Genetics and Hypertension (2015)

Colloquium series on Colloquium series on
CHENG • JOE
ISSN
ISSN 2154-560X
2154-560X
integrated systems Physiology: integrated systems Physiology:

from moleCule to funCtion to disease from moleCule to funCtion to disease
Series Editors: D. Neil Granger, Louisiana State University
Series Editors: D. Neil Granger & Joey Granger
Joey Granger, University of Mississippi School of Medicine
Physiological
Genetics and Hypertension
Physiological Genomics
Xi
Xi Cheng
Genomics Program,
Cheng and
Program, Center
and Bina
Center for
Bina Joe
Joe
for Hypertension
Hypertension and
and Personalized
Personalized Medicine,
Medicine,
Genetics and
Hypertension
Department
Department of
of Physiology
Physiology and
and Pharmacology,
Pharmacology, University
University of
of Toledo
Toledo College
College of
of Medicine
Medicine and
and Life
Life Sciences
Sciences
Hypertension,
Hypertension, or or elevated
elevated blood
blood pressure,
pressure, is
is aa major
major risk
risk factor
factor for
for various
various cardiovascular,
cardiovascular, renal
renal diseases,
diseases,
GENETICS AND HYPERTENSION

and
and stroke.
stroke. The
The form
form of of hypertension
hypertension withwith no no identifiable
identifiable cause
cause isis referred
referred toto as
as Essential
Essential Hyperten-
Hyperten-
sion.
sion. Familial
Familial studies
studies indicate
indicate that
that Essential
Essential Hypertension
Hypertension is is heritable
heritable and,
and, thereby,
thereby, classical
classical genetic
genetic
approaches
approaches havehave been
been applied
applied onon both
both human
human and and other
other mammalian
mammalian modelsmodels ofof hypertension
hypertension to to map
map the
the
locations
locations ofof the
the allelic
allelic variants
variants within
within quantitative
quantitative trait
trait loci
loci for
for blood
blood pressure.
pressure.The
The post
post genome
genome era era has
has
further
further elevated
elevated this
this area
area of
of research
research into
into large-scale
large-scale genomewide
genomewide association
association studies
studies ofof hypertension
hypertension
in
in humans.
humans. Collectively,
Collectively, these
these studies
studies have
have resulted
resulted in
in the
the prioritization
prioritization andand cataloging
cataloging ofof several
several ge-
ge-
nomic
nomic regions
regions containing
containing allelic
allelic variants
variants as
as candidates
candidates linked
linked oror associated
associated with
with essential
essential hypertension.
hypertension.
Further,
Further, they
they are
are providing
providing evidence
evidence toto suggest
suggest thatthat the
the inheritance
inheritance of of hypertension
hypertension is is rather
rather complex,
complex,
encompassing
encompassing multiple
multiple variants
variants both
both within
within protein-coding
protein-coding and and non-coding
non-coding annotations,
annotations, each
each ofof which
which
may
may act
act independently
independently or or interactively
interactively with
with other
other genes
genes and/or
and/or environmental
environmental factors
factors to
to differentially
differentially
regulate
regulate blood
blood pressure.
pressure. This
This book
book provides
provides an an overview
overview of of the
the various
various methods
methods employed
employed to to study
study the
the
genetics
genetics of
ute
ute towards
of hypertension
hypertension and
towards individualized
and discuss
discuss the
individualized clinical
the progress
progress and
clinical management
management of
and prospects
prospects of
of hypertension
hypertension in
of this
this area
in the
area of
of research
the future.
future.
research that
that may
may contrib-
contrib- Xi Cheng
Bina Joe
This
This volume
volume isis aa printed
printed version
version of
of aa work
work that
that appears
appears in
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Colloquium Series on
Integrated Systems Physiology:
From Molecule to Function to Disease
Editors
D. Neil Granger, Louisiana State University Health Sciences Center
Joey P. Granger, University of Mississippi Medical Center
Physiology is a scientific discipline devoted to understanding the functions of the body. It addresses
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Genetics and Hypertension

Xi Cheng and Bina Joe
www.morganclaypool.com
ISBN: 9781615046942 paperback
ISBN: 9781615046959 ebook
DOI: 10.4199/C00131ED1V01Y201506ISP062
A Publication in the
Colloquium Series on Integrated Systems Physiology: From Molecule to

Function to Disease
Lecture #62
Series Editor: D. Neil Granger, LSU Health Sciences Center, and Joey P. Granger, University of Mississippi
Medical Center
Series ISSN
ISSN 2154-560X print
ISSN 2154-5626 electronic
Genetics of Hypertension
Xi Cheng and Bina Joe
Physiological Genomics Program
Center for Hypertension and Personalized Medicine
Department of Physiology and Pharmacology
University of Toledo College of Medicine and Life Sciences
COLLOQUIUM SERIES ON Integrated Systems Physiology:

From Molecule to Function to Disease #62
Abstract
Hypertension, or elevated blood pressure, is a major risk factor for various cardiovascular, renal
diseases, and stroke. The form of hypertension with no identifiable cause is referred to as Essen-
tial Hypertension. Familial studies indicate that Essential Hypertension is heritable and, thereby,
classical genetic approaches have been applied on both human and other mammalian models of
hypertension to map the locations of the allelic variants within quantitative trait loci for blood
pressure. The post genome era has further elevated this area of research into large-scale genome-
wide association studies of hypertension in humans. Collectively, these studies have resulted in the
prioritization and cataloging of several genomic regions containing allelic variants as candidates
linked or associated with essential hypertension. Further, they are providing evidence to suggest
that the inheritance of hypertension is rather complex, encompassing multiple variants both within
protein-coding and non-coding annotations, each of which may act independently or interactively
with other genes and/or environmental factors to differentially regulate blood pressure. This book
provides an overview of the various methods employed to study the genetics of hypertension and
discuss the progress and prospects of this area of research that may contribute towards individual-
ized clinical management of hypertension in the future.
Key Words
linkage, GWAS, variant, non coding, rat, congenic, zinc-finger nucleases, ncRNA, miRNA,
lncRNA, gene, microbiota, genome
vii
Contents
1 Introduction........................................................................................................1
2 Linkage Analyses for Essential Hypertension in Humans.......................................5
3 Genome-wide Association Studies of Human Hypertension..................................7
4 Rat Models in Hypertension Research................................................................ 11
5 Genetic Linkage Analysis Using Hypertensive Rat Models.................................. 13
6 Substitution Mapping Using Congenic Strains.................................................... 15
7 Genome-Editing Tools for Rat Hypertension Research....................................... 17

7.1 Overview of Genome-Editing Techniques......................................................... 17
7.2 The Application of Genome-Editing Tools in Hypertensive Rat Models ........ 20
8 Beyond Protein-Coding Genes- “Missing Heritability”........................................ 25
9 MicroRNA and Hypertension............................................................................. 27

9.1 What is MicroRNA?.......................................................................................... 27
9.2 The Role of MicroRNA in Blood Pressure Regulation...................................... 29
9.2.1 Polymorphisms in MicroRNA Binding Sites......................................... 29
9.2.2 Polymorphisms within MicroRNAs....................................................... 32
10 Long Non-coding RNA and Hypertension.......................................................... 33

10.1 What is Long Non-coding RNA?...................................................................... 33
10.2 The Role of Long Non-coding RNA in Blood Pressure Regulation.................. 35
11 Gut Microbiota and Hypertension...................................................................... 39
12 Summary and Perspectives.................................................................................. 41
References.................................................................................................................. 43
Author Biographies..................................................................................................... 55

chapter 1
Introduction
Hypertension, also known as high blood pressure, is a serious disorder in which blood pressure is
consistently elevated in the arteries. Blood pressure is the force exerted by circulating blood against
the walls of arteries when the heart pumps blood to the body. Blood pressure is usually measured
by systolic blood pressure (SBP) and diastolic blood pressure (DBP) in the arteries. The SBP is
referred to as the maximum arterial pressure when the heart contracts and the DBP is referred to
as the minimum arterial pressure during the dilation of the heart. Normal SBP is in the range of
100–140 mm mercury (mmHg) and normal DBP is in the range of 60–90 mmHg. Hypertension is
defined as a SBP at or above 140 mmHg and a DBP at or above 90 mmHg.
Hypertension in most patients is classified as either essential (or primary) hypertension or
secondary hypertension. Essential hypertension is not associated with any obvious medical cause
and accounts for about 95% cases of hypertension. The remaining ~5% cases are classified under
secondary hypertension, which has an identifiable medical cause, such as pre-existing kidney dis-
ease, diabetes, or obesity.
Hypertension is referred to as the “silent killer” because it often has no warning signs or
symptoms. However, hypertension remains a major risk factor for cardiovascular diseases, such as
cardiomyopathy [7], coronary artery diseases [8], and peripheral vascular diseases [9]. In the United
States, nearly 78 million adults (1 in every 3) have high blood pressure [10], but only about half
(47%) of people with high blood pressure have their condition under control. Hypertension con-
tributes to over 200,000 deaths annually [11]. The annual medical costs attributable to hyperten-
sion are projected to increase by 186% from $150 billion to $274 billion between 2015 and 2030
(Figure 1) [1].
Essential hypertension is characterized as a multi-factorial disease (Figure 2). It is estimated
that ~70–80% is due to environmental factors including stress, high salt intake and poor physical
activity. While hypertension can be controlled with lifestyle choices that render favorable environ-
mental factors for the maintenance of blood pressure, it is also clear that making these beneficial
lifestyle choices alone is insufficient to control the development and progression of hypertension.
In addition, clinical management of hypertension is limited by the fact that knowledge regarding
the primary factors that cause the onset of hypertension is lacking. This brings us to the question of
genetics of hypertension
FIGURE 1: Projected total costs of cardiovascular disease (CVD), 2015–2030 (2012 $ in billions) in
the United States. CHD: coronary heart disease; CHF, congestive heart failure; and HBP, high blood
pressure [1]. Used with permission by AHA, Wolters Kluwer [1].
what other factors influence the development of hypertension. A notable feature of hypertension is
that it is heritable in families.
Familial and twin studies show that 30–50% of the phenotypic variation of blood pressure
is attributable to genetic heritability [12]. Estimated from familial studies, the heritability of hy-
pertension is between 31% and 68% [13, 14]. Despite these well-documented observations on the
definitive role of genetics in hypertension, the identities of the precise genetic elements confer-
ring susceptibility to the development of essential hypertension remain largely unknown. Recent
research using both human population studies and experimental models of hypertension indicates
that the genetic control of blood pressure is complex and facilitated by the concerted action of a
largely underestimated number of genes, each of which may function individually or interactively.
The historical progression of classical genetic approaches applied to both human and experimental
animal model research that lead to this overall conclusion and a perspective on how such research
is poised to shed light on the “blueprint” of the genetics of hypertension, will be presented in this
book.
introduction
FIGURE 2: The influence of genetic and environmental factors on hypertension.
• • • •

chapter 2
Linkage Analyses for Essential

Hypertension in Humans
Using DNA from related subjects, linkage studies were the early genome-wide studies conducted to
identify the genetic loci associated with human hypertension. The approach of linkage studies is to
look for linkage (or a correlation) between a genetic marker used to denote a region on a chromo-
some and the extent of blood pressure. The genetic marker is an allelic variation of either a single or
a continuous stretch of many nucleotides. In simple terms, within a family, the allele associated with
hypertension is expected to be inherited by the individuals who have hypertension and the alternate
allele at the same locus is inherited by the individuals who do not present with hypertension.
Several linkage studies of hypertension have been performed in humans of different races.
Examples include a linkage study of 1054 individuals from 188 rural families from Nigeria, wherein
several significant loci were identified to be linked to blood pressure on human chromosome 6
and 7 [15]. A study of 328 Chinese individuals, comprising 111 hypertensive probands and their
siblings, identified significant SNPs associated with hypertension on chromosome 5 and also identi-
fied several regions associated with systolic and diastolic blood pressure on chromosomes 2 and 5
[16] and a large meta-analysis of nine genome wide scans from Caucasian populations [17], which
detected linkage to blood pressure of two regions on chromosome 2 (2p12-q22.1) and chromosome
3 (3p14.1-q12.3).
A more organized approach to linkage analyses was applied in 1995 by the National Heart,
Lung, and Blood Institute (NHLBI) of the National Institutes of Health (NIH), which funded the
Family Blood Pressure Program (FBPP) as a research resource to study the genetics of hypertension.
FBPP consisted of four independent multicenter networks—GenNet (University of Michigan),
GENOA (University of Texas at Houston), HyperGEN (University of Utah), and SAPPHIRe
(Stanford)—each of which recruited the subjects from the African American, Mexican Ameri-
can, Asian, and non-Hispanic white populations and aimed to study the genetic determinants of
inter-individual BP variation [18]. One important linkage study by using the FBPP resource was
published by Rao et al. in 2011 [19]. This study includes a total of 13,044 individuals consisting of
4,226 African American, 2,154 Asian, 4,229 Caucasian, and 2,435 Mexican American participants.
This study identified five quantitative trait loci (QTLs) on chromosomes 6p22.3, 8q23.1, 20q13.12,
21q21.1, and 21q21.3 [19].
Data collected from linkage studies have provided insights into novel loci on the human
genome that are inherited and thereby interpreted to causally influence the development of hyper-
tension. However, linkage studies have limitations, the major one being that the regions identified
are large encompassing several genes. In order to achieve a greater precision in mapping regions by
linkage, one has to rely on meiotic recombinations to occur within shorter intervals, which requires
a larger population size. Even so, there are regions on the genome that are relatively resistant to
recombinations, which makes linkage analyses particularly challenging and thus thwarts the identi-
fication of each of the allelic variants underlying the large segments identified as novel quantitative
trait loci for blood pressure.
• • • •

chapter 3
Genome-wide Association Studies

of Human Hypertension
Since the human genome sequence was deciphered, genome-wide association studies (GWAS)
gained importance as a means to extensively probe the genome at the resolution of single nucleotide
polymorphisms and their association with various human disorders including hypertension. In a
GWAS, information regarding the status of allelic variants are gathered through whole genome
sequencing of individuals and associated with diseases. For example, through comparing the whole
genome sequences between hypertensive patients and normotensive individuals, we may find that
hundreds or thousands of single nucleotide polymorphisms (SNPs) occur more frequently in the
hypertensive patients than normotensive individuals, by which we recognize that these SNPs may
be associated with the development of hypertension.
The first GWAS for hypertension was conducted in 2007 by the Wellcome Trust Case Con-
trol Consortium suing 14,000 individuals [20]. This study did not detect any loci with significant
association to hypertension. Two years later, a large GWAS study including 34,433 subjects of Euro-
pean ancestry from the Global Blood Pressure Genetics (Global BPgen) consortium was conducted
to test 2.5 million imputed SNPs [21]. This study identified eight loci associated with systolic blood
pressure (SBP) or diastolic blood pressure (DBP) and the variants in these eight regions were near
the CYP17A1, CYP1A2, FGF5, SH2B3, MTHFR, c10orf107, ZNF652, and PLCD3 genes. In the
same year, another large GWAS study including 21,136 subjects from the Cohorts for Heart and
Aging Research in Genome Epidemiology (CHARGE) Consortium was reported by Levy et al
[22]. From 2.5 million imputed SNPs, this study identified 13 SNPs associated with SBP, 20 SNPs
associated with DBP, and 10 SNPs associated with hypertension. The authors further tested the
GWAS significant SNPs in 34,433 independent subjects from the Global BPgen Consortium. The
joint meta-analysis identified four loci significantly associated with SBP, six loci significantly associ-
ated with DBP and one locus significantly associated with hypertension. These novel loci included
ATP2B1 (for SBP, DBP, and hypertension), SH2B3 (for DBP), and TBX3/TBX5 (for DBP). An-
other GWAS study including 200,000 individuals of European descent identified 16 novel loci
associated with blood pressure regulation [23]. In this study, 6 of these 16 loci were identified as
being near the genes previously reported to be associated with blood pressure [23]. Despite the
genetic loci associated with SBP and DBP, one GWAS study with a discovery-phase sample of
74,064 individuals reported four novel loci associated with pulse pressure (PP), two novel loci as-
sociated with mean arterial pressure (MAP) and one locus associated with both PP and MAP [24].
Another meta-analysis of GWAS study including 19,608 East Asian participants from the AGEN-
BP consortium identified five novel loci associated with SBP or DBP and also confirmed seven loci
previously reported in European descent [25]. Together, all these studies are accumulating a large
body of evidence regarding locations on the genome that are associated with hypertension. As newer
GWAS are constantly reported in the literature for all disorders including hypertension, up-to-date
information on published GWAS studies can be obtained from an online catalog of all GWAS
studies maintained by the National Institutes of Health at (https://www.genome.gov/26525384).
Although much work has been done using GWAS as a strategy, researchers are divided in
their opinions about the contributions of GWAS to the progress in our understanding of the ge-
netic contributions to human diseases. Among many reasons debated [26, 27], a prominent one is
that GWAS does not take into account the heritability aspect because it is a study of large groups of
individuals that are not all related. Another drawback of GWAS is that it lacks the power to differ-
entiate between causal factors and casually associated factors. Validation studies were badly needed
to substantiate the loci identified by GWAS. This need has prompted post-GWAS validation stud-
ies that shed further light on some of the GWAS prioritized candidate genes for hypertension. For
example, the SH2B adaptor protein 3 (SH2B3) is one of the genes verified by several GWAS stud-
ies and further studied for its molecule significance in hypertension. SH2B3 was reported to be as-
sociated with blood pressure in previous GWAS studies [28, 29]. Based on computational GWAS
prediction, the experimental procedures have been performed to study and verify the physiological
role of SH2B3 in regulating blood pressure. Rudemiller et al. targeted SH2B3 for mutation in the
genetic background of the hypertensive Dahl salt-sensitive (S) rat by using the genome-editing tool
zincfiger nucleases [30]. The mutation in the Src homology 2 (SH2) domain of SH2B3 significantly
decreased the blood pressure in the mutant S rat, which confirmed the association of SH2B3 with
hypertension in GWAS study. The authors also demonstrated SH2B3 may influence the blood pres-
sure through mediating the immune system as its potential molecular mechanism [30]. Levy et al.
further confirmed the SNP rs3184504 in SH2B3 could function as a trans-regulator of another six
transcripts associated with blood pressure [31] and also demonstrated that the expression levels of
several genes predicted to be mediated by SH2B3 were changed in SH2B3 knockout mice. An-
other GWAS study from the HYPERGENES Project including a discovery phase of 1,865 hyper
tensive cases and 1,750 controls identified a novel locus (rs3918226) in the promoter region of the
Genome-wide Association Studies of Human Hypertension
endothelial NO synthase gene [32]. The endothelial NO synthase is important for blood pressure
control and the variation in its promoter region may influence the gene transcription and thereby
cause the blood pressure variation. These studies and several more that will be detailed later in this
book demonstrate that GWAS serve as a screening step and thereby enable us to prioritize variants
that may regulate blood pressure.
• • • •
11
chapter 4
Rat Models in Hypertension Research
Driven by the ease to conduct custom breeding using smaller mammals such as rats and mice, prior
to the systematic genetic investigation of blood pressure in humans, experimental research in the
genetic analysis of blood pressure, particularly in rats, was underway. Over the past 50 years, several
animal models of essential hypertension, predominantly in the rat, have been developed that mimic
essential hypertension. Several inbred strains of hypertensive rat models are widely used genetic
models of identify genes involved in hypertension. A list and brief introduction of rat models for
hypertension research is given below.
1. Spontaneously hypertensive rats (SHR). Spontaneously hypertensive rat (SHR) can de-
velop essential hypertension around 5–6 weeks of age and it was initially bred for high
blood pressure without the involvement of any dietary or environmental intervention by
Okamoto and Aoki [33]. The normotensive Wistar-Kyoto rats (WKY ) are the “control”
stocks of SHR.
2. Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. Dahl salt-sensitive (S) and Dahl
salt-resistant (R) rats were developed from Sprague Dawley rats that were bred on the
basis of their blood pressure after being fed a high-salt (8% NaCl) diet [34, 35]. The S rat
develops hypertension even at low-salt diet and fatal hypertension is developed when fed
with high-salt diet. The R rat remains normotensive even at a high-salt diet. The original
selected rats were maintained as outbred stocks and the inbred S and R rats were developed
from these outbred stocks by the Professor John Paul Rapp at the Medical College of Ohio
in Toledo (now the University of Toledo College of Medicine and Life Sciences) in the
United States [36].
3. Milan hypertensive strain of rats (MHS), and Milan normotensive strain of rats (MNS).
The Milan rat strains were selected for Milan hypertensive strain of rats (MHS) and Mi-
lan normotensive strain of rats (MNS) without the involvement of any special dietary or
environmental intervention [37].
4. Sabra hypertension-prone (SBH) rats and Sabra hypertension-resistant (SBN) rats. Sa-
bra hypertension-prone (SBH) rats and Sabra hypertension-resistant (SBN) rats were bred
12 genetics of hypertension
on the basis of the blood pressure following unilateral nephrectomy and treatment with
deoxycorticosterone acetate and 1% NaCl (sodium chloride) [38].
5. Fawn-hooded (FH) rats. The fawn-hooded (FH) rat develops hypertension spontane-
ously and the elevation in systolic blood pressure starts at five weeks of age [39]. FH rat is
also characterized as heavy proteinuria [39], mild breeding disorder [40], and glomerular
sclerosis [41].
6. Genetically hypertensive (GH) rats. The genetically hypertensive (GH) rats were selec-
tively bred on the basis of blood pressure without the involvement of any special dietary or
environmental intervention [42].
7. Lyon hypertensive (LH), Lyon low blood pressure (LL), and Lyon normotensive (LN)
rats. The Lyon strains of rats were selectively bred for high (LH), low (LL), or normal
(LN) blood pressure without the involvement of any special dietary or environmental in-
tervention [43].
8. Prague hypertensive rat (PHR) and the Prague normotensive rat (PNR). The Prague
hypertensive rat (PHR) and the Prague normotensive rat (PNR) were selectively bred from
a single pair of breeders.
Each of these rat strains above represents an individual genetic pool of naturally occurring al-
leles that construct an overall phenotype of hypertension. Therefore, these rat models serve as useful
alternates for the genetic analysis of the alleles that are causative of hypertension and understanding
the potential molecular mechanisms of BP control in humans. The genetic experiments of using
these hypertensive rat models are discussed later.
• • • •
13
chapter 5
Genetic Linkage Analysis Using

Hypertensive Rat Models
The first stage is to perform the linkage analysis to identify statistically significant genomic region
associated with BP control. Linkage analysis using rat models of hypertension have been success-
fully applied in searching the genomic regions that may contain genes involved in BP control and
these genomic regions are also known as quantitative trait loci (QTL). Linkage analysis of BP QTL
is usually performed by using the hypertensive rat models typically involving the generation of a
segregating population using a hypertensive rat stain and another inbred strain with a contrasting
phenotype, i.e., a normotensive rat strain. On occasions, the genetic linkage analysis of BP QTL
was also performed on populations generated from two hypertensive strains [44, 45]. Using this ap-
proach, the information on the chromosomal location, the magnitude of BP effect, and the mode of
inheritance of each causative locus can be obtained. To date, multiple genetic linkage analysis have
been performed and the results from these studies show that there are a number of genomic regions
containing BP QTL throughout the rat genome. The detailed BP QTL information is summarized
at the following website: http://rgd.mcw.edu/qtls/.
The major findings from multiple linkage analysis by using hypertensive rat models are listed
as follows.
• Each rat chromosome contains at least one BP QTL, suggesting that the comprehen
sive genetic control of BP is complex and involves the co-regulation of multiple genetic
elements.
• Some BP QTLs are repeatedly detected in multiple genetic linkage analysis by using dif-
ferent hypertensive rat strains, suggesting that these BP QTLs might play more important
roles in BP regulation.
• The type of segregating population indicating the genetic background used for linkage
analysis has an important effect on whether a given QTL would be detected or not.
• Environmental factors, such as diet (salt-feeding) and stress, can influence the result of
linkage analysis.
• BP QTL can be sex dependent. For example, some BP QTLs were detected in males but
not in females, or vice versa.
• BP QTL might depend on the developmentally-specific genetic factors. For example,
Some BP QTLs were detected only at specific ages.
Linkage analysis is an important tool for searching genomic regions that contain BP QTL.
However, similar to linkage studies in humans, there are two recognizable disadvantages: (1) the
detection of BP QTL through linkage analysis relies heavily on the predictions based on statistical
analysis; and (2) the resolution of linkage analysis is too low to identify the actual BP controlling
gene. Linkage analysis results in the identification of broad genomic regions and one BP QTL may
contain hundreds of genes, and therefore it would be very difficult to find the gene(s) responsible
for BP control by simply relying on linkage analysis. However, since the whole-genome sequencing
approaches are increasingly becoming cost-effective, linkage analysis is once again emerging in the
literature as a valuable and powerful tool for understanding the etiology of diseases [46].
• • • •
15
chapter 6
Substitution Mapping Using

Congenic Strains
Initial low-resolution linkage analysis is only the first step for the identification of broad genomic
regions associated with BP control. Therefore, further corroborative genetic experiments are re-
quired to confirm and refine BP QTL for searching the actual gene(s) involved in BP regulation.
To this end, consomic or congenic strains of rats are constructed and studied. The construction of
congenic strains involves the substitution of a segment of a chromosome, or in the case of consomic
strains, the entire chromosome, from one inbred strain (the donor strain) into another inbred strain
(the recipient strain). This process is termed substitution mapping.
First, the donor and recipient strains are cross-bred to produce F1 animals and then F1 are
cross-bred to recipient strains (first backcross). The offspring from this first backcross are genotyped
by using tail biopsy DNA for markers across a putative QTL containing region on a chromo-
some. Because chromosomal crossovers occur between the donor and recipient chromosomes in
the meioses of the F1 animals, recombinant chromosomal regions can be detected in some of the
offspring, which means that an offspring receiving the donor chromosomal segment can be easily
identified. Then the F1 offspring carrying the desired donor chromosomal segment (heterozygous)
are backcrossed-bred again to the recipient strain, and the F2 offspring carrying the specific donor
chromosomal segment are again selected. According to the established protocol constructing the
congenic strains, the procedure is usually repeated at least eight times. At each backcross, about half
of the unlinked heterozygous loci outside the selected (congenic) region become homozygous for
the recipient allele, meaning that the genetic background of the new congenic strain is progressively
becoming that of the recipient strain. After eight backcrosses, rats are selectively bred to fix the
donor chromosomal segment in the homozygous state on the background of the recipient strain, by
which congenic strain is successfully constructed.
After the BP QTL is confirmed in the congenic strain initially generated, new congenic
strains with smaller donor chromosomal segment will be further constructed to refine the location
of the BP QTL to a small genomic interval. This step is usually an iterative process and continues
until the genomic interval contains a small number of genes. To date, many BP QTLs have only
been confirmed using large congenic strains and very few have been localized to small genomic
intervals.
Congenic strains are usually indicated by the following notation: “Recipient strain. Donor
strain”, wherein, “recipient strain” is the strain to whose genome a known genomic region from the
“donor strain” is introgressed. For example, SHR.BN refers to the introgression of a segment of
Brown Norway (BN) rats into the SHR genetic background.
Extensive congenic strains haven been developed to confirm BP QTLs disclosed by linkage
analysis. The origins and progress of most, if not all, of these studies were reviewed in detail by Rapp
in 2000 [42]. Further progress since 2000 were summarized by Joe in 2005 [47].
While substitution mapping has its merits in that one can reliably obtain definitive evidence
for a segment to be harboring a locus that confers a change in blood pressure through the alleles lo-
cated within its physical genomic limits, this method has its limitations. In order to achieve a greater
precision in mapping regions by using substitution mapping, one has to rely on meiotic recombina-
tions to occur within shorter intervals, which requires a larger population size. Even so, there are
regions on the genome that are relatively resistant to recombinations. This renders substitution
mapping particularly challenging, expensive, time-consuming, and thus thwarts the identification
of each of the allelic variants underlying the large segments identified as novel quantitative trait
loci for blood pressure. For almost five years (from 2003–2008) from the year the rat genome was
sequenced, there was no means by which one could further localize blood pressure quantitative trait
loci other than by high-resolution substitution mapping. However, in 2008, the zinc-finger nuclease
based generation of a targeted gene-disruption model was published. Since then, this and several
other gene-disruption strategies have been applied as complementary methods to validate the blood
pressure loci identified by high resolution substitution mapping.
• • • •
17
chapter 7
Genome-Editing Tools for

Rat Hypertension Research
7.1 Overview of Genome-Editing Techniques
In the recent past, several novel genome-editing tools have been developed for target disruption of
a specific gene on the genome. Therefore, these techniques could be used to disrupt (or knock out)
a candidate gene within a substitution-mapped BP QTL and thereby serve as a validation step to
identify a specific gene responsible for BP regulation. Current popular techniques are through the
use of zinc finger nucleases (ZFNs), transcription Activator-Like Effector Nucleases (TALENs),
and the latest CRISPR/Cas9 system. In the case of a high-resolution mapped blood pressure locus,
the basic idea is to use any one of these genetic engineering techniques to construct a rat model with
a single gene “deleted” through a targeted mutation on the genome of the hypertensive rat strain
(knock-out rat) or substituted with alleles from a normotensive strain (knock-in rat). The next step
is to compare the BP of the genetically-engineered rat model with that of wild-type rat model,
which allows for the evaluation of the role of a specific gene in BP regulation. The mechanisms of
the above three genome-editing tools will be briefly introduced below.
1. Zinc finger nucleases (ZFNs). Zinc finger nucleases are artificial proteins consisting of
a DNA-binding zinc finger domain and a DNA cleavage domain of FokI restriction en-
donuclease. The DNA-binding zinc finger domain can be engineered to precisely target
specific desired DNA sequences and the domain is composed of eukaryotic transcription
factors and 3–6 fingers. The DNA cleavage domain is responsible for the DNA cleavage
at the target site. The site is then repaired through non-homologous end-joining, a repair
process which is inherently erroneous resulting in either the introduction or deletion of
nucleotides. This erroneous repair process is exploited through the ZFN-based genome
editing method to disrupt the coding sequence of the gene (Figure 3).
2. Transcription Activator-Like Effector Nucleases (TALENs). The principle of tran-
scription Activator-Like Effector Nucleases (TALENs) is very similar to that of ZFNs.
FIGURE 3: The principle of ZFNs-mediated gene editing.
TALENs are synthetic restriction enzymes consisting of a TAL effector DNA-binding

domain and a DNA cleavage domain. The TAL effector DNA-binding domain can be
engineered to specifically bind to the desired DNA sequence and the domain is composed
of tandem 33-34 amino acid repeat module. The DNA cleavage domain is responsible for
cutting DNA strain at the target site (Figure 4). TALENs have more targeted specificity
than ZFNs because TALENs have longer DNA-binding domain to ensure specificity.
3. CRISPR/Cas9 system. CRISPR stands for clustered regularly interspaced short palin-
dromic repeats. CRISPR was first shown to work as a genome engineering/editing tool
in human cell culture [48]. Figure 5 describes the principle of the CRSPR /Cas9 system: a
single guide RNA (gRNA) consists of a crRNA sequence that is specific to the DNA target,
and a tracrRNA sequence. tracrRNA links crRNA to the Cas9 protein by interacting with
Cas9. Cas9 has DNA endonuclease activity. The resulting complex is guided by crRNA to
the target site and causes target-specific double-stranded DNA cleavage by Cas9. The cleav-
age site will be repaired by the non-homologous end joining (NHEJ) DNA repair pathway.
If a donor oligonucleotide or donor plasmid is injected together with Cas9 mRNA and
gRNA, the cleavage site will be repaired by the homologous recombination (HR).
Genome-Editing Tools for Rat Hypertension Research 19
FIGURE 4: The principle of TALENs-mediated gene editing.
FIGURE 5: The principle of CRISPR/Cas9-mediated gene editing.

7.2 The Application of Genome-Editing Tools in

Hypertensive Rat Models
Out of the three methods described above, to date, we and others have used the ZFN method for
the purpose of validating genes for hypertension that were previously prioritized through other
classic genetic approaches. In our laboratory, we used the TALEN approach to generate a single rat
model for the gene Rififylin (a candidate gene prioritized by substitution mapping of rat chromo-
some 10), the homozygous rat generated by this approach was not viable (Data unpublished). Be-
fore the TALEN technology could be further applied for hypertension research, the CRISPR /Cas9
system was described as a much cheaper and efficient system. Therefore, in our laboratory (and
perhaps several other laboratories), the CRISPR /Cas9 method is currently being used to generate
both knock-out and knock-in rat models of hypertension. The progress made as a result of the de-
velopment of these genome-edited tools for hypertension research will be discussed below.
Adamts16
One of the first genes to be tested using the ZFN approach was A disintegrin-like metalloprotein-
ase with thrombospondin motifs-16 (Adamts16), which was a candidate gene for BP regulation
mapped by our laboratory using the linkage and substitution mapping approaches. Custom ZFNs
with proprietory rights were designed by SIGMA and injected into the Dahl rat embryos to gen-
erate a Dahl S rat model with targeted disruption of the gene Adamts16. Figure 6(A) showed the
PCR-amplified products from the genomic DNA of different mutants. The allele of wild-type Dahl
S rat is 307 bp and the mutant allele of one mutant rat is 290 bp, wherein this 17 bp deletion oc-
curred in the first exon of Adamts16, resulting a stop codon in the transcript (Figure 6B) [3].
The Adamts16 mutant model showed a significantly lower systolic and diastolic blood pres-
sure compared with the wild-type S rats, suggesting an important role of Adamts16 in the develop-
ment of hypertension (Figure 7) [3]. The translational significance of this work was ascertained
by the observation that several allelic variants of human ADAMTS16 were associated with two
independent cohorts, GenNet and the Quebec Family Study [49].
Renin
The second gene studied using the ZFN approach in rats is the gene renin. The renin-angiotensin
system is known to play an important role in BP control. To define the role of renin in the context
of the Dahl rat genome, ZFNs targeting the renin gene were designed and microinjected into
SS/JrHsdMcwi (SS) rat embryos to construct a transgenic SS/JrHsdMcwi (SS) rat model with a
renin knock-out [50]. ZFNs caused a 10bp deletion in the exon 5 of renin, resulting in a frameshift
mutation. In the renin knock-out rat, plasma renin activity was not detected and renin protein was
FIGURE 6: (A) The PCR-amplified genomic DNA on the agarose gel indicated the genotype of dif-
ferent mutants. One mutant had 17 bp deletion. (B) The DNA sequencing result confirmed the 17 bp
deletion, resulting in a premature stop codon indicated by TGA within the first exon of Adamts16 [3].
Used with permission by PNAS [3].
FIGURE 7: The comparison of blood pressure (BP) among homozygous Adamts16mutant, heterozygous
Adamts16mutant, and wild-type S rats. (A) Mean systolic BP effect ± SEM by the tail-cuff method. (B and
C) Systolic and diastolic BP of the same animals measured by radiotelemetry transmitters. Data plotted
are the recordings obtained once every 5 min continuously for 24 h and averaged for 4 h intervals; **P <
0.01, ***P < 0.001 [3]. Used with permission by PNAS [3].
missing in the juxtaglomerular cells in the kidney. The authors found that blood pressure of the
renin knock-out SS rats was significantly lower compared with that of wild-type SS rats [50], thus
confirming the important role of renin in hypertension. Note, however, that the renin gene, which is
located very near but not within a BP QTL on rat chromosome 13, was not particularly a candidate
gene whose alleles are inherited to cause hypertension of the Dahl S rat. Thus, this study proves
that renin remains an important gene, whose protein product has significant function in the control
of blood pressure.
p67(phox)
The third gene studied using the ZFN approach is the gene coding for a subunit of the NAD(P)H
oxidase, p67(phox). NAD(P)H oxidase is involved in the development of salt-sensitive hypertension
and a subunit of NAD(P)H oxidase, p67(phox), was reported to be overexpressed in the outer renal
medulla of the Dahl salt-sensitive (S) rat when fed with a high-salt diet [51]. The p67(phox) was
disrupted in the S rat by using zinc-finger nucleases and a significant reduction of salt-sensitive hy-
pertension was observed in those rats, which confirmed the important role of the p67(phox) subunit
of NAD(P)H oxidase in blood pressure regulation [51].
Agtrap-Plod1 locus
ZFN rat models have been used to test and validate a single locus bearing six candidate genes
for blood pressure. All six genes at the Agtrap-Plod1 locus, Agtrap, Mthfr, Clcn6, Nppa, Nppb, and
Plod1, were tested by generating six individual ZFN rat models for each of the genes. The results
indicated that mutations of Nppa, Plod1, and Mthfr increased blood pressure whereas mutations
within Agtrap and Clcn6 decreased the risk of hypertension. Reanalysis of the human AGTRAP-
PLOD1 locus also implied that disease-associated haplotype blocks with polygenic effects were
highly plausible. These data collectively demonstrate that multiple modifiers of hypertension can
cosegregate at a single GWAS locus [52].
Cd247
Human genetic studies showed that the gene CD3 ζ chain (CD247), involved in T-cell signaling,
was associated with systolic and diastolic blood pressure in hypertensive individuals [53]. To further
validate this finding using a rat model, ZFNs were introduced to target the rat Cd247 gene, which
resulted in a premature stop codon due to a 11 bp frameshift deletion in exon 1 of Cd247 (54). In the
Cd247 mutant rat model, the reduced infiltration of CD3(+) T cells into the kidney was associated
with the decreased mean arterial blood pressure, which confirmed the human genetic studies [54].
Plekha7
PLEKHA7 (pleckstrin homology domain containing family A member 7), a candidate gene for
human hypertension was investigated for its influence on the pathogenesis of salt-sensitive hy-
pertension by mutating Plekha7 in the Dahl salt-sensitive (SS/JrHsdMcwi) rat using zinc-finger
nuclease technology [55]. Homozygous mutant rats had lower mean arterial blood pressure and
improved renal and vascular function compared with WT littermates. Indeed, both flow-mediated
dilation and endothelium-dependent vasodilation in response to acetylcholine were improved in
isolated mesenteric resistance arteries of Plekha7 mutant rats compared with WT and these vascular
improvements were correlated with changes in intracellular calcium handling, resulting in increased
nitric oxide bioavailability in mutant vessels [55]. Thus, Plekha7 may contribute to blood pressure
regulation and cardiovascular function through its effects on the vasculature.
Nr2f 2
The transcription factor, Nuclear receptor 2, factor 2, is yet another example of validation of a hu-
man gene associated with hypertension using a rat model. GWAS studies showed that NR2F2
was associated with essential hypertension in humans [56]. A targeted Nr2f 2-edited rat model was
constructed by using ZFNs, wherein a 15bp region was deleted in the hinge region of Nr2f 2 [57].
The systolic and diastolic blood pressures of the Nr2f 2 mutant rat were significantly lower than
controls. This study, which was reported from our laboratory further demonstrated that the interac-
tion between Nr2f 2 and another transcription factor, Friend of Gata2 (Fog2), critically influences
blood pressure [57].
Besides the genes discussed above, genetically engineered mice have been used by Graham
et al to obtain evidence for uromodulin as a protein-coding gene contributing to the inheritance
of hypertension [58]. Several other genes linked to the trait of blood pressure have been reviewed
elsewhere and represented as an update to the 2014 update to the Paige model of blood pressure
regulation. The diagram for this updated model is reproduced below and readers are redirected to
the review article by Padmanabhan et al. for further details on the Paige model (Figure 8) [6].
FIGURE 8: The 2014 updated Paige mosaic model of blood pressure (BP) regulation [6]. Used with
permission by AHA, Wolters Kluwer [6].
• • • •
25
chapter 8
Beyond Protein-Coding
Genes- “Missing Heritability”
From the current findings on the genetics of hypertension, it is apparent that the small number of
positionally cloned protein-coding genes explain only a minority of the inheritance of hyperten-
sion. This means that we are missing key pieces to comprehend the contribution of the genome
in its entirety to the trait of blood pressure. Current thinking is that there could be two possibili-
ties for accounting for missing heritability: (1) epistasis and (2) yet undiscovered variants. Lander
and colleagues [59] also consider a third possibility based on the very definition of heritability as
reproduced below: The proportion of heritability explained by a set of variants is the ratio of (i)
the heritability due to these variants (numerator), estimated directly from their observed effects, to
(ii) the total heritability (denominator), inferred indirectly from population data. The prevailing
view has been that the explanation for missing heritability lies in the numerator—that is, in as-yet
undiscovered variants. While many variants surely remain to be found, a substantial portion of miss-
ing heritability could arise from overestimation of the denominator, creating “phantom heritability.”
In any case, we and others have provided evidence for both the themes, i.e., epistasis (or genetic
interactions) and non-coding RNA contributing to the genetics of hypertension [60–73].
During the decades following the genome sequencing period of the early 2000’s, more and
more non-coding RNA are being functionally annotated in areas of the genome previously referred
to as “Junk DNA.” These non-coding RNA molecules are emerging as molecules—allelic varia
tions within which play crucial roles in several diseases. Before addressing the question of the rel-
evance of variants within non-coding RNA molecules as pertinent to hypertension, we will review
the definitions and types of non-coding RNAs.
A non-coding RNA (ncRNA) is a functional RNA molecule that does not encode a pro-
tein [74]. The genome-wide studies have shown that the human genome is pervasively transcribed
and produces many thousands of regulatory non-protein-coding RNAs (ncRNAs), including micro
RNAs (miRNAs), long non-coding RNAs (lncRNAs), small interfering RNAs, and PIWI-
interacting RNAs [75]. It is now clear that these RNAs play critical roles in the transcriptional and
post-transcriptional regulation of gene expression [75]. Currently, the most widely studied classes
of ncRNAs are miRNAs and lncRNAs.
• • • •
27
chapter 9
MicroRNA and Hypertension
9.1 What is MicroRNA?
A microRNAs (miRNA) is a small non-coding RNA molecule containing ~22 nucleotides. A large
number of miRNAs were found in animals, plants, and some viruses, and function as both tran-
scriptional and post-transcriptional regulators of gene expression [76].
MiRNAs are usually transcribed by RNA polymerase II into primary miRNAs (pri-miRNAs)
from genomic DNA in the nucleus. Pri-mRNAs are long RNA molecules (100–1000 nucleotides)
and may contain several precursor-miRNAs (pre-miRNAs). Pri-miRNAs are further processed
into pre-miRNAs in the nucleus by the microprocessor complex Drosha/Dgcr8. In the Drosha/
Dgcr8 complex, DGCR8 functions as a molecular anchor for the recognition of pri-miRNAs and
orients the endonuclease Drosha to cleave pri-miRNAs and release shorter pre-miRNA molecules
(70–90 nucleotides) in the nucleus. Pre-miRNAs are then exported by Exportin 5 from the nucleus
to the cytoplasm for further processing into ~22 nucleotide mature miRNAs by the endonuclease
Dicer (Figure 9) [5].
Currently, most studies focus on miRNAs as post-transcriptional regulators in the cytoplasm
by degrading mRNAs of target genes. In the cytoplasm, animal miRNAs assemble into RNA-
induced silencing complex (RISC) containing the Argonaute (Ago) protein. RISC uses miRNA as
a template for recognizing complementary mRNA through perfect or near perfect base pairing with
a site in the 3' untranslated region (UTR) of mRNA. When miRNA binds to the complementary
strand of its target mRNA, RISC will degrade target mRNA by activating Ago and cleaving the
mRNA (Figure 10) [4].
It has been reported that miRNAs are not only restricted to function in the cytoplasm, they
can also act as transcriptional regulators to regulate gene expression in the nucleus [4]. Intriguingly,
functional RISC activity was also detected in the nucleus [77–79]. Studies show that Ago-miRNA
complex can be imported into the nucleus by binding to Importin 8 and Ago-miRNA complex can
bind to chromosomal DNA sequences (Figure 10) [4]. MicroRNA-10a (miR-10a) can target the
promoter region of the hoxd4 gene and represses its transcription in human breast cancer cells [80].
FIGURE 9: The processing of miRNAs [5]. Used with permission by Nature Publishing Group [5].
Recently, it is becoming evident for miRNA-mediated regulation of other classes of ncRNAs in

the nucleus. For example, microRNA-9 (miR-9) has been identified to target the long non-coding
RNA MALAT1 for degradation through an AGO2-mediated manner in the nucleus [81]. There-
fore, miRNAs can function as both transcriptional and post-transcriptional regulators in the cyto-
plasm or nucleus to regulate the expression of protein-coding genes and other classes of ncRNAs.
MicroRNA and Hypertension 29
FIGURE 10: Actions of miRNAs in the nucleus and cytoplasm [4]. Used with permission by Taylor
& Francis [4].
9.2 The Role of MicroRNA in Blood Pressure

Regulation
9.2.1 Polymorphisms in MicroRNA Binding Sites

MiRNAs can be involved in the development of hypertension through their interaction with 3' un
translated region (3'UTR) of their target genes which are implicated in the regulation of blood
pressure. The single nucleotide polymorphisms (SNPs) located in the miRNA binding sites may
interfere with the interaction between miRNAs and their target genes. For example, if a miRNA
binds more efficiently to one 3'UTR allele (A) of its target gene compared to another 3'UTR allele
(T), the miRNA may more efficiently suppress the expression of the target gene containing the al-
lele A. If the lower expression of this gene is associated with the development of hypertension, the
aberrant interaction between the miRNA and its target gene due to the SNP located in the 3'UTR
could play an important role in the development of high blood pressure. More than 20,000 SNPs
of miRNA binding sites have been identified to be associated with different diseases including
hypertension [82].
The human angiotensin II type 1 receptor (AGTR1) is associated with hypertension and one
study reported that a SNP rs5186 located in the 3'-UTR of AGTR1 alters the binding efficiency
of hsa-miR-155 to AGTR1 mRNA [83]. Specifically, the study showed that hsa-miR-155 could
bind more efficiently to the 1166A allele of rs5186, but not the 1166C allele, and 1166C allele has
been identified in a significant number of patients with essential hypertension. Therefore, the SNP
located in the 3'-UTR of AGTR1 may prevent the binding of hsa-miR-155 and thereby cause the
increased AGTR1 levels, which increases blood pressure in hypertensive patients [83]. Interestingly,
another study from the same group reported that the down-regulation of hsa-mir-155 was nega-
tively correlated with increased AGTR1 protein expression [84]. The decreased level of hsa-mir-
155 and the increased AGTR1 protein expression were observed in 19 hypertensive subjects with
the homozygous C/C allele in comparison to 25 and 20 hypertensive subjects with the homozygous
A/A allele and the heterozygous A/C allele, respectively. This suggests that the aberrant expression
of miRNAs and the altered interaction between miRNAs and their target genes may contribute
to the development of hypertension [84]. Population studies established that the polymorphism
rs5068 (A /G) in the 3'UTR of the NPPA gene encoding atrial natriuretic peptide (ANP) was as-
sociated with blood pressure phenotype [85]. One miRNA, miR-425, was demonstrated to bind
the 3'UTR with the allele A, but not the allele G, and thereby cause the differential ANP expres-
sion and different downstream blood pressure effect [86]. Similarly, a polymorphism of 28 base
pairs (bp) in the 3'UTR of fibrinogen alpha gene (FGA ) was reported to be associated with chronic
thromboembolic pulmonary hypertension. The miR-759 can interact with this 28 bp polymorphic
3’UTR region and thereby regulate the expression of FGA, which was associated with the suscepti-
bility to chronic thromboembolic pulmonary hypertension [87]. Chromogranin A (Chga) is impor-
tant for blood pressure homeostasis and it was reported that the dysregulation of Chga was observed
in the spontaneously hypertensive rat (SHR). One 3'UTR polymorphism at the Chga locus in SHR
was demonstrated to affect the miR-22 binding ability and thereby was considered to be associ-
ated with high blood pressure in SHR. Interestingly, through in vivo administration of miR-22
antagomir, which was used to disrupt the miR-22 binding ability, decreased blood pressure was ob-
served in SHR [88]. The study above also provides the potential feasibility of miRNA-based thera-
peutic strategy for hypertension. Similarly, another polymorphism C+87T (rs7610) identified in the
Chga 3'UTR was also reported to be associated with hypertension through its altered interaction
with miR-107 [89].
MicroRNA and Hypertension 31
MiRNAs could participate in a signaling pathway and be involve in the regulation of multiple
genes implicated in blood pressure regulation. One example is that of a minor allele rs17228616
existing in the 3'UTR of acetylcholinesterase (AChE) that caused the weakened interaction be-
tween miR-608 and the AchE 3'UTR, and therefore increased the AchE expression. However, this
weakened interaction enhanced the miR-608 binding to its other target genes and subsequently
decreased the expression of these genes including CDC42 and interleukin-6 (IL6), which was dem-
onstrated to be associated with the observed blood pressure increasing effect in the subjects contain-
ing the minor allele [90].
The catecholamine release-inhibitor catestatin and its precursor Chga contributes to blood
pressure regulation. One study showed that the 3'-UTR polymorphismT+3246C (rs938671) from
the vacuolar H(+)-ATPase subunit gene (ATP6V0A1) preferred the binding of the miRNA hsa-
miR-637, which was associated with the downstream catestatin processing from Chga for subse-
quent blood pressure regulation [91]. This also provides a case that miRNAs may be involved in a
signaling pathway and indirectly regulate the expression of downstream genes responsible for blood
pressure regulation.
The polymorphism(s) located in the 3'UTR of hypertension-related genes may change the
binding sites for miRNAs and thereby cause the differential gene expression. A polymorphism
ss52051869 in the 3'UTR of the human solute carrier family 7 (SLC7A1) gene was previously re-
ported to be associated with a genetic predisposition to develop essential hypertension and this poly-
morphism may alter the binding ability of the miR-122 to the SLC7A1 3'UTR, thereby causing the
abnormal SLC7A1 gene expression in hypertensive subjects [92]. Specifically, the authors found that
the minor allele of ss52051869 was more likely associated with a long 3'UTR variant of SLC7A1,
which contains four potential miR-122 binding sites. In contrast, the major allele of ss52051869 was
more likely associated with a short 3'UTR variant of SLC7A1, which just contains three potential
miR-122 binding sites. The study demonstrated that the SLC7A1 variant containing long 3'UTR
was much lower expressed compared with the variant containing short 3'UTR, which suggests that
the different number of miR-122 binding sites, associated with a polymorphism ss52051869 in the
SLC7A1 3'UTR, may cause the differential gene expression and regulate blood pressure [92].
The examples discussed above demonstrate that the polymorphism in 3'UTR of hypertension-
related genes may cause changes in blood pressure due to their altered interactions with miRNAs.
9.2.2 Polymorphisms within MicroRNAs

Another possible mechanism linking miRNAs to hypertension is through the aberrant miRNA ex-
pression due to polymorphisms in microRNA sequences. The dysregulation of miRNA expression
levels may subsequently alter the expression of their target genes responsible for blood pressure reg-
ulation. For example, through comparing the genotype of a miRNA, miR-143, from blood samples
of 156 patients with essential hypertension and 187 healthy individuals, a polymorphism rs4705342
located in the promoter of miR-143 was identified to be associated with essential hypertension,
which suggests that the altered expression level of miR-143 due to the polymorphism in its pro-
moter may contribute to the downstream blood pressure difference [93]. One study showed that one
mutant allele rs2168518 in the seed region of miR-4513, significantly associated with systolic and
diastolic pressure, resulted in the decreased expression of miR-4513 [94]. Similarly, a recent clini-
cal report demonstrated that genetic polymorphisms in pre-miRNAs, e.g., miR-4513 rs2168518,
miR-499 rs3746444, and miR-423 rs6505162, were associated with blood pressure [95].
• • • •
33
chapter 1 0
Long Non-coding RNA and

Hypertension
10.1 What is Long Non-coding RNA?
Compared with microRNAs which are short RNA molecules, long non-coding RNAs (lncRNAs)
are a large and diverse class of ncRNAs and as the name suggests, longer RNA molecules that are
by definition longer than 200 nucleotides. LncRNAs are usually transcribed by RNA polymerase II
or III [96]. Unlike miRNAs, lncRNAs are poorly conserved and they can be tissue- and cell-type
specific.
A significant majority of lncRNAs are located exclusively in the nucleus, but their biological
functions have remained elusive. LncRNAs may regulate gene expression either by binding to DNA
or RNA in a sequence specific manner or by binding to proteins [97]. LncRNAs are not restricted
into a common mode of action, but can exert their functions in a number of different ways. For
example, lncRNAs can participate in the epigenetic regulation by binding to chromatin modifying
complexes and recruiting them to specific genomic loci to regulate gene expression. The lncRNA
HOTAIR can recruit and direct the Polycomb Repressive Complex 2 (PRC2) to the HOXD locus,
by which the histone H3 lysine-27 residues (me3K27) is trimethylated and thereby the gene expres-
sion is repressed [98] (Figure 11).
LncRNAs can also be involved in the regulation of the transcriptional process through differ-
ent mechanisms. For example, the lncRNA transcribed from the upstream promoter of the human
dihydrofolate reductase gene can form a stable triplex with the major promoter, which restricts the
binding of the transcription factor TFIID and thereby repress the transcription of the dihydrofolate
reductase (Figure 12) [99].
Moreover, lncRNAs can also regulate the post-transcriptional process. In some cases, lnc
RNAs are expressed as overlapped antisense RNAs from protein-coding genes, also known as natu
ral antisense transcripts (NATs). For example, in the case of Zeb2 [100], NAT expression can inhibit
FIGURE 11: The lncRNA, HOTAIR, can direct the Polycomb Repressive Complex 2 (PRC2) and cause
the trimethylation of the histone H3 lysine-27 residues (me3K27) in the HOXD locus.
the splicing of an internal ribosome entry site (IRES)-containing 5' UTR intron by forming RNA-
RNA duplexes. The 5' UTR intron maintenance results in efficient binding of the translational
machinery to IRES, and therefore NAT expression indirectly facilitates Zeb2 expression (Figure 13).
However, the common mechanism by which antisense lncRNAs influence splicing process still
remains unclear.
FIGURE 12: LncRNA can repress the transcription of dihydrofolate reductase (DHFR) by forming a
stable triplex with the major promoter of DHFR and thereby restricting the binding of the transcription
factor TFIID.
FIGURE 13: Overlapped antisense lncRNAs from protein-coding genes can influence splicing patterns
of pre-mRNA.
10.2 The Role of Long Non-coding RNA in Blood

Pressure Regulation
Research into the physiological role of lncRNAs in the regulation of blood pressure is still at a
very early stage. There are no human studies that link essential hypertension with lncRNAs. In
our laboratory, we conducted the lncRNA sequencing and lncRNA transcriptome analysis in three
widely used rat strains for hypertension research: the hypertensive Dahl salt-sensitive rat (S), the
spontaneously hypertensive rat (SHR), and the normotensive Dahl salt-resistant rat (R) [2]. By
comparing the lncRNA profiling among the hypertensive S rat, the hypertensive SHR rat and the
normotensive R rat, we expected to identity lncRNAs implicated in blood pressure regulation. We
found that 186 lncRNAs are expressed in S but not in R, whereas 199 lncRNAs are expressed in R
but not in S (Figure 14). Similarly, 199 lncRNAs are expressed in S but not in SHR, whereas 208
lncRNAs are expressed in SHR but not in S (Figure 14) [2].
Our results also showed that a total of 273 lncRNAs were differentially expressed between
S and R and a total of 749 lncRNAs were differentially expressed between S and SHR (Figure 15)
[2]. We also found that the differential expression of lncRNAs was associated with several dif-
ferentially expressed protein-coding genes. For example, four genes, Asb3, Chac2, Pex11b, and Sp5,
were differentially expressed between S and R, which were inversely correlated to the differentially
expressed lncRNAs (Figure 16) [2]. Other than this study, Wang et al. recently surveyed lncRNAs
in Brown Norway, Dahl S, and SS.13 (BN26) congenic rats, which also suggests a role of lncRNAs
in hypertension [101]. Interestingly, in this study, two of the differentially expressed lncRNAs were
with several sequence variants and within the congenic segment, indicating their prioritization for
further research as candidates for conferring susceptibility to hypertension [101]. Collectively, these
two studies constitute the first two reports that point to lncRNAs as genetic determinants of blood
pressure.
Long Non-coding RNA and Hypertension 37
FIGURE 14: Long non-coding RNAs (lncRNAs) were detected in different rat chromosomes in hy-
pertensive Dahl salt-sensitive rat (Dahl S), the normotensive Dahl salt-resistant rat (Dahl R) and the
spontaneously hypertensive rat (SHR) [2]. Used with permission by AHA, Wolters Kluwer [2].
FIGURE 15: The differential expression of long non-coding RNAs (lncRNAs) among the hypertensive
Dahl salt-sensitive rat (S), the normotensive Dahl salt-resistant rat (R), and the spontaneously hyper
tensive rat (SHR). (e.g., S Vs R up: higher levels in S compared with R); down: down-regulated (e.g., S
Vs R down: lower levels in S compared with R) [2]. Used with permission by AHA, Wolters Kluwer [2].
FIGURE 16: The differential expression of long non-coding RNAs (lncRNAs) was inversely correlated
with the differential expression of protein-coding genes through comparing the lncRNA and gene ex-
pression patterns between the hypertensive Dahl salt-sensitive rat (Dahl S) and the normotensive Dahl
salt-resistant rat (Dahl R). **P < 0.01; ***P < 0.001; and NS, not significant [2]. Used with permission
by AHA, Wolters Kluwer [2].
• • • •
39
chapter 1 1
Gut Microbiota and Hypertension
Beyond the genome of the host, because mammals host a variety of gut microbes, the gut microbi-
ome is emerging as an additional factor conferring susceptibility to many diseases. Although studies
addressing the relevance of gut microbiota in human essential hypertension are scanty, there are re
ports emerging in the literature with models used for hypertension research that provide a definitive
link between the microbiome and the genetics of hypertension. Hypertension in the two most ex-
tensively used rat models for hypertension, the Dahl S, and the SHR, have both been demonstrated
to have links to the gut microbiota [102, 103].
• • • •
41
chapter 1 2
Summary and Perspectives
This book is focused on the genetic influence on blood pressure. The content of this work captures
the many genetic experimental approaches have been applied over several decades to investigate and
identify inherited elements on the genome that confer susceptibility to the development of hyper-
tension. Human linkage studies identified many genomic loci associated with hypertension by in-
vestigating large families in which hypertension is inherited among family members. However, the
identified genomic loci are usually large and include many genes, which is a rather low-resolution
and thereby a shortcoming of linkage analysis. In contrast, recent human GWAS studies, which
rely heavily on statistical analysis, have higher resolution and aims to identify SNPs associated with
hypertension. To date, several large meta-analysis of GWAS studies have identified many novel
variants and genes implicated in hypertension. Beyond these human studies, several experimental
rat models of hypertension have been developed and studied. Similar to human linkage studies, but
with improved options for controlled environmental factors and breeding, multiple genomic regions
have been identified, many of which are within regions homologous to those identified by linkage in
humans. Beyond linkage analyses, experimental rat models served as tools for substitution mapping
through the construction of consomic and congenic rat models of hypertension. These strategies
allowed for the fine-mapping of blood pressure quantitative trait locus. Further, novel genome-
editing tools, such as ZFNs, TALENs, and CRISPR /Cas9 systems, have been developed to gen-
erate genetically-engineered rat models of hypertension to verify the role of specific prioritized
candidate genes in regulating blood pressure.
Beyond the identification of protein-coding genes on different genomic loci as important
genes regulating blood pressure, in recent years, more novel ncRNAs, such as miRNAs and lnc
RNAs, are gradually identified and demonstrated to be involved in the regulation of blood pres-
sure, which make the etiology of hypertension more complicated. Further, recent reports on the
importance of the gut microbiota and their microbiotal genomic interactions with the host genome
are becoming apparent as important contributors to the extent of hypertension [102–106]. These
findings and the emerging role of other epigenetic factors [107, 108] are further enhancing the
complexity of the genetic regulation of hypertension. Due to this complexity, it is very challenging
for biomedical scientists to come up with simple therapeutic strategies to cure hypertensive patients.
At the very best, the identification of select genetic elements as reviewed in this book constitutes the
beginnings of our understandings of the genetic ‘blueprint’ of hypertension. Over the next few years
and decades, with the discovery of more and more genes and genetic elements, the allelic variants
of which differentially influence blood pressure and the expected expansion into including the host
genomic interactions with the microbiome, this body of knowledge is highly likely to cumulatively
point to the possibility that genetic contribution to the development of hypertension differs from
person to person and that contemplation of personalized medicine will be required for better clinical
management of hypertension.
Funding
This work was supported by Grants from the National Heart Lung and Blood Institute of the Na-
tional Institutes of Health to BJ (HL076709 and HL020176).
• • • •
43
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55
Author Biographies
Bina Joe is a Professor and Chair of Physiology and Pharmacology

and the founding Director of the Center for Hypertension and Per-
sonalized Medicine at the University of Toledo College of Medicine.
She is also a Fellow of the American Heart Association and the Car-
diovascular section of the American Physiological Society. She is an
international leading authority in the research area of Genetics of
Hypertension, and has identified several novel genetic factors linked
to the inheritance of hypertension. She is the recipient of several Re-
search Awards including the Young Scholar Award from the American
Society of Hypertension, the Lewis K. Dahl Memorial Lecture Award from the American Heart
Association Council on Hypertension and the Outstanding Researcher Award from the University
of Toledo. Her research work is published in several top tier journals including PNAS, Cell and
Nature Communications. Besides being an internationally recognized Researcher, she has taken on
multiple leadership positions within the American Physiological Society and the American Heart
Association. Notably, she has contributed substantially to the development of the Physiological
Genomics group of the American Physiological Society and was recognized with a Distinguished
Service Award. She has served on the Editorial Board of Hypertension and is the current Editor of
the Journal, Physiological Genomics.
Xi Cheng is a pre-doctoral fellow in Biomedical Sciences at the

University of Toledo College of Medicine and Life Sciences. His re-
search focus is in the area of Cardiovascular and Metabolic Diseases.
He received his Bachelor’s degree in Bioengineering from the Fuzhou
University Zhicheng College in China in 2012. Besides obtaining a
Graduate Certificate in Bioinformatics and Biomarkers, since 2013,
he has been researching with Dr. Bina Joe to investigate the contribu-
tions of non-coding RNAs to the inheritance of hypertension using
novel congenic and gene-edited rat models of hypertension. He has been invited to deliver podium
presentations of his research at various Research conferences including the 2015 Midwest Graduate
Symposium held in Toledo, OH, USA and the 2015 Annual Federation of American Societies for
Experimental Biology meeting held in Boston, USA and the 42nd Annual Pharmacology Research
Colloquium held in Toledo, OH, USA.

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Colloquium series on Colloquium series on

integrated systems Physiology: integrated systems Physiology:

GENETICS AND HYPERTENSION

For the full list of available titles, please visit:

Please e-mail info@morganclaypool.com for more information.

For Published titles please see the website, www.morganclaypool.com/toc/isp/1/1.

Genetics and Hypertension

ISBN: 9781615046942 paperback

ISBN: 9781615046959 ebook

Colloquium Series on Integrated Systems Physiology: From Molecule to

COLLOQUIUM SERIES ON Integrated Systems Physiology:

2 Linkage Analyses for Essential Hypertension in Humans.......................................5

3 Genome-wide Association Studies of Human Hypertension..................................7

4 Rat Models in Hypertension Research................................................................ 11

5 Genetic Linkage Analysis Using Hypertensive Rat Models.................................. 13

6 Substitution Mapping Using Congenic Strains.................................................... 15

7 Genome-Editing Tools for Rat Hypertension Research....................................... 17

8 Beyond Protein-Coding Genes- “Missing Heritability”........................................ 25

9 MicroRNA and Hypertension............................................................................. 27

10 Long Non-coding RNA and Hypertension.......................................................... 33

11 Gut Microbiota and Hypertension...................................................................... 39

12 Summary and Perspectives.................................................................................. 41

FIGURE 2: The influence of genetic and environmental factors on hypertension.

Linkage Analyses for Essential

Genome-wide Association Studies

Rat Models in Hypertension Research

Genetic Linkage Analysis Using

Substitution Mapping Using

Genome-Editing Tools for

7.1 Overview of Genome-Editing Techniques

FIGURE 3: The principle of ZFNs-mediated gene editing.

TALENs are synthetic restriction enzymes consisting of a TAL effector DNA-binding

FIGURE 4: The principle of TALENs-mediated gene editing.

FIGURE 5: The principle of CRISPR/Cas9-mediated gene editing.

7.2 The Application of Genome-Editing Tools in

MicroRNA and Hypertension

9.1 What is MicroRNA?

Recently, it is becoming evident for miRNA-mediated regulation of other classes of ncRNAs in

9.2 The Role of MicroRNA in Blood Pressure

9.2.1 Polymorphisms in MicroRNA Binding Sites

9.2.2 Polymorphisms within MicroRNAs

Long Non-coding RNA and

10.1 What is Long Non-coding RNA?

10.2 The Role of Long Non-coding RNA in Blood

Gut Microbiota and Hypertension

Summary and Perspectives

attenuates Dahl salt-sensitive hypertension via inflammatory modulation. Hypertension.

2009;10:12. PubMed PMID: 19232136. Pubmed Central PMCID: 2680403. doi: 10

Hypertension. 2014 Apr;63(4):827-38. PubMed PMID: 24420542. Pubmed Central PM-

Bina Joe is a Professor and Chair of Physiology and Pharmacology

Xi Cheng is a pre-doctoral fellow in Biomedical Sciences at the

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