTTTTTTTCTTT -2110 WKY -2159 TGTGGTGACCACCACACATCCCTCCAACCATAAACTTATTTTTTTTCTTT -2110 RefSeq -2159 TGTGGTGACCACCACACATCCCTCCAACCATAAACTTATTTTTTTTCTTT -2110

SHR -2109 CGATGTCACAACTCTAATTTTGCTACTGTTAAGAACCGTAATTTAAATAT -2060 WKY -2109 CGATGTCACAACTCTAATTTTGCTACTGTTAAGAACCGTAATTTAAATAT -2060 RefSeq -2109 CGATGTCACAACTCTAATTTTGCTACTGTTAAGAACCGTAATTTAAATAT -2060

SHR -2059 CTGTATTTCCAAACTGTTTTGGGTGATCCTTGTGAATGAGTCATTCAACT -2010 WKY -2059 CTGTATTTCCAAACTGTTTTGGGTGATCCTTGTGAATGAGTCATTCAACT -2010 RefSeq -2059 CTGTATTTCCAAACTGTTTTGGGTGATCCTTGTGAATGAGTCATTCAACT -2010

SHR -2009 CCTCCCCCACAAGTGGGCCACAGCCCACAAGTTGAGACCCTCTGTTCTAA -1960 WKY -2009 CCTCCCCCACAAGTGGGCCACAGCCCACAAGTTGAGACCCTCTGTTCTAA -1960 RefSeq -2009 CCTCCCCCACAAGTGGGCCACAGCCCACAAGTTGAGACCCTCTGTTCTAA -1960

SHR -1959 AGGGTCATTAAGCAACTGGGTCATTGGCCCTTCTGACAAGACCCTGATTG -1910 WKY -1959 AGGGTCATTAAGCAACTGGGTCATTGGCCCTTCTGACAAGACCCTGATTG -1910 RefSeq -1959 AGGGTCATTAAGCAACTGGGTCATTGGCCCTTCTGACAAGACCCTGATTG -1910

SHR -1909 TGCCACCAGTCTACACAAAATGAATCTCATAAACCGGCTGCATTTGCATT -1860 WKY -1909 TGCCACCAGTCTACACAAAATGAATCTCATAAACCGGCTGCATTTGCATT -1860 RefSeq -1909 TGCCACCAGTCTACACAAAATGAATCTCATAAACCGGCTGCATTTGCATT -1860

SHR -1859 ACTTCCAATGTGTTCTATATTGGGCACAGGGGCTTTTCAAAGGGCAGTTC -1810 WKY -1859 ACTTCCAATGTGTTCTATATTGGGCACAGGGGCTTTTCAAAGGGCAGTTC -1810 RefSeq -1859 ACTTCCAATGTGTTCTATATTGGGCACAGGGNNNNNNNNNNNNNNNNNNN -1810

SHR -1809 TTCCTTCCCTCTGACTGCTAATCACCAGCCCTCAATGATCAGTTCTGAGG -1760 WKY -1809 TTCCTTCCCTCTGACTGCTAATCACCAGCCCTCAATGATCAGTTCTGAGG -1760 RefSeq -1809 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1760

SHR -1759 CGGCCTTGTGAAAAATCTTTGTAACAGCCCTTCACCTCTCCTCTATGCAG -1710 WKY -1759 CGGCCTTGTGAAAAATCTTTGTAACAGCCCTTCACCTCTCCTCTATGCAG -1710 RefSeq -1759 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1710

SHR -1709 CTGCTGGCAGGGGACTGGCCATTTGGAGCTGAGATGTGCAAGCTGGTGCC -1660 WKY -1709 CTGCTGGCAGGGGACTGGCCATTTGGAGCTGAGATGTGCAAGCTGGTGCC -1660 RefSeq -1709 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1660

202

SHR -1659 CTTCATACAGAAGGCTTCTGTGGGGATCACAGTGTTGAGTCTATGTGCTC -1610 WKY -1659 CTTCATACAGAAGGCTTCTGTGGGGATCACAGTGTTGAGTCTATGTGCTC -1610 RefSeq -1659 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1610

SHR -1609 TAAGTATTGACAGGTAAGAGTCCATTCTTAGGCCAAGGAGATCCTAACCC -1560 WKY -1609 TAAGTATTGACAGGTAAGAGTCCATTCTTAGGCCAAGGAGATCCTAACCC -1560 RefSeq -1609 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1560

SHR -1559 ACCATATAACTGCTCTGTCACTTAGGAGATGAACTATGCAATTCAATATA -1510 WKY -1559 ACCATATAACTGCTCTGTCACTTAGGAGATGAACTATGCAATTCAATATA -1510 RefSeq -1559 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1510

SHR -1509 ACCAAGCCCTACCTGCCTGAGAGATAATTCTGTTTACGTCTCCAGATATC -1460 WKY -1509 ACCAAGCCCTACCTGCCTGAGAGATAATTCTGTTTACGTCTCCAGATATC -1460 RefSeq -1509 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1460

SHR -1459 GAGCTGTTGCTTCTTGGAGTCGAATTAAAGGAATTGGGGTTCCAAAATGG -1410 WKY -1459 GAGCTGTTGCTTCTTGGAGTCGAATTAAAGGAATTGGGGTTCCAAAATGG -1410 RefSeq -1459 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1410

SHR -1409 ACAGCAGTAGAAATTGTTTTAATTTGGGTGGTCTCTGTGGTTCTGGCTGT -1360 WKY -1409 ACAGCAGTAGAAATTGTTTTAATTTGGGTGGTCTCTGTGGTTCTGGCTGT -1360 RefSeq -1409 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1360

SHR -1359 CCCTGAAGCCATAGGTTTTGATGTGATTACGTCGGACTACAAAGGAAAGC -1310 WKY -1359 CCCTGAAGCCATAGGTTTTGATGTGATTACGTCGGACTACAAAGGAAAGC -1310 RefSeq -1359 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1310

SHR -1309 CCCTAAGGGTCTGCATGCTTAATCCCTTTCAGAAAACAGCCTTCATGCAG -1260 WKY -1309 CCCTAAGGGTCTGCATGCTTAATCCCTTTCAGAAAACAGCCTTCATGCAG -1260 RefSeq -1309 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1260

SHR -1259 GTAAGTTCACTTCGCCTGTTCTTCCTGCACTTTCCTTATAAATATTTAAC -1210 WKY -1259 GTAAGTTCACTTCGCCTGTTCTTCCTGCACTTTCCTTATAAATATTTAAC -1210 RefSeq -1259 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1210

SHR -1209 TATCTCCCCCAGTTCCCCACCTTGGTAAGCTATTGATTTATTACTACCTC -1160 WKY -1209 TATCTCCCCCAGTTCCCCACCTTGGTAAGCTATTGATTTATTACTACCTC -1160 RefSeq -1209 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1160

SHR -1159 TGGCATAAATTAAGAGTTTGCATCCCAAGACTGTGAGGTTAACCATCACT -1110 WKY -1159 TGGCATAAATTAAGAGTTTGCATCCCAAGACTGTGAGGTTAACCATCACT -1110 RefSeq -1159 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1110

SHR -1109 AATGACCCGACCTACAAGAGTCAGGATAATTATTCCTCCAAGAACACCCT -1060 WKY -1109 AATGACCCGACCTACAAGAGTCAGGATAATTATTCCTCCAAGAACACCCT -1060 RefSeq -1109 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1060

SHR -1059 GGGGATCCAGATAAATCAAGTTCCTACAAGAATGGCTGTGACTTTATGGC -1010 WKY -1059 GGGGATCCAGATAAATCAAGTTCCTACAAGAATGGCTGTGACTTTATGGC -1010 RefSeq -1059 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -1010

203

SHR -1009 TTTTGTGTCTAAGGTCCCACAGGATTGATTATTATTGTGTAGTCTACACA -960 WKY -1009 TTTTGTGTCTAAGGTCCCACAGGATTGATTATTATTGTGTAGTCTACACA -960 RefSeq -1009 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -960

SHR -959 AGACAACCCAACATTGTCAGAGAAGCAGATGGTTTAACATATGTTTAGAT -910 WKY -959 AGACAACCCAACATTGTCAGAGAAGCAGATGGTTTAACATATGTTTAGAT -910 RefSeq -959 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -910

SHR -909 AGAAGTTGCCTAATTGTAGTAGATTTGGAACAGCAGCCTGACCCTTCGTC -860 WKY -909 AGAAGTTGCCTAATTGTAGTAGATTTGGAACAGCAGCCTGACCCTTCGTC -860 RefSeq -909 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -860

SHR -859 TCTGACGGTAAAAGAAAAGCTGTCTTGCAAAATAATATCAAATTTTGGCC -810 WKY -859 TCTGACGGTAAAAGAAAAGCTGTCTTGCAAAATAATATCAAATTTTGGCC -810 RefSeq -859 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -810

SHR -809 ACCTACTAAAGAACTTCTGATCAGAAAATAGGAAAGCATGGGGATATTAG -760 WKY -809 ACCTACTAAAGAACTTCTGATCAGAAAATAGGAAAGCATGGGGATATTAG -760 RefSeq -809 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -760

SHR -759 TCTCCAAATGATAGATCCTAAGTATAAGATTTATTTTAGGCAGGATTGGT -710 WKY -759 TCTCCAAATGATAGATCCTAAGTATAAGATTTATTTTAGGCAGGATTGGT -710 RefSeq -759 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -710

SHR -709 TAATACAAAGGGGGTTGATTGGTCTGAATATAACCAAGGATTATTTTCTA -660 WKY -709 TAATACAAAGGGGGTTGATTGGTCTGAATATAACCAAGGATTATTTTCTA -660 RefSeq -709 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -660

SHR -659 TTTTACATGTAAAAGACTATAAAAACTTTATGAGCTACACATTCGACAAT -610 WKY -659 TTTTACATGTAAAAGACTATAAAAACTTTATGAGCTACACATTCGACAAT -610 RefSeq -659 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -610

SHR -609 CAGTGAAGGAGCAAAGTGGGTCACCAATGCAGAGTTGAGAAGAGGTTGTT -560 WKY -609 CAGTGAAGGAGCAAAGTGGGTCACCAATGCAGAGTTGAGAAGAGGTTGTT -560 RefSeq -609 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -560

SHR -559 GTTTACCAGAAGGGGAATATAGCACATTGGGGACATAAAATTCTTTCCAA -510 WKY -559 GTTTACCAGAAGGGGAATATAGCACATTGGGGACATAAAATTCTTTCCAA -510 RefSeq -559 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN -510

SHR -509 CGTAGATATATCTGAATCGATAAGGTTTTCTGAAAGGTAACAGTAGAAAA -460 WKY -509 CGTAGATATATCTGAATCGATAAGGTTTTCTGAAAGGTAACAGTAGAAAA -460 RefSeq -509 NNNNNNNNNNNNNNNNNNNNNNNNGTTTTCTGAAAGGTAACAGTAGAAAA -460

SHR -459 GAACACAGGAAAAGTGTAACTCTTGATTGACCTTTCATGTAGACACTACT -410 WKY -459 GAACACAGGAAAAGTGTAACTCTTGATTGACCTTTCATGTAGACACTACT -410 RefSeq -459 GAACACAGGAAAAGTGTAACTCTTGATTGACCTTTCATGTAGACACTATT -410

SHR -409 GACTTAAATGGCCAGTACAGACCTCTTCATACCTGGTTTTCACACTAAAC -360 WKY -409 GACTTAAATGGCCAGTACAGACCTCTTCATACCTGGTTTTCACACTAAAC -360 RefSeq -409 GACTTAAATGGCCAGTACAGACCTCTTCATACCTGGTTTTCACACTAAAC -360

SHR -359 ACACTTAATAGAAAGGGAAAACATCACCTAACACTGAAATAGGTTTATAT -310 WKY -359 ACACTTAATAGAAAGGGAAAACATCACCTAACACTGAAATAGGTTTATAT -310 RefSeq -359 ACACTTAATAGAAAGGGAAAACATCACCTAACACTGAAATAGGTTTATAT -310

204

SHR -309 ACTTAACACCTAACATTTGAAATACCTGGTTATTCAGTGTTATCTACACA -260 WKY -309 ACTTAACACCTAACATTTGAAATACCTGGTTATTCAGTGTTATCTACACA -260 RefSeq -309 ACTTAACACCTAACATTTGAAATACCTGGTTATTCAGTGTTATCTACACA -260

SHR -259 TAAGAATTTTGCTCAGCTGCCTACAGGTTACTATACTGTCCCAAAGAATT -210 WKY -259 TAAGAATTTTGCTCAGCTGCCTACAGGTTACTATACTGTCCCAAAGAATT -210 RefSeq -259 TAAGAATTTTGCTCAGCTGCCTACAGGTTACTATACTGTCCCAAAGAATT -210

SHR -209 GAAATATGTTCTGTAATATTTCTTTAATTTAATGCAGGACTTTAATTATG -160 WKY -209 GAAATATGTTCTGTAATATTTCTTTAATTTAATGCAGGACTTTAATTATG -160 RefSeq -209 GAAATATGTTCTGTAATATTTCTTTAATTTAATGCAGGACTTTAATTATG -160

SHR -159 TGTTCAGTAGACAGGGCCCATGAAATTCTATACAGTACATAGACGGAAGG -110 WKY -159 TGTTCAGTAGACAGGGCCCATGAAATTCTATACAGTACATAGACGGAAGG -110 RefSeq -159 TGTTCAGTAGACAGGGCCCATGAAATTCTATACAGTACATAGACGGAAGA -110

SHR -109 CGTTACCAAAATCATTGTCCTTTGATGAATATTTTATTTTTAGTTGACAT -60 WKY -109 CGTTACCAAAATCATTGTCCTTTGATGAATATTTTATTTTTAGTTGACAT -60 RefSeq -109 CGTTACCAAAATCATTGTCCTTTGATGAATATTTTATTTTTAGTTGACAT -60

SHR -59 TTGATACATAAGGTGCTTTAAGCAGGAGATATTAATCATAAACATTTCTT -10 WKY -59 TTGATACATAAGGTGCTTTAAGCAGGAGATATTAATCATAAACATTTCTT -10 RefSeq -59 TTGATACATAAGGTGCTTTAAGCAGGAGATATTAATCATAAACATTTCTT -10  +1|-1  SHR -9 TCTTCCCATAGTTTTACAAGACAGCCAAAGACTGGTGGCTGTTCAGTTTC +41 WKY -9 TCTTCCCATAGTTTTACAAGACAGCCAAAGACTGGTGGCTGTTCAGTTTC +41 RefSeq -9 TCTTCCCATAGTTTTACAAGACAGCCAAAGACTGGTGGCTGTTCAGTTTC +41

SHR +42 TACTTCTGCTTGCCGCTAGCCATCACTGCGATCTTTTACACCCTAATGAC +91 WKY +42 TACTTCTGCTTGCCGCTAGCCATCACTGCGATCTTTTACACCCTAATGAC +91 RefSeq +42 TACTTCTGCTTGCCGCTAGCCATCACTGCGATCTTTTACACCCTAATGAC +91

SHR +92 CTGTGAGATGCTCAGAAAGAAAAGTGGTATGCAGATTGCCTTGAATGACC +141 WKY +92 CTGTGAGATGCTCAGAAAGAAAAGTGGTATGCAGATTGCCTTGAATGACC +141 RefSeq +92 CTGTGAGATGCTCAGAAAGAAAAGTGGTATGCAGATTGCCTTGAATGACC +141  EXON 1| SHR +142 ACTTAAAGCAGGTAAGAGGATGGGAGAATCCAGTAGAGGGAAGCCCTAAC +191 WKY +142 ACTTAAAGCAGGTAAGAGGATGGGAGAATCCAGTAGAGGGAAGCCCTAAC +191 RefSeq +142 ACTTAAAGCAGGTAAGAGGATGGGAGAATCCAGTAGAGGGAAGCCCTAAC +191

SHR +192 TATGGTCAGATGGAAGTCATGATGATGGTGATGATGGTGGTGATG----- +241 WKY +192 TATGGTCAGATGGAAGTCATGATGATGGTGATGATGGTGGTGATG----- +241 RefSeq +192 TATGGTCAGATGGAAGTCATGATGATGGTGATGATGGTGGTGATGGTGAT +241

SHR +242 -ATGATGATGATGATGATGATGATGATGATGTCACCAAAAATGATGATAA +291 WKY +242 -ATGATGATGATGATGATGATGATGATGATGTCACCAAAAATGATGATAA +291 RefSeq +242 GATGATGATGATGATGATGATGATGATGATGTCACCAAAAATGATGATAA +291

SHR +292 ACTAATATTAGACACTAGAACATATTTTCACTTTCATCCTTCATTCTATT +341 WKY +292 ACTAATATTAGACACTAGAACATATTTTCACTTTCATCCTTCATTCTATT +341 RefSeq +292 ACTAATATTAGACACTAGAACATATTTTCACTTTCATCCTTCATTCTATT +341

205

SHR +342 TCTGTAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +391 WKY +342 TCTGTAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +391 RefSeq +342 TCTGTAACAAAAAAACATTTTATATTACTTGTTTAGAGACTATATCATCT +391

SHR +392 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +441 WKY +392 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +441 RefSeq +392 ACAATGATCTAAAGAGAATCATTGCCTTCTGCTGTTGTTTTAAGCTAGAA +441

SHR +442 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +491 WKY +442 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +491 RefSeq +442 TCTCAATTAGCCCAGGCTAGACTTGAATTCAGCATGTAGCCAGAGATAAC +491

SHR +492 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +541 WKY +492 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +541 RefSeq +492 CTTGGATTCCAGATCCATTTGCTTCTGACTCCCAAATGATGGTAGTACAG +541

SHR +542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +591 WKY +542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +591 RefSeq +542 ACCTGTGCTATTTTTCCAGTTTCACTCACATATGCGTGCATGCTCTCTCT +591

SHR +592 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +641 WKY +592 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +641 RefSeq +592 CTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCACACACA +641

SHR +642 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +691 WKY +642 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +691 RefSeq +642 CACACACACACACACACACACACACAAAATGGTTGTATAATATATTTTTA +691

SHR +692 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +741 WKY +692 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +741 RefSeq +692 GATTTATTTTATGTATGTGAATGTTTTGCTTGTGTGCGTGTGTGTGTGCC +741

SHR +742 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +791 WKY +742 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +791 RefSeq +742 TGGTGCCCATGGGGTTCAGAGTAAGGGCATCAGATTCTATGAAACTGGCG +791

SHR +792 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +841 WKY +792 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +841 RefSeq +792 TGATAGATGATTATTGACCACTATGGAGGTACTGGCAATCGAACCTATTT +841

SHR +842 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +891 WKY +842 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +891 RefSeq +842 CTTTTGCAAAACAAGTGCTCTAAGCTGCTGAGCTGTCTGTCCATCCCACT +891

SHR +892 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +941 WKY +892 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +941 RefSeq +892 CCCCCAAACCCTTAGTTTTAAAACATGATCCCTAGCGATTTTAGGTCTAA +941

SHR +942 NNNNNNNNNNNNNNNNNNNNNNNNGCTCATTGGCCCAAGTCTTTATTTGT +991 WKY +942 NNNNNNNNNNNNNNNNNNNNNNNNGCTCATTGGCCCAAGTCTTTATTTGT +991 RefSeq +942 GTCGCACTAATTATCTAACTGAAAGCTCATTGGCCCAAGTCTTTATTTGT +991

SHR +992 GTGAATACGATTAAGTATCTCTGTGATAAGAGTGTGTGTTAGCTTTATAA +1041 WKY +992 GTGAATACGATTAAGTATCTCTGTGATAAGAGTGTGTGTTAGCTTTATAA +1041 RefSeq +992 GTGAATACGATTAAGTATCTCTGTGATAAGAGTGTGTGTTAGCTTTATAA +1041

206

|EXON 2  SHR +1042 GGATCCGTTGTTCTGTTTCAGAGACGAGAAGTGGCCAAGACAGTATTCTG +1091 WKY +1042 GGATCCGTTGTTCTGTTTCAGAGACGAGAAGTGGCCAAGACAGTATTCTG +1091 RefSeq +1042 GGATCCGTTGTTCTGTTTCAGAGACGAGAAGTGGCCAAGACAGTATTCTG +1091

SHR +1092 CCTGGTCCTCGTGTTTGCCCTCTGTTGGCTTCCCCTTCACCTCAGCAGGA +1141 WKY +1092 CCTGGTCCTCGTGTTTGCCCTCTGTTGGCTTCCCCTTCACCTCAGCAGGA +1141 RefSeq +1092 CCTGGTCCTCGTGTTTGCCCTCTGTTGGCTTCCCCTTCACCTCAGCAGGA +1141

SHR +1142 TTCTGAAGCTCACCCTTTATGACCAGAGCAATCCTCAGAGGTGTGAACTT +1191 WKY +1142 TTCTGAAGCTCACCCTTTATGACCAGAGCAATCCTCAGAGGTGTGAACTT +1191 RefSeq +1142 TTCTGAAGCTCACCCTTTATGACCAGAGCAATCCTCAGAGGTGTGAACTT +1191

 EXON 2| SHR +1192 CTGAGGTAAGAAAAGACAGTAGAACACATGTGACATGGAAAGAAATGTGG +1241 WKY +1192 CTGAGGTAAGAAAAGACAGTAGAACACATGTGACATGGAAAGAAATGTGG +1241 RefSeq +1192 CTGAGGTAAGAAAAGACAGTAGAACACATGTGACATGGAAAGAAATGTGG +1241

SHR +1242 CAGCTATCTGGGGAAAGGTAGAGGGAGGTAGGAGGGGGTGCTGAGGGGGA +1291 WKY +1242 CAGCTATCTGGGGAAAGGTAGAGGGAGGTAGGAGGGGGTGCTGAGGGGGA +1291 RefSeq +1242 CAGCTATCTGGGGGAAGGTAGAGGGAGGTAGGAGGGGGTGCTGAGGGGGA +1291

SHR +1292 GAGGTAAAGAAAGACCAAAACGCAATGACATGTATGTCATAACATGTATG +1341 WKY +1292 GAGGTAAAGAAAGACCAAAACGCAATGACATGTATGTCATAACATGTATG +1341 RefSeq +1292 GAGGTAAAGAAAGACCAAAACGCAATGACATGTATGTCATAACATGTATG +1341

SHR +1342 TGATAACATGTATGTATGAAACTCAGTAATCTGTGCTAACTTAGGTTGAA +1391 WKY +1342 TGATAACATGTATGTATGAAACTCAGTAATCTGTGCTAACTTAGGTTGAA +1391 RefSeq +1342 TGATAACATGTATGTATGAAACTCAGTAATCTGTGCTAACTTAGGTTGAA +1391

SHR +1392 AATAAGAGACTAGAAATAATTATGAAATTGTCTTCCTGATGTTTCTCCAT +1441 WKY +1392 AATAAGAGACTAGAAATAATTATGAAATTGTCTTCCTGATGTTTCTCCAT +1441 RefSeq +1392 AATAAGAGACTAGAAATAATTATGAAATTGTCTTCCTGATGTTTCTCCAT +1441

SHR +1442 TATCAATCCGTCTAATAGGCAGAAAGAATACTATTTTCTTATCCACAGAN +1491 WKY +1442 TATCAATCCGTCTAATAGGCAGAAAGAATACTATTTTCTTATCCACAGAN +1491 RefSeq +1442 TATCAATCCGTCTAATAGGCAGAAAGAATACTATTTTCTTATCCACAGAG +1491

SHR +1492 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1541 WKY +1492 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1541 RefSeq +1492 GCCAATTAAGACTCCCCATTTTCCCTTTACTCCTGCTCTAGATGGGTAAA +1541

SHR +1542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1591 WKY +1542 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1591 RefSeq +1542 TTAGAGGGATTGGGAAACCTTGGGAATGTGGGCACAGTTGTCAGAATGAT +1591

SHR +1592 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1641 WKY +1592 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1641 RefSeq +1592 GAGTGACAAGATGAAAAGTGTCAGATCCGAGATGTCACTGTTCACATCAG +1641

SHR +1642 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1691 WKY +1642 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1691 RefSeq +1642 TCCCCTCTCTTCTGAACACCCCGTCACTTCCAAACGAGAACCTTACTTTT +1691

207

SHR +1692 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1741 WKY +1692 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1741 RefSeq +1692 CAAAGAAAAAGCTGGATGTGGGAGATGAAGGCAGTGGAGATGCCAAAGAC +1741

SHR +1742 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1791 WKY +1742 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1791 RefSeq +1742 AGAGAGAGAGAAAGAGAGAGACAGACAAAGAGAGAGAGAGAAAGAGAGAG +1791

SHR +1792 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1841 WKY +1792 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1841 RefSeq +1792 AGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAATGGCAG +1841

SHR +1842 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1891 WKY +1842 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1891 RefSeq +1842 GCAGAGCAGCAGACAGGTGCTTTACAGGCAAATCGGTGGTTAAGCAAATC +1891

SHR +1892 NNNNNNNNNNNNNNNNNNNNNNNNNNNTTTGGGTTTTCTTCATAAACACA +1941 WKY +1892 NNNNNNNNNNNNNNNNNNNNNNNNNNNTTTGGGTTTTCTTCATAAACACA +1941 RefSeq +1892 GGTGGTCAAATCATTGCGTGCACGTAATTTGGGTTTTCTTCATAAACACA +1941

SHR +1942 CAAAGGTGTGCATTAAATCCACACTTGGAGGAATGTAATCTTTACAGACA +1991 WKY +1942 CAAAGGTGTGCATTAAATCCACACTTGGAGGAATGTAATCTTTACAGACA +1991 RefSeq +1942 CAAAGGTGTGCATTAAATCCACACTTGGAGGAATGTAATCTTTACAGACA +1991 |EXON 3  SHR +1992 CATTTGGGTTTCTTGCAGTTTTTTGCTGGTTTTGGACTACATTGGTATCA +2041 WKY +1992 CATTTGGGTTTCTTGCAGTTTTTTGCTGGTTTTGGACTACATTGGTATCA +2041 RefSeq +1992 CATTTGGGTTTCTTGCAGTTTTTTGCTGGTTTTGGACTACATTGGTATCA +2041

SHR +2042 ACATGGCTTCTTTGAATTCCTGCATTAATCCAATCGCTCTGTATTTGGTG +2091 WKY +2042 ACATGGCTTCTTTGAATTCCTGCATTAATCCAATCGCTCTGTATTTGGTG +2091 RefSeq +2042 ACATGGCTTCTTTGAATTCCTGCATTAATCCAATCGCTCTGTATTTGGTG +2091  EXON 3| SHR +2092 AGCAAGAGATTCAAAAACTGCTTTAAGGTAAGGGATTCTTCTAAGATAAA +2141 WKY +2092 AGCAAGAGATTCAAAAACTGCTTTAAGGTAAGGGATTCTTCTAAGATAAA +2141 RefSeq +2092 AGCAAGAGATTCAAAAACTGCTTTAAGGTAAGGGATTCTTCTAAGATAAA +2141

SHR +2142 AAGTATCCTGTGATCTGGCATCCAATATGGGCTTTAAAAAAACACTAATA +2191 WKY +2142 AAGTATCCTGTGATCTGGCATCCAATATGGGCTTTAAAAAAACACTAATA +2191 RefSeq +2142 AAGTATCCTGTGATCTGGCATCCAATATGGGCTTTAAAAAAACACTAATA +2191

SHR +2192 TCCCTATCTAGAGAGAGCTGGAAAATTGCTAATTTTTCCTGCTTTGTAAT +2241 WKY +2192 TCCCTATCTAGAGAGAGCTGGAAAATTGCTAATTTTTCCTGCTTTGTAAT +2241 RefSeq +2192 TCCCTATCTAGAGAGAGCTGGAAAATTGCTAATTTTTCCTGCTTTGTAAT +2241

SHR +2242 ATATGTGATGTTTTTAAATATTGCATTTCCACGACAGGACATATTTGTAT +2291 WKY +2242 ATATGTGATGTTTTTAAATATTGCATTTCCACGACAGGACATATTTGTAT +2291 RefSeq +2242 ATATGTGATGTTTTTAAATATTGCATTTCCACGACAGGACATATTTGTAT +2291

SHR +2292 ATTTGTCAGGAGTCAGCCATGCCCTGATACCTTAGCCACTCACCGATACN +2341 WKY +2292 ATTTGTCAGGAGTCAGCCATGCCCTGATACCTTAGCCACTCACCGATACN +2341 RefSeq +2292 ATTTGTCAGGAGTCAGCCATGCCCTGATACCTTAGCCACTCACCGATACA +2341

SHR +2342 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +4891 WKY +2342 NNNNNNNNNNNNNNN...... NNNNNNNNNNNNNNN +4891 RefSeq +2342 ATTAGACTTGATCAG...... GAGGGAACCATAATC +4891

208

SHR +4892 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCCCCAGAAGAGTGGTGGCC +4941 WKY +4892 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGCCCCAGAAGAGTGGTGGCC +4941 RefSeq +4892 CCACACTGGCAAGGACAGTCAGGGCCCCAAGCCCCAGAAGAGTGGTGGCC +4941 |EXON 4  SHR +4942 GTGTGCCTGAGTAAATGTGTAACGGCCGTTCTGTTTTATACAGTCGTGTT +4991 WKY +4942 GTGTGCCTGAGTAAATGTGTAACGGCCGTTCTGTTTTATACAGTCGTGTT +4991 RefSeq +4942 GTGTGCCTGAGTAAATGTGTAACGGCCGTTCTGTTTTATACAGTCGTGTT +4991

SHR +4992 TGTGCTGCTGGTGCCAAACGTTTGAGGAAAAACAGTCCTTAGAGGAGAAG +5041 WKY +4992 TGTGCTGCTGGTGCCAAACGTTTGAGGAAAAACAGTCCTTAGAGGAGAAG +5041 RefSeq +4992 TGTGCTGCTGGTGCCAAACGTTTGAGGAAAAACAGTCCTTAGAGGAGAAG +5041

SHR +5042 CAATCCTGCTTGAAGTTCAAAGCTAACGATCACGGATACGACAACTTCCG +5091 WKY +5042 CAATCCTGCTTGAAGTTCAAAGCTAACGATCACGGATACGACAACTTCCG +5091 RefSeq +5042 CAATCCTGCTTGAAGTTCAAAGCTAACGATCACGGATACGACAACTTCCG +5091 |3’UTR  SHR +5092 CTCCAGCAATAAATACAGCTCATCTTGAaggaaggaacactcactgaatc +5141 WKY +5092 CTCCAGCAATAAATACAGCTCATCTTGAaggaaggaacactcactgaatc +5141 RefSeq +5092 CTCCAGCAATAAATACAGCTCATCTTGAaggaaggaacactcactgaatc +5141

SHR +5142 tcattgtcctcatcgtggacagatagcattaaaacaaaatgaaacctttg +5191 WKY +5142 tcattgtcctcatcgtggacagatagcattaaaacaaaatgaaacctttg +5191 RefSeq +5142 tcattgtcctcatcgtggacagatagcattaaaacaaaatgaaacctttg +5191

SHR +5192 ccaaacccaaacggaaaaccgtgcttgcggaaaggtgtgcacgcatggga +5241 WKY +5192 ccaaacccaaacggaaaaccgtgcttgcggaaaggtgtgcacgcatggga +5241 RefSeq +5192 ccaaacccaaacggaaaaccgtgcttgcggaaaggtgtgcacgcatggga +5241

SHR +5242 gagggattgttttttaaccgttctaactttccacacctgatatttcacgg +5291 WKY +5242 gagggattgttttttaaccgttctaactttccacacctgatatttcacgg +5291 RefSeq +5242 gagggattgttttttaaccgttctaactttccacacctgatatttcacgg +5291

SHR +5292 gctgtttacaacctaagaaagccatgggaatgaatgaagcctcgggaaag +5341 WKY +5292 gctgtttacaacctaagaaagccatgggaatgaatgaagcctcgggaaag +5341 RefSeq +5292 gctgtttacaacctaagaaagccatgggaatgaatgaagcctcgggaaag +5341

SHR +5342 cacttagattcttagtcagcacttcagcacggctcttaaaagccctcact +5391 WKY +5342 cacttagattcttagtcagcacttcagcacggctcttaaaagccctcact +5391 RefSeq +5342 cacttagattcttagtcagcacttcagcacggctcttaaaagccctcact +5391

SHR +5392 gcactcacagcccacttacatttaaaaacaagaactcaaactctattcag +5441 WKY +5392 gcactcacagcccacttacatttaaaaacaagaactcaaactctattcag +5441 RefSeq +5392 gcactcacagcccacttacatttaaaaacaagaactcaaactctattcag +5441

SHR +5442 gggtttattatccagtcctatgaatctggatacaggaatgcatgacattg +5491 WKY +5442 gggtttattatccagtcctatgaatctggatacaggaatgcatgacattg +5491 RefSeq +5442 gggtttattatccagtcctatgaatctggatacaggaatgcatgacattg +5491

SHR +5492 caaaacaattcttaaagcaaagtttcaattgctcgatttgagacaaaaaa +5541 WKY +5492 caaaacaattcttaaagcaaagtttcaattgctcgatttgagacaaaaaa +5541 RefSeq +5492 caaaacaattcttaaagcaaagtttcaattgctcgatttgagacaaaaaa +5541

209

 EXON 4| SHR +5542 caaaacAAAAAAAAAAAA +5559 WKY +5542 caaaacAAAAAAAAAAAA +5559 RefSeq +5542 caaaacAAAACAAAAAAA +5559

210

Neuropeptide Y (Npy)

SHR -1660 TTGCTTCTATGAGCTGCCAATGGTTACAGGAAACTGCGGAAGGGAGGGGT -1611 WKY -1660 TTGCTTCTATGAGCTGCCAATGGTTACAGGAAACTGCGGAAGGGAGGGGT -1611 RefSeq -1660 TTGCTTCTATGAGCTGCCAATGGTTACAGGAAACTGCGGAAGGGAGGGGT -1611

SHR -1610 TAATTGATGGAGTGGTATCGGAGAATGACAGCATGTTAAGAGCCCAAGGC -1561 WKY -1610 TAATTGATGGAGTGGTATCGGAGAATGACAGCATGTTAAGAGCCCAAGGC -1561 RefSeq -1610 TAATTGATGGAGTGGTATCGGAGAATGACAGCATGTTAAGAGCCCAAGGC -1561

SHR -1560 CAGATTTGACAAGGAGAAGAACAGGGTATAAGTGACATTCTGGGCACGCC -1511 WKY -1560 CAGATTTGACAAGGAGAAGAACAGGGTATAAGTGACATTCTGGGCACGCC -1511 RefSeq -1560 CAGATTGGACAAGGAGAAGAACAGGGTATAAGTGACATTCTGGGCACGCC -1511

SHR -1510 CTCCCTTGACTTCATTTTCCAAATGCTGAAGGGAAAAGTTCTTGTTCTCA -1461 WKY -1510 CTCCCTTGACTTCATTTTCCAAATGCTGAAGGGAAAAGTTCTTGTTCTCA -1461 RefSeq -1510 CTCCCTTGACTTCATTTTCCAAATGCTGAAGGGAAAAGTTCTTGTTCTCA -1461

SHR -1460 AGCACCAACATTATAAAGTAAGTTAATCAGATTCCCAGAGTCCCTTGCTC -1411 WKY -1460 AGCACCAACATTATAAAGTAAGTTAATCAGATTCCCAGAGTCCCTTGCTC -1411 RefSeq -1460 AGCACCAACATTATAAAGTAAGTTAATCAGATTCCCAGAGTCCCTTGCTC -1411

SHR -1410 AAAAGTGGCACCCCTACCCCTACCCCCTCCGTGTACTTTCTCCCAGATTC -1361 WKY -1410 AAAAGTGGCACCCCTACCCCTACCCCCTCCGTGTACTTTCTCCCAGATTC -1361 RefSeq -1410 AAAAGTGGCACCCCTACCCCTACCCCCTCCGTGTACTTTCTCCCAGATTC -1361

SHR -1360 TGTGACGCCTGCTGGACCCAGGTTTTGACCGATGTTACTCCCTGATTCAC -1311 WKY -1360 TGTGACGCCTGCTGGACCCAGGTTTTGACCGATGTTACTCCCTGATTCAC -1311 RefSeq -1360 TGTGACGCCTGCTGGACCCAGGTTTTGACCGATGTTACTCCCTGATTCAC -1311

SHR -1310 TACAACAGGAAGATTACTTTATTGAACAGCAGTGTGTGCCTTCCTCCTTA -1261 WKY -1310 TACAACAGGAAGATTACTTTATTGAACAGCAGTGTGTGCCTTCCTCCTTA -1261 RefSeq -1310 TACAACAGGAAGATTACTTTATTGAACAGCAGTGTGTGCCTTCCTCCTTA -1261

SHR -1260 CCAGAGCGCTCTTGTTAGATGCCTTTCTCTTAGCAAAGGTTCTCAGGAGG -1211 WKY -1260 CCAGAGCGCTCTTGTTAGATGCCTTTCTCTTAGCAAAGGTTCTCAGGAGG -1211 RefSeq -1260 CCAGAGCGCTCTTGTTAGATGCCTTTCTCTTAGCAAAGGTTCTCAGGAGG -1211

SHR -1210 GCACAAGAGAAACTCTGACAGCTCAGTGTTTGTGGCCTGGTGCAGGTTTC -1161 WKY -1210 GCACAAGAGAAACTCTGACAGCTCAGTGTTTGTGGCCTGGTGCAGGTTTC -1161 RefSeq -1210 GCACAAGAGAAACTCTGACAGCTCAGTGTTTGTGGCCTGGTGCAGGTTTC -1161

SHR -1160 TGACATAGGGAGAGTGCAGTCTTCCAGCTGCGAGGGATGGGATATTGGTG -1111 WKY -1160 TGACATAGGGAGAGTGCAGTCTTCCAGCTGCGAGGGATGGGATATTGGTG -1111 RefSeq -1160 TGACATAGGGAGAGTGCAGTCTTCCAGCTGCGAGGGATGGGATATTGGTG -1111

SHR -1110 TTCAGGATTATCATTTGATCACATAGTGTCTCATATTCATTCTCTCTCTC -1061 WKY -1110 TTCAGGATTATCATTTGATCACATAGTGTCTCATATTCATTCTCTCTCTC -1061 RefSeq -1110 TTCAGGATTATCATTTGATCACATAGTGTCTCATATTCATTCTCTCTCTC -1061

SHR -1060 TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC--ACTAAAAGTTAATT -1011 WKY -1060 TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCACTAAAAGTTAATT -1011 RefSeq -1060 TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCACTAAAAGTTAATT -1011

211

SHR -1010 CATCCCTATTTAAACAATGCACAAAGTGACACTTGAATGAAATCATTTAT -961 WKY -1010 CATCCCTATTTAAACAATGCACAAAGTGACACTTGAATGAAATCATTTAT -961 RefSeq -1010 CATCCCTATTTAAACAATGCACAAAGTGACACTTGAATGAAATCATTTAT -961

SHR -960 TTTAGCTTTGAAATAACGCTTTAAAACAAACTTTAGCTTTAAAATAAACT -911 WKY -960 TTTAGCTTTGAAATAACGCTTTAAAACAAACTTTAGCTTTAAAATAAACT -911 RefSeq -960 TTTAGCTTTGAAATAACGCTTTAAAACAAACTTTAGCTTTAAAATAAACT -911

SHR -910 CCACTTTAGCAACATACCATTTTACCTAGATTCAAACTTCCAGAGGCATT -861 WKY -910 CCACTTTAGCAACATACCATTTTACCTAGATTCAAACTTCCAGAGGCATT -861 RefSeq -910 CCACTTTAGCAACATACCATTTTACCTAGATTCAAACTTCCAGAGGCATT -861

SHR -860 AACTCAAGCGTACCTTGTAAAAATCCCCATCTGGTAAACATAATTTCAAT -811 WKY -860 AACTCAAGCGTACCTTGTAAAAATCCCCATCTGGTAAACATAATTTCAAT -811 RefSeq -860 AACTCAAGCGTACCTTGTAAAAATCCCCATCTGGTAAACATAATTTCAAT -811

SHR -810 ATCGTTCATATTTATTCAACAGGTTTAACGCGAGGAGCACAGTGGCGTTG -761 WKY -810 ATCGTTCATATTTATTCAACAGGTTTAACGCGAGGAGCACAGTGGCGTTG -761 RefSeq -810 ATCGTTCATATTTATTCAACAGGTTTAACGCGAGGAGCACAGTGGCGTTG -761

SHR -760 GTCTTTTAATCTGCTCCATTTCAATCTAAAAGCAAGTGTTCATTCGGGCG -711 WKY -760 GTCTTTTAATCTGCTCCATTTCAATCTAAAAGCAAGTGTTCATTCGGGCG -711 RefSeq -760 GTCTTTTAATCTGCTCCATTTCAATCTAAAAGCAAGTGTTCATTCGGGCG -711

SHR -710 TTAGCCGAGAACACTCGGGACTTCACAAAGCTTACAGATAGGGGCTCGAA -661 WKY -710 TTAGCCGAGAACACTCGGGACTTCACAAAGCTTACAGATAGGGGCTCGAA -661 RefSeq -710 TTAGCCGAGAACACTCGGGACTTCACAAAGCTTACAGATAGGGGCTCGAA -661

SHR -660 TCGCTGCCACTTTCCGCTGTAGATACAGAAGCCTCCTGGACCGCCGGCTC -611 WKY -660 TCGCTGCCACTTTCCGCTGTAGATACAGAAGCCTCCTGGACCGCCGGCTC -611 RefSeq -660 TCGCTGCCACTTTCCGCTGTAGATACAGAAGCCTCCTGGACCGCCAGCTC -611

SHR -610 CCCGTGGCAGCCTTAGGGAGTTTCTGGCTGAGGCCGAGCCTGGCTAGCAG -561 WKY -610 CCCGTGGCAGCCTTAGGGAGTTTCTGGCTGAGGCCGAGCCTGGCTAGCAG -561 RefSeq -610 CCCGTGGCAGCCTTGGGGAGTTTCTGGCTGAGGCCGAGCCTGGCTAGCAG -561

SHR -560 CGTTGGGGAGTGTGCTTGGGGAAGGGTCCACCTTTGGTGGGGGAGACCAG -511 WKY -560 CGTTGGGGAGTGTGCTTGGGGAAGGGTCCACCTTTGGTGGGGGAGACCAG -511 RefSeq -560 CGTTGGGGAGTGTGCTTGGGGAAGGGTCCACCTTTGGTGGGGGAGACCAG -511

SHR -510 TAGGTCCAGTAGGTCCAGTAGGTCTAAGAAAGCCGCTGGGGACCTTGCGG -461 WKY -510 TAGGTCCAGTAGGTCCAGTAGGTCTAAGAAAGCCGCTGGGGACCTTGCGG -461 RefSeq -510 TAGGTCCAGTAGGTCCAGTAGGTCTAAGAAAGCCGCTGGGGACCTTGCGG -461

SHR -460 TCCGGGACACCTGCTCCGGGAGCGGGAAAAACCTTGCTCGACTGCTTCCC -411 WKY -460 TCCGGGACACCTGCTCCGGGAGCGGGAAAAACCTTGCTCGACTGCTTCCC -411 RefSeq -460 TCCGGGACACCTGCTCCGGGAGCGGGAAAAACCTTGCTCGACTGCTTCCC -411

SHR -410 TCCCAGCGCTCGCAGTTGTCCCAGAGATGCTCCCCAAGTACCGTGTCTGG -361 WKY -410 TCCCAGCGCTCGCAGTTGTCCCAGAGATGCTCCCCAAGTACCGTGTCTGG -361 RefSeq -410 TCCCAGCGCTCGCAGTTGTCCCAGAGATGCTCCCCAAGTACAGTGTCTGG -361

212

SHR -360 TCCCTACAGACCCGCGCGCAGACAGCAGGCAATTCCCGCGACAGGCAATC -311 WKY -360 TCCCTACAGACCCGCGCGCAGACAGCAGGCAATTCCCGCGACAGGCAATC -311 RefSeq -360 TCCCTACAGACCCGCGCGCAGACAGCAGGCAATTCCCGCGACAGGCAATC -311

SHR -310 TAAGCGGTCCCTGCTTTATCTTTCTCTCTGGCAGCGGGACTCGACGGGGA -261 WKY -310 TAAGCGGTCCCTGCTTTATCTTTCTCTCTGGCAGCGGGACTCGACGGGGA -261 RefSeq -310 TAAGCGGTCCCTGCTTTATCTTTCTCTCTGGCAGCGGGACTCGACGGGGA -261

SHR -260 GAAGTAAAGAGGGATCTGGGGGATGCTCACTCTTGGATGTTCCCTTCTCC -211 WKY -260 GAAGTAAAGAGGGATCTGGGGGATGCTCACTCTTGGATGTTCCCTTCTCC -211 RefSeq -260 GAAGTAAAGAGGGATCTGGGGGATGCTCACTCTTGGATGTTCCCTTCTCC -211

SHR -210 TCTCAGAGCGGGCTGCCTGGAATTGGGGTGTGGGTGGCTCCAGACGCCGC -161 WKY -210 TCTCAGAGCGGGCTGCCTGGAATTGGGGTGTGGGTGGCTCCAGACGCCGC -161 RefSeq -210 TCTCAGAGCGGGCTGCCTGGAATTGGGGTGTGGGTGGCTCCAGACGCCGC -161

SHR -160 CACTCGAGCGGCTGTGGCTCCAGCCTCCTCCCCCGCTGCTGGGGGCGGGA -111 WKY -160 CACTCGAGCGGCTGTGGCTCCAGCCTCCTCCCCCGCTGCTGGGGGCGGGA -111 RefSeq -160 CACTCGAGCGGCTGTGGCTCCAGCCTCCTCCCCCGCTGCTGGGGGCGGGA -111

SHR -110 AGTGGCTGTGGGAGTCACCCGGGCGTGACTGCCCCCGAGGCCCCTCCTGC -61 WKY -110 AGTGGCTGTGGGAGTCACCCGGGCGTGACTGCCCCCGAGGCCCCTCCTGC -61 RefSeq -110 AGTGGCTGTGGGAGTCACCCGGGCGTGACTGCCCCCGAGGCCCCTCCTGC -61

SHR -60 CGCGACAAGGGCGCTCCATAAAAGCCCGTTGGCGACCCGCTCTACGCATC -11 WKY -60 CGCGACAAGGGCGCTCCATAAAAGCCCGTTGGCGACCCGCTCTACGCATC -11 RefSeq -60 CGCGACAAGGGCGCTCCATAAAAGCCCGTTGGCGACCCGCTCTACGCATC -11  +1|-1  SHR -10 CCACCGGTGGagctcattcctcgcagaggcgcccagagcagagcacccgc +40 WKY -10 CCACCGGTGGagctcattcctcgcagaggcgcccagagcagagcacccgc +40 RefSeq -10 CCACCGGTGGagctcattcctcgcagaggcgcccagagcagagcacccgc +40  EXON 1| SHR +41 tgcgcagagaccacagcccgcccgccATGGTGAGTGCCAGGACCAACTGG +90 WKY +41 tgcgcagagaccacagcccgcccgccATGGTGAGTGCCAGGACCAACTGG +90 RefSeq +41 tgcgcagagaccacagcccgcccgccATGGTGAGTGCCAGGACCAACTGG +90

SHR +91 GACAGCGGTGCGGGCCCCTAGACTCCCTTGAACTTGCCCTGCAGCCGGTC +140 WKY +91 GACAGCGGTGCGGGCCCCTAGACTCCCTTGAACTTGCCCTGCAGCCGGTC +140 RefSeq +91 GACAGCGGTGCGGGCCCCTAGACTCCCTTGAACTTGCCCTGCAGCCGGCC +140

SHR +141 CCCTGAGCTTGTTCTGCCAACTTGACACCCNNNNNNNNNNNNNNNNNNNN +190 WKY +141 CCCTGAGCTTGTTCTGCCAACTTGACACCCNNNNNNNNNNNNNNNNNNNN +190 RefSeq +141 CCCTGAGCTTGTTCTGCCAACTTGACACCCAGCTCTTTGGGGCAGCTAAA +190

SHR +191 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +240 WKY +191 NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +240 RefSeq +191 TTTCACTTGCTGGACTCTGGTTCGACCCTCCACCTGTCCTTCTCCGGAGC +240

SHR +241 NNNNNNNNNNNNNNN ...... NNNNNNNNNNNNNNN +690 WKY +241 NNNNNNNNNNNNNNN ...... NNNNNNNNNNNNNNN +690 RefSeq +241 CCTCCTATCTAGGCG ...... GGCGGAGCAGAGGGG +690

SHR +691 CCAGGTCCGGGCAAGTGGTTCTCTAAGGCTCTGGAAGTGGAGCCTGCCCA +740 WKY +691 CCAGGTCCGGGCAAGTGGTTCTCTAAGGCTCTGGAAGTGGAGCCTGCCCA +740 RefSeq +691 CCAGGTCCGGGCAAGTGGTTCTCTAGGGCTCTGGAAGTGGAGCCTGCCCA +740

213

SHR +741 ATCTGGGCTTTTTTTCCTAGGGTCTGGGATGGGAATAGATGAGGGTCTAG +790 WKY +741 ATCTGGGCTTTTTTTCCTAGGGTCTGGGATGGGAATAGATGAGGGTCTAG +790 RefSeq +741 ATCTGGGCTTTTTTTCCTAGGGTCTGGGATGGGAATAGATGAGGGTCTAG +790

SHR +791 AGTGGTAGCTGGAATCGGGACAAAGGCGAGCATTCTCTGCATCTCCAAGT +840 WKY +791 AGTGGTAGCTGGAATCGGGACAAAGGCGAGCATTCTCTGCATCTCCAAGT +840 RefSeq +791 AGTGGTAGCTGGAATCGGGACAAAGGCGAGCATTCTCTGCATCTCCAAGT +840 |EXON 2  SHR +841 CTGAGCCTTCTGTATCCACAGATGCTAGGTAACAAACGAATGGGGCTGTG +890 WKY +841 CTGAGCCTTCTGTATCCACAGATGCTAGGTAACAAACGAATGGGGCTGTG +890 RefSeq +841 CTGAGCCTTCTGTATCCACAGATGCTAGGTAACAAACGAATGGGGCTGTG +890

SHR +891 TGGACTGACCCTCGCTCTATCCCTGCTCGTGTGTTTGGGCATTCTGGCTG +940 WKY +891 TGGACTGACCCTCGCTCTATCCCTGCTCGTGTGTTTGGGCATTCTGGCTG +940 RefSeq +891 TGGACTGACCCTCGCTCTATCCCTGCTCGTGTGTTTGGGCATTCTGGCTG +940

SHR +941 AGGGGTACCCCTCCAAGCCGGACAATCCGGGCGAGGACGCGCCAGCAGAG +990 WKY +941 AGGGGTACCCCTCCAAGCCGGACAATCCGGGCGAGGACGCGCCAGCAGAG +990 RefSeq +941 AGGGGTACCCCTCCAAGCCGGACAATCCGGGCGAGGACGCGCCAGCAGAG +990

SHR +991 GACATGGCCAGATACTACTCCGCTCTGCGACACTACATCAATCTCATCAC +1040 WKY +991 GACATGGCCAGATACTACTCCGCTCTGCGACACTACATCAATCTCATCAC +1040 RefSeq +991 GACATGGCCAGATACTACTCCGCTCTGCGACACTACATCAATCTCATCAC +1040  EXON 2| SHR +1041 CAGACAGAGGTGGGTGTATCCGCGGCTGGTATCTCGAGCCCCAAAAAACT +1090 WKY +1041 CAGACAGAGGTGGGTGTATCCGCGGCTGGTATCTCGAGCCCCAAAAAACT +1090 RefSeq +1041 CAGACAGAGGTGGGTGTATCCGCGGCTGGTATCTCGAGCCCCAAAAAACT +1090

SHR +1091 GCGGTTCTGGGAATCTTGGACGCCAGAGACCATTTCTTTCTCCTTGTTCT +1140 WKY +1091 GCGGTTCTGGGAATCTTGGACGCCAGAGACCATTTCTTTCTCCTTGTTCT +1140 RefSeq +1091 GCGGTTCTGGGAATCTTGGACGCCAGAGACCATTTCTTTCTCCTTGTTCT +1140

SHR +1141 GTCCCAGAATAGGACAGGATCCGGCATATATTCAGCTCCAGATAAATATG +1190 WKY +1141 GTCCCAGAATAGGACAGGATCCGGCATATATTCAGCTCCAGATAAATATG +1190 RefSeq +1141 GTCCCAGAATAGGACAGGATCCGGCATATATTCAGCTCTAGATAAATATG +1190

SHR +1191 TGAGATGGGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1240 WKY +1191 TGAGATGGGTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +1240 RefSeq +1191 TGAGATGGGTACACACGCCACAAACTGGATACATTGTCTTTGTCACTAGA +1240

SHR +1241 NNNNNNNNNNNNNNN ...... NNNNNTTTTTCTCCT +5090 WKY +1241 NNNNNNNNNNNNNNN ...... NNNNNTTTTTCTCCT +5090 RefSeq +1241 CTTTCTCTCCAACCC ...... CTCCCTTTTTCTCCT +5090

SHR +5091 ATGGTTACTCCAAGCTTACTTTATAAAACCTTGATTTTACTTTCTTGTTT +5140 WKY +5091 ATGGTTACTCCAAGCTTACTTTATAAAACCTTGATTTTACTTTCTTGTTT +5140 RefSeq +5091 ATGGTTACTCCAAGCTTACTTTATAAAACCTTGATTTTACTTTCTTGTTT +5140 |EXON 3  SHR +5141 CAGATATGGCAAGAGATCCAGCCCTGAGACACTGATTTCAGATCTCTTAA +5190 WKY +5141 CAGATATGGCAAGAGATCCAGCCCTGAGACACTGATTTCAGATCTCTTAA +5190 RefSeq +5141 CAGATATGGCAAGAGATCCAGCCCTGAGACACTGATTTCAGATCTCTTAA +5190

214

 EXON 3| SHR +5191 TGAGAGAAAGCACAGAAAATGCCCCCAGAACAAGGTATGGCCAAGCTAGG +5240 WKY +5191 TGAGAGAAAGCACAGAAAATGCCCCCAGAACAAGGTATGGCCAAGCTAGG +5240 RefSeq +5191 TGAGAGAAAGCACAGAAAATGCCCCCAGAACAAGGTATGGCCAAGCTAGG +5240

SHR +5241 GATGGAGATGTTGCTACAGAGCTTAAGGTGCCAGGCAAGGAGATCTAGGG +5290 WKY +5241 GATGGAGATGTTGCTACAGAGCTTAAGGTGCCAGGCAAGGAGATCTAGGG +5290 RefSeq +5241 GATGGAGATGTTGCTACAGAGCTTAAGGTGCCAGGCAAGGAGATCTAGGG +5290

SHR +5291 GAGTCTGTGTGAACATTAGGATAGCAGCATCCGGGAAAGAGGGGTTAGGG +5340 WKY +5291 GAGTCTGTGTGAACATTAGGATAGCAGCATCCGGGAAAGAGGGGTTAGGG +5340 RefSeq +5291 GAGTCTGTGTGAACATTAGGATAGCAGCATCCGGGAAAGAGGGGTTAGGG +5340

SHR +5341 TAGACACAGGGGATGGAGAAGTATACATGAAAGACCTTGCATTTCATGCA +5390 WKY +5341 TAGACACAGGGGATGGAGAAGTATACATGAAAGACCTTGCATTTCATGCA +5390 RefSeq +5341 TAGACACAGGGGATGGAGAAGTATACATGAAAGACCTTGCATTTCATGCA +5390

SHR +5391 TTCACAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +5440 WKY +5391 TTCACAGCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN +5440 RefSeq +5391 TTCACAGCAAATCTCAACTTTCAAGGCATATTGTTTTCTAATACATATTA +5440

SHR +5441 NNNNNNNNNNNNNNN ...... CAGGAGGCTTCACAATAG +6890 WKY +5441 NNNNNNNNNNNNNNN ...... CAGGAGGCTTCACAATAG +6890 RefSeq +5441 TTTGGTGTCACTTTT ...... CAGGAGGCTTCACAATAG +6890

SHR +6891 CTTCTGATTATCTTGTAGCTCAGGGTGGAGAGTCTCACTCTTGTAAGACC +6940 WKY +6891 CTTCTGATTATCTTGTAGCTCAGGGTGGAGAGTCTCACTCTTGTAAGACC +6940 RefSeq +6891 CTTCTGATTATCTTGTAGCTCAGGGTGGAGAGTCTCACTCTTGTAAGACC +6940 |EXON 4  SHR +6941 CCGGGAGCTCCCATGTTTTGCCTCTTGTGTTTTACAGGCTTGAAGACCCT +6990 WKY +6941 CCGGGAGCTCCCATGTTTTGCCTCTTGTGTTTTACAGGCTTGAAGACCCT +6990 RefSeq +6941 CCGGGAGCTCCCATGTTTTGCCTCTTGTGTTTTACAGGCTTGAAGACCCT +6990 |3’UTR  SHR +6991 TCCATGTGGTGAtgggaaatgaaacttgctctcctgacttttcctagttt +7040 WKY +6991 TCCATGTGGTGAtgggaaatgaaacttgctctcctgacttttcctagttt +7040 RefSeq +6991 TCCATGTGGTGAtgggaaatgaaacttgctctcctgacttttcctagttt +7040

SHR +7041 ccccccacatctcatctcatcctgtgaaaccagtctgcctgtcccaccaa +7090 WKY +7041 ccccccacatctcatctcatcctgtgaaaccagtctgcctgtcccaccaa +7090 RefSeq +7041 ccccccacatctcatctcatcctgtgaaaccagtctgcctgtcccaccaa +7090

SHR +7091 tgcatgccaccaccaggctggattccgacccatttcccttgttgtcgttg +7140 WKY +7091 tgcatgccaccaccaggctggattccgacccatttcccttgttgtcgttg +7140 RefSeq +7091 tgcatgccaccaccaggctggattccgacccatttcccttgttgtcgttg +7140  EXON 4| SHR +7141 tatatatgtgtgtttaaataaagtatcatgcattcAAAATTGTGTTCTCT +7190 WKY +7141 tatatatgtgtgtttaaataaagtatcatgcattcAAAATTGTGTTCTCT +7190 RefSeq +7141 tatatatgtgtgtttaaataaagtatcatgcattcAAAATTGTGTTCTCT +7190

SHR +7191 GTGAATAATCTGCTATCACAATAGAAAGGATTAGGTTAGCCCTTAAATTA +7240 WKY +7191 GTGAATAATCTGCTATCACAATAGAAAGGATTAGGTTAGCCCTTAAATTA +7240 RefSeq +7191 GTGAATAATCTGCTATCACAATAGAAAGGATTAGGTTAGCCCTTAAATTA +7240

215

SHR +7241 TAATCAGCCTCAAGTAACAAACTCAGTTTGTTTCACCAACATTAAGCAAT +7290 WKY +7241 TAATCAGCCTCAAGTAACAAACTCAGTTTGTTTCACCAACATTAAGCAAT +7290 RefSeq +7241 TAATCAGCCTCAAGTAACAAACTCAGTTTGTTTCACCAACATTAAGCAAT +7290

SHR +7291 GGTCATGAAGAGTAAATAAATAATTATGCTGCCCTTGGAAGAATTTATCT +7340 WKY +7291 GGTCATGAAGAGTAAATAAATAATTATGCTGCCCTTGGAAGAATTTATCT +7340 RefSeq +7291 GGTCATGAAGAGTAAATAAATAATTATGCTGCCCTTGGAAGAATTTATCT +7340

SHR +7341 CTTAGAACTTTGTGCAAAATGTCTCATGTCCATCTATCTACTATAGCA +7388 WKY +7341 CTTAGAACTTTGTGCAAAATGTCTCATGTCCATCTATCTACTATAGCA +7388 RefSeq +7341 CTTAGAACTTTGTGCAAAATGTCTCATGTCCATCTATCTACTATAGCA +7388

216

Phenylethanolamine-N-methyltransferase (Pnmt)

Data not available: unpublished data from a collaborator.

Appendix C: Mutagenesis Primers

217 218

The oligonucleotide primers used to create polymorphic variants of the

Chga cDNA/EAP or Pnmt promoter/luciferase constructs are listed below.

Chga cDNA mutagenesis primers

Effect SNP on Primer Primer Sequence target SNP 5’-AAAGAGGAAGAGGAGGAGGAGAAAGAGGAGAA Deletion rCGA_Glu_Mut_F GGCGATCGCCAGAGAGAAGGCT-3’ GLN of 8 GLN 5’-CTCCTCCTCTTCCTCTTTCTCCTCTTGCTTGGCT repeat residues rCGA_Glu_Mut_R TTTCTGGCTTGCTGCTGGGCACTGGGACC-3’ (24 bp)

Pnmt promoter mutagenesis primers

Effect SNP on Primer Primer Sequence (5’3’) target SNP PnmtSNP1_F AGGCCAGAACAGAGTGTCCTCTCTGAAGGAGGATAG BNSHR T-548C PnmtSNP1_R CTATCCTCCTTCAGAGAGGACACTCTGTTCTGGCCT (TC)

PnmtSNP2_F AGAAGGGGAGTTTGTAAGGGTACCCCGAGAG In/Del A- BNSHR PnmtSNP2_R CTCTCGGGGTACCCTTACAAACTCCCCTTCT 476 (A--)

PnmtSNP3_F CTGGGACTGGGAACACGAGCTAGCTCAGACCTTGG BNSHR T-423C PnmtSNP3_R CCAAGGTCTGAGCTAGCTCGTGTTCCCAGTCCCAG (TC)

PnmtSNP4_F ACTGGGAACATGAGCTAGTTCAGACCTTGGGAAAGAG BNSHR C-415T PnmtSNP4_R CTCTTTCCCAAGGTCTGAACTAGCTCATGTTCCCAGT (CT)

PnmtSNP5_F CTGGACGCTGGATGGAGTCTGTGGGAGG BNSHR C-370T PnmtSNP5_R CCTCCCACAGACTCCATCCAGCGTCCAG (CT)

PnmtSNP1b_F AGGCCAGAACAGAGTGTCCTTTCTGAAGGAGGATAG SHRBN T-548C PnmtSNP1b_R CTATCCTCCTTCAGAAAGGACACTCTGTTCTGGCCT (CT)

PnmtSNP2b_F AGAAGGGGAGTTTGTAAAGGGTACCCCGAGAG In/Del A- SHRBN PnmtSNP2b_R CTCTCGGGGTACCCTTTACAAACTCCCCTTCT 476 (--A)

PnmtSNP3b_F CTGGGACTGGGAACATGAGCTAGTTCAGACCTTGG SHRBN T-423C PnmtSNP3b_R CCAAGGTCTGAACTAGCTCATGTTCCCAGTCCCAG (CT)

PnmtSNP4b_F ACTGGGAACACGAGCTAGCTCAGACCTTGGGAAAGAG SHRBN C-415T PnmtSNP4b_R CTCTTTCCCAAGGTCTGAGCTAGCTCGTGTTCCCAGT (TC)

PnmtSNP5b_F CTGGACGCTGGACGGAGTCTGTGGGAGG SHRBN C-370T PnmtSNP5b_R CCTCCCACAGACTCCGTCCAGCGTCCAG (TC)