GENETIC DEFECTS OF CALCIUM AND POTASSIUM SIGNALING IN ...

GENETIC DEFECTS OF CALCIUM AND POTASSIUM SIGNALING IN INHERITED VENTRICULAR

ARRHYTHMIAS

Päivi Laitinen

Department of Medicine, and

Research Program in Molecular Medicine, Biomedicum Helsinki,

University of Helsinki,

Finland

Academic dissertation

To be publicly discussed with the permission of the Faculty of Medicine,


in the Lecture Hall 2 of the Biomedicum Helsinki, Haartmaninkatu 8,

on 26 March 2004, at 12 noon.

Helsinki 2004

ISBN 952-91-6949-3 (paperback)

ISBN 952-10-1721-X (pdf)

Yliopistopaino Oy

Helsinki 2004

Supervised by Professor Kimmo Kontula

Department of Medicine,


Helsinki, Finland

Reviewed by Docent Katarina Pelin

Department of Biological and

Environmental Sciences,


Helsinki, Finland

Docent Anu Wartiovaara

Programme of Neurosciences and

Department of Neurology,


Helsinki, Finland

To be publicly discussed with Docent Ann-Christine Syvänen

Department of Medical Sciences,

University of Uppsala,

Uppsala, Sweden

CONTENTS

3

LIST OF ORIGINAL PUBLICATIONS 5

ABBREVIATIONS 6

ABSTRACT 7

INTRODUCTION 9

REVIEW OF THE LITERATURE 10

1 ELECTRICAL AND CONTRACTILE FUNCTIONS OF THE HEART CELL 101.1 Cardiac action potential 101.2 Potassium and calcium currents of the heart 111.3 Excitation-contraction coupling–from plasma membrane to contraction apparatus 131.4 Cardiac Ca2+ signaling proteins involved in calcium-induced calcium release 14

2 GENETIC BASIS OF INHERITED ARRHYTHMIC DISORDERS 162.1 Electric inherited arrhythmias 162.2 Arrhythmogenic disorders associated with structural abnormalities 26

3 ION CHANNEL DISEASES 303.1 Ryanodine receptor defects–examples of diseases of calcium metabolism 303.2 Diseases of potassium and sodium signaling 31

4 MOLECULAR DIAGNOSIS IN INHERITED ARRHYTHMIC DISEASES 32

AIMS 34

PATIENTS AND METHODS 35

1 PATIENTS AND CONTROLS 352 MOLECULAR GENETIC STUDIES 36

2.1 DNA extraction and polymerase chain reaction 362.2 Mutation screening 362.3 Microsatellite analysis 362.4 Linkage and haplotype analysis 362.5 Biocomputing 372.6 Specific detection methods for DNA alterations 37

3 FUNCTIONAL ANALYSES 374 STATISTICAL ANALYSES 38

RESULTS 40

1 IDENTIFICATION OF THE GENE CAUSING DOMINANT CPVT 401.1 Fine-mapping the CPVT locus (I) 401.2 Screening of candidate genes (I) 411.3 Identification of RYR2 mutations in CPVT patients (I, III) 411.4 Phenotypic characteristics of RYR2 mutations (I, II) 421.5 Functional properties of mutant RYR2 channels (II) 43

CONTENTS

4

CONTENTS

2 SCREENING OF THE CASQ2 GENE IN THE FINNISH CPVT PROBANDS (III) 443 SURVEY OF THE HERG GENE IN FINNISH LQTS PATIENTS 45

3.1 Identification of private HERG mutations and the HERG-Fin founder mutation (IV, V) 453.2 Phenotypic characteristics of HERG mutation carriers (IV, V) 453.3 Functional properties of the HERG-L552S mutation in vitro (IV) 463.4 Common amino acid polymorphism in the HERG gene (V) 47

DISCUSSION 48

1 IDENTIFICATION OF GENES UNDERLYING CPVT 481.1 Mutations of the ryanodine receptor gene in dominant CPVT 481.2 Functional and phenotypic properties of RYR2 mutations 501.3 CASQ2–the second gene for CPVT 52

2 HERG ALTERATIONS IN LQTS FAMILIES 532.1 Point mutations and small deletions 532.2 Phenotypic effects of K897T polymorphism 55

3 IMPLICATIONS FOR MOLECULAR DIAGNOSIS IN THE VENTRICULAR ARRHYTHMIA DISEASES CPVT AND LQTS 564 CONTINUING SEARCH FOR ARRHYTHMIA GENES 58

CONCLUSIONS 60

ACKNOWLEDGEMENTS 62

REFERENCES 64

1 PRINTED REFERENCES 642 INTERNET REFERENCES 75

LIST OF ORIGINAL PUBLICATIONS

5

LIST OF ORIGINAL PUBLICATIONS

This thesis is based on the following original articles, which are referred to in the text by

Roman numerals I-V. In addition, some unpublished data are presented.

I Laitinen PJ*, Brown KM*, Piippo K, Swan H, Devaney JM, Brahmbhatt B,

Donarum EA, Marino M, Tiso N, Viitasalo M, Toivonen L, Stephan DA, Kontula K.

Mutations of the cardiac ryanodine receptor (RyR2) gene in familial polymorphic

ventricular tachycardia. Circulation 2001; 103: 485-490.

II Lehnart SE*, Wehrens XHT*, Laitinen PJ*, Reiken SR, Deng S-X, Cheng Z,

Landry DW, Kontula K, Swan H, Marks AR. Molecular mechanism of familial

polymorphic ventricular tachycardia: calcium release channel (ryanodine receptor)

leak. Submitted.

III Laitinen PJ, Swan H, Kontula K. Molecular genetics of exercise-induced

polymorphic ventricular tachycardia: identification of three novel cardiac ryanodine

receptor mutations and two common calsequestrin 2 amino acid polymorphisms.

European Journal of Human Genetics 2003; 11: 888-891.

IV Piippo K, Laitinen P, Swan H, Toivonen L, Viitasalo M, Pasternack M, Paavonen

K, Chapman H, Wann KT, Hirvelä E, Sajantila A, Kontula K. Homozygosity for

a HERG potassium channel mutation causes a severe form of long QT syndrome:

identification of an apparent founder mutation in the Finns. Journal of the American

College of Cardiology 2000; 35: 1919-1925.

V Laitinen P, Fodstad H, Piippo K, Swan H, Toivonen L, Viitasalo M, Kaprio J,

Kontula K. Survey of the coding region of the HERG gene in long QT syndrome

reveals six novel mutations and an amino acid polymorphism with possible

phenotypic effects. Human Mutation, Mutation in Brief 2000; # 334: 1-6.

* Equal contribution

Study IV also appears in the thesis on Kirsi Piippo (2001).

6

ABBREVIATIONS

ABBREVIATIONS

Mg2+ magnesium ionms millisecondsNa+ sodium ionPKA protein kinase APCCD progressive cardiac conduction defectQT

c QT interval corrected for heart rate

RWS Romano-Ward syndromeRYR1 ryanodine receptor, type 2RYR1 gene for ryanodine receptor, type 1RYR2 ryanodine receptor, type 2RYR2 gene for ryanodine receptor, type 2RYR3 ryanodine receptor, type 3RYR3 gene for ryanodine receptor, type 3SCN5A cardiac voltage-gated sodium channelSCN5A gene for sodium channel, voltage-

gated, type V, alphaSERCA sarco/endoplasmic reticulum calcium

ATPaseSR sarcoplasmic reticulumWPW Wolf-Parkinson-White syndrome

In addition, standard one letter abbreviations for nucleotides and amino acids are used.

AF atrial fibrillationAnkB the gene for ankyrin BAP action potentialARVD arrhythmogenic right ventricular

dysplasiaATP adenosine triphosphatebpm beats per minuteBS Brugada syndromeCa2+ calcium ionCACNA1C cardiac L-type calcium channelcAMP cyclic adenosine monophosphateCASQ2 calsequestrin, type 2CASQ2 the gene for calsequestrin, type 2CCD central core diseasecM centiMorganCPVT catecholaminergic polymorphic

ventricular tachycardiaDCM dilated cardiomyopathyDHPLC denaturing high-performance liquid

chromatographyECG electrocardiogramFKBP12.6 FK506 binding protein 1B, 12.6 kDaFKBP1B gene for FKBP12.6HCM hypetrophic cerdiomyopathyHERG cardiac voltage-gated potassium

channelHERG human ether a-go-go related geneI

Ca cardiac calcium current

IKs

slow component of the delayed rectifier current

IKr

rapid component of the delayed rectifier current

INa

cardiac sodium currentJLNS Jervell and Lange-Nielsen syndromeK+ potassium ionKCNE1 gene for potassium voltage-gated

channel, Isk-related family, member 1KCNE2 gene for potassium voltage-gated

channel, Isk-related family, member 2KCNQ1 cardiac voltage-gated potassium

channelKCNQ1 gene for potassium voltage-gated

channel, KQT-like subfamily, member 1

LQTS long QT syndromeMH malignant hyperthermia

ABSTRACT

7

ABSTRACT

morphological or histological abnormalities

of the cardiac muscle are observed in affected

individuals. Both autosomal dominant and

recessive inheritance have been described,

with the dominant form more prevalent, yet

very rare. At the beginning of this study,

the gene underlying this life-threatening

disorder had been mapped to chromosome

1q42-43, but the molecular pathogenesis of

the disease was completely unknown.

The present study aimed at dissecting

the genetic background of LQTS and

CPVT. The HERG gene known to underlie

LQTS was screened for mutations by direct

sequencing. The chromosomal location of

CPVT locus was further delineated with

a dense panel of microsatellite markers.

Mutation screening was performed for

several positional and functional candidate

genes.

As one of the principal results of this

study, the gene coding for ryanodine

receptor type 2 (RYR2) was shown to

underlie CPVT. Six mutations: V2306I,

P2328S, Q4201R, V4653F, P4902L,

and R4959Q were detected in Finnish

families and were shown to co-segregate

with the disease phenotype. Functional

consequences of the mutations P2328S,

Q4201R, and V4653F were studied in

single-channel experiments. The mutated

Inherited arrhythmias as disorders are

rare, but have major clinical importance.

The best known of these diseases is long

QT syndrome (LQTS). It manifests with

prolonged QT interval in electrocardiogram,

ventricular tachycardias, syncope (sudden

loss of consciousness), and sudden death.

The trait can be inherited as autosomal

dominant (Romano-Ward syndrome, RWS)

or autosomal recessive (Jervell and Lange-

Nielsen syndrome, JLNS). Of these, RWS

is more common, having an approximate

prevalence of 1:5 000. In the extremely rare

JLNS, the cardiac manifestations are more

severe and are accompanied by congenital

deafness. Seven genes have been identified

as underlying the LQTS phenotypes. Six of

these, including KCNQ1, HERG, SCN5A,

KCNE1, KCNE2, and KCNJ2, encode

cardiac ion channels, whereas the gene

product of AnkB is a membrane-targeting

protein. LQTS displays considerable

genetic and phenotypic heterogeneity as

well as variable penetrance.

Catecholaminergic polymorphic

ventricular tachycardia (CPVT) has its

onset in adolescence and is characterized

by ventricular tachyarrhythmias induced by

physical or emotional stress. It is inherited

with almost complete penetrance and

if left untreated, has high mortality. No

8

ABSTRACT

RYR2 channels showed reduced binding

affinity to the regulatory protein FKBP12.6

and when phosphorylated, displayed

significantly increased activities consistent

with a gain-of-function abnormality.

In addition, the mutant channels were

resistant to inhibition by physiological

concentrations of magnesium.

Furthermore, genetic analysis of the

HERG gene disclosed a total of nine

mutations (453delC, R176W, P451L,

1631delAG, L552S, Y569H, G584S,

G601S, and T613M) in LQTS patients.

One of these mutations, L552S, was

detected in several families and represented

a Finnish founder mutation (HERG-

Fin). In functional studies, HERG-Fin

displayed a marked increase in its rate

of deactivation, which is suggested to

result in decreased channel activity at

the end of the repolarization phase. The

first common amino acid polymorphism

(K897T) of this gene was also described in

this study and its effect on the QT interval

examined in a cohort of LQTS mutation

carriers. A suggestive genotype-phenotype

relationship was demonstrated.

The results of this study can be applied

to the molecular diagnosis of affected

family members. Because the Finnish

founder mutation HERG-Fin accounts for

approximately 5% of all Finnish LQTS

cases, it is presently used in routine

screening of all LQTS probands. The

identification of the gene underlying

CPVT has enabled early presymptomatic

diagnosis in the families. Furthermore,

the pathological mechanism resulting in

this devastating disease is revealed, with

direct implications for pharmacological

interventions suggested.

INTRODUCTION

9

INTRODUCTION

Sudden cardiac death constitutes one of

the leading causes of mortality in western

countries. A minor but clinically significant

portion of these deaths is the consequence

of an inherited predisposition to cardiac

arrhythmias. During the last decade, the

genetic basis of these arrhythmic diseases

has been studied intensively. The first gene

to be localized in the genome was the one

that underlies type 1 long QT syndrome

(Keating et al. 1991). The first actual

disease-causing gene identified was the

HERG gene, whose mutations result in type

2 long QT syndrome (Curran et al. 1995).

To date, more than ten genes and hundreds

of mutations underlying these fatal diseases

have been characterized (Vincent 1998;

Chiang and Roden 2000).

As the knowledge of the genetic factors

underlying arrhythmic diseases constantly

increases, it is becoming evident that most,

if not all, proteins taking part in cardiac

action potential, excitation-contraction

coupling, and formation of the myocardial

sarcomere can harbor defects due to

mutations in their corresponding genes.

Identification of these genes and their

mutations has enabled the biochemical and

electrophysiological investigation of these

key components of myocardial function. In

turn, important functional aspects effective

in the normal and diseased state of the cell

have been revealed. Thus, disease-causing

mutations provide us not only with new

disease mechanisms, but also with new

insights into cell biology.

Most of the arrhythmic diseases known

thus far are monogenic and inherited as

autosomal dominant traits. This picture is

complicated by findings that one disorder

can be due to mutations in several distinct

genes (locus heterogeneity), and mutations

in a single gene can lead to a variety of

clinical phenotypes (allelic heterogeneity).

Considerable phenotypic and genetic

heterogeneity suggests the presence of

modifying genes and challenges the

traditional clinical diagnosis.

This study aimed at investigating

the genetic background of two inherited

arrhythmic diseases, the catecholaminergic

polymorphic ventricular tachycardia and

the long QT syndrome. The study led

to identification of the gene underlying

catecholaminergic polymorphic ventricular

tachycardia. Several previously unknown

mutations, including a Finnish founder

mutation resulting in long QT syndrome

were characterized. Functional studies on

these genetic alterations elucidated the

pathogenic mechanisms underlying the

clinical phenotypes.

10

REVIEW OF THE LITERATURE


1 ELECTRICAL AND CONTRACTILE FUNCTIONS OF THE HEART CELL

1.1 CARDIAC ACTION POTENTIAL

A pulse of electrical excitation in a group of

specialized pacemaker cells initiates each

heartbeat (Marban 2002). This electrical

impulse subsequently spreads throughout

the heart and is known as the action

potential (AP). The AP of ventricular cells

is characterized by a prolonged plateau

phase. This long plateau is important in

two ways: calcium ions (Ca2+) that are

necessary to initiate contraction enter the

cell, and sodium channels are held in the

inactive state until the depolarizing wave

has spread to the whole electrically coupled

myocardium (Gargus 2003).

The plasma membrane of a resting

cardiac myocyte is highly permeable to

K+ and impermeable to Na+ (Roden et al.

2002). Because resting potential of the

plasma membrane is formed by open K+

channels, it is approximately –70 mV,

close to the Nernst potential of potassium

(Ackerman and Clapham 1997). Because

a propagating impulse from the sinus

node changes the potential, Na+ channels

Figure 1. Ventricular action potential (top), the underlying ionic currents and the genes encoding the corresponding channels. Currents are depicted as function of time, depolarizing currents below the base line and repolarizing currents above the base line. I

Na, cardiac

sodium current; ICa,L

, cardiac L-type calcium current; I

Na/Ca, sodium/

calcium exhanger; IK1

, pacemaker current; I

To1 and I

To2, transient

outward currents; IKr

, rapid component of the delayed rectifier current; I

Ks, slow component of the

delayed rectifier current; IKp

, time-independent background current.


11

alter their conformation and thus open.

This results in rapid influx of Na+ ions and

depolarization of the membrane (Figure 1,

phase 0). Balance of an inward, depolarizing

Ca2+ current and an outward, repolarizing

K+ current maintains the long plateau phase

(phase 1-2). The outward potassium current

produced by delayed rectifier K+ channels

and inactivation of Ca2+ channels (phase

3) complete the repolarization. The final

repolarization (phase 4) is due to the inward

rectifier K+ channels’ producing an outward

K+ current (see Ackerman and Clapham

1997 and Roden et al. 2002 for review).

1.2 POTASSIUM AND CALCIUM

CURRENTS OF THE HEART

All voltage-gated channels share several

fundamental functional modules (Terlau

and Stuhmer 1998; Roden et al. 2002).

These include a voltage sensor sensing

the changes in membrane potential, an

activation gate opening in response to

the voltage sensor, and a selectivity filter

favoring permeation of a specific ion.

In potassium channels transmembrane

segment S4 acts as the voltage sensor (Bao

et al. 1999), whereas the S5 segment takes

part in the gating process (Shieh et al. 1997)

(Figure 2).

The action potential is the result of the

coordinated function of numerous voltage-

gated ion channels and the ionic currents

they produce (Marban 2002). Some

important ionic currents of ventricular AP

are depicted in Figure 1. Here, only the

major potassium and calcium currents and

the respective ion channels are reviewed.

Delayed rectifier K+ currents activate

with some delay following the initial

depolarization. The HERG (human ether a-

go-go related) gene encodes the α subunit

Figure 2. Schematic representation of the predicted transmembrane topology of voltage-gated K+, Na+, and Ca2+ channels. Arrows indicate the ion-permeating pore regions. The potassium channel is formed by four

identical α-subunits, each comprising six transmembrane domains. In sodium and calcium channels, the same overall structure consists of four repeats (I-IV) of six transmembrane domains.

12


of the potassium channel underlying IKr

, the

rapid component of the delayed rectifier

potassium current (Sanguinetti et al. 1995).

The HERG gene is the human homolog

of the Drosophila eag-gene, identified by

the leg-shaking phenotype of the fly. The

mature channel is a tetramer formed by four

HERG subunits and possibly modulated by

the auxiliary β subunit, which is encoded by

the KCNE2 gene (Abbott et al. 1999). Each

α subunit comprises six transmembrane

regions and an ion-permeating pore domain

(Warmke and Ganetzky 1994; Roden and

George 1997) (Figure 2). The main function

of the HERG channel is in the beginning of

the repolarization phase, when it produces a

large outward current that further promotes

rapid repolarization. The unique features

of this channel are fast inactivation and

slow deactivation (Sanguinetti et al. 1995).

Many drugs block the HERG channel

(Vandenberg et al. 2001).

The KCNQ1 gene encodes the slow

component (IKs

) of the delayed rectifier.

The KCNQ1 channel is a tetramer,

which, in addition to four α subunits,

contains auxiliary β subunits (Sanguinetti

et al. 1996b). The KCNE1 gene codes

for this auxiliary β subunit (Murai et al.

1989). Slow activation and deactivation

characterize IKs

, which functions at the end

of the repolarization phase.

The L-type (long-lasting) and T-type

(transient, tiny or high threshold) are the

major cardiac Ca2+ currents (ICa). As

T-type channels are mostly present in

specialized structures of the heart, such

as sinus and atrioventricular nodes and

the conduction system, and as they do not

contribute to ventricular AP, ICa

usually

refers to the L-type current.

The L-type Ca2+ current has two

important functions: it is a major contributor

in the inward current sustaining the long

cardiac action potential and triggers the

Ca2+ release needed for muscle contraction.

The calcium is released from the

sarcoplasmic reticulum (SR), a specialized

type of endoplasmic reticulum (Roden et al.

2002). L-type calcium channels are located

on the plasma membrane invaginations

(T-tubules). Although ICa

is activated

by membrane depolarization, calcium-

dependent inactivation at the cytoplasmic

side limits the amount of Ca2+ entering the

cell during the action potential (Peterson et

al. 1999). This important feedback function

is mediated by calmodulin, the ubiquitous

Ca2+ sensor of cells.

A single large α subunit (Schultz et al.

1993), together with an auxiliary β subunit

(Chien et al. 1995), forms the cardiac L-

type Ca2+ channel which is also called the

dihydropyridine receptor. The CACNA1C

gene encodes the alpha subunit (Mikami

et al. 1989) consisting of four repeats of

six transmembrane domains (Terlau and

Stuhmer 1998) (Figure 2).


13

1.3 EXCITATION-CONTRACTION

COUPLING–FROM PLASMA

MEMBRANE TO CONTRACTION

APPARATUS

Depolarization of the plasma membrane of

cardiac muscle cells initiates the sequence

of electrical and biological functions

leading to the contraction of the cardiac

myocyte and the beating of the heart. The

L-type calcium channels located in the

sarcolemmal SR junctions (Figure 3) allow

the entry of a small quantity of Ca2+ ions

to the cell (Bers 2002). As the cytoplasmic

calcium concentration rises, Ca2+ binds to

the cardiac ryanodine receptor (RYR2)

channels located on the SR and activates

the channels. The mechanism underlying

this activation and the termination of

Ca2+ release from the SR has been termed

calcium-induced calcium release (Figure

3).

SR is an important reservoir of Ca2+ ions.

Activated RYR2 channels release a massive

Ca2+ spark from the SR (Wier and Balke

1999), and the elevated Ca2+ concentration

allows troponin C to bind Ca2+. This leads

to conformational changes in troponin C

protein and thereby releases the inhibition

between actin and myosin, which results in

contraction of the myocyte. All the proteins

comprising this signal transduction

apparatus are in the vicinity of each other.

This allows the calcium concentration to

rise locally without the disturbance of other

signaling functions in the cell.

Ca2+ ions must be removed from the

Figure 3. Calcium transport in ventricular myocytes. RYR2, ryanodine receptor type 2; CASQ2, cardiac calsequestrin; PLB, phospholamban; SERCA2, cardiac SR calcium ATPase; PMCA, plasma membrane calcium ATPase; NCX, Na+/Ca2+ exchanger.

14


cytoplasm in order to lower the calcium

concentration and thereby allow relaxation.

Rapid reuptake of calcium back into the SR

is achieved by a large number of cardiac

SR calcium ATPase pumps which are

negatively regulated by phospholamban

(Frey et al. 2000; Stokes and Wagenknecht

2000). A smaller amount of Ca2+ is also

extruded from the cell by plasma membrane

calcium ATPase and by Na+/Ca2+ exchanger.

This extrusion is necessary to maintain the

calcium balance of the cell, since some of

the Ca2+ present in the cytoplasm initially

entered through the L-type channels.

Together, these two systems remove 98%

of the Ca2+, whereas the remaining 2% is

removed by sarcolemmal Ca2+-ATPase and

mitochondrial Ca2+ uniporter (Bers 2002).

1.4 CARDIAC CA2+ SIGNALING PROTEINS

INVOLVED IN CALCIUM-INDUCED

CALCIUM RELEASE

Numerous proteins contribute to the

delicately regulated process of calcium-

induced calcium release. Here, only those

proteins relevant for this work are discussed

in detail.

Ryanodine receptor, type 2 (RYR2)

is a calcium-release channel expressed

in the heart and brain (Otsu et al. 1990;

McPherson and Campbell 1993). Four 565

kDa subunits form the functional channel

(Tunwell et al. 1996; Marx et al. 2000).

The N-terminal part corresponding to

approximately four-fifths of the channel

subunit is located in the cytoplasm, while

the remaining one-fifth (1 000 amino

acids) form the transmembrane domains

(Figure 4). The number of transmembrane

segments is disputed, estimates ranging

from 4 to 12 (Otsu et al. 1990; Tunwell et

al. 1996; Stokes and Wagenknecht 2000).

Recent experiments on the skeletal muscle

homolog RYR1 suggest the presence of

eight transmembrane domains (Du et al.

2002). The large cytoplasmic “foot” domain

of the channel functions as a scaffold for a

number of regulatory proteins (Stokes and

Wagenknecht 2000).

The functional channel is a

macromolecular complex of four RYR2

subunits and four FKBP12.6 (also called

calstabin 2) proteins. In addition, cAMP-

dependent protein kinase A (PKA), the

protein phosphatases PP1 and PP2A, and

the anchoring protein mAKAP6 have been

shown to belong to the complex (Marx

et al. 2000). Calcium release through

RYR2 occurs in a coordinated manner,

because RYR2 channels are functionally

and physically coupled. Removal of the

regulatory protein FKBP12.6 removes the

functional but not the physical coupling.

(Marx et al. 2001)

During exercise, the sympathetic

nervous system stimulation leads to

catecholaminergic activation of beta-

adrenergic receptors (Rockman et al.

2002). These are coupled to G-proteins to

activate the adenylyl cyclase that elevates


15

calcium release (channel openings), while

the low affinity binding site inhibits it.

Free magnesium inactivates the channels

at 1 mM concentration, which is the

physiologic level of this ion (Zucchi and

Ronca-Testoni 1997; Copello et al. 2002).

ATP, as well as other adenine nucleotides,

acts as an activator of RYR2, increasing

the channel’s open probability (Zucchi and

Ronca-Testoni 1997).

The importance of the RYR2 channel

is underlined by the findings that mutant

mice lacking RYR2 develop morphological

abnormalities in the heart tube and die

at approximately embryonic day 10

(Takeshima et al. 1998). In failing human

hearts, RYR2 channels have been proposed

to be hyperphosphorylated by PKA (Marx

et al. 2000). This is predicted to result in

defective function of the channels due

to increased sensitivity to Ca2+-induced

activation (Marks 2000; Marx et al. 2000).

Figure 4. Schematic model of the cardiac ryanodine receptor and its suggested interactions with triadin, junctin and calsequestrin. Proteins not drawn to scale. RYR2, ryanodine receptor type 2; CASQ2, cardiac calsequestrin.

Cytoplasm

SR lumen

N

C

RYR2 pore

N

C

N

C

JunctinTriadin

CASQ2

N

C

N

C

CASQ2

RYR2

intracellular cAMP concentration, that in

turn activates cAMP-dependent protein

kinase A (PKA) (Brodde and Michel

1999). As a result, RYR2 channels are

phosphorylated by PKA. Phosphorylation of

the RYR2 channels dissociates FKBP12.6,

which in turn results in an increase in the

open probability of the channel.

Calcium, magnesium, and ATP

modulate RYR2 channels (Valdivia

et al. 1995). Of these, calcium is the

physiological channel activator (Coronado

et al. 1994; Zucchi and Ronca-Testoni

1997). The Ca2+ dependence of the

channel is biphasic: low (micromolar)

cytoplasmic [Ca2+] activates the channels,

and high (millimolar) cytoplasmic [Ca2+]

inactivates them. The biphasic response to

calcium has been hypothesized to result

from the presence of both high- and low-

affinity Ca2+ binding sites in the channel:

the high-affinity binding site stimulates

16


Calsequestrin 2 (CASQ2) protein is

located inside the SR. It binds calcium

with high capacity and moderate affinity,

buffering Ca2+ inside the SR and preventing

its intra-sarcoplasmic precipitation

(Mitchell et al. 1988). Crystallization of

the skeletal muscle isoform CASQ1 reveals

that calsequestrin forms three domains,

each with a thioredoxin fold (Wang et

al. 1998). Calcium binds mostly between

these domains and to the protein surface.

This mechanism differs from that of other

Ca2+-binding proteins, since most of them

have EF-hand structures (motifs formed by

two helixes, E and F) that mediate binding.

The acidic carboxyterminus of CASQ2

plays a critical role in calcium binding

and in interactions with other proteins

(MacLennan and Reithmeier 1998). As

Ca2+ is bound, calsequestrin polymerizes

into extended soluble structures (Wang et

al. 1998).

RYR2 and CASQ2 form a complex

through triadin and junctin (Figure 4).

These two membrane proteins anchor

calsequestrin molecules at the inner face of

the junctional SR membrane (Zhang et al.

1997). Together, RYR2, CASQ2, triadin,

and junctin form the junctional complex

essential for calcium-induced calcium

release. L-type calcium channels in the

T-tubular regions and RYR2 channels in

the SR are located close to each other, and

thereby the activation of RYR2 channels is

under the tight control of L-type channels,

resulting in the rapid and efficient coupling

of membrane depolarization and SR Ca2+

release (Wier and Balke 1999).

2 GENETIC BASIS OF INHERITED ARRHYTHMIC DISORDERS

A number of familial disorders are known

to manifest with cardiac arrhythmias.

Symptoms of cardiac myopathies may be

associated with structural changes in the

heart muscle, while in other disorders, the

heart muscle itself is intact, but its electrical

properties are compromised. Although

at population level these arrhythmic

disorders are rare, their clinical importance

is significant. They may have severe

health consequences (syncope, ventricular

fibrillation, sudden death) and usually affect

young, otherwise healthy individuals.

During recent years, many genes

underlying these fatal diseases have been

identified, mainly through positional

cloning strategy. Several genes remain

unknown, however. As initial research

has focused on diseases with high clinical

impact, arrhythmic diseases with a milder

phenotype or a complex inheritance have

probably been neglected.

2.1 ELECTRIC INHERITED ARRHYTHMIAS

2.1.1 Long QT syndromes

In 1957, Jervell and Lange-Nielsen

described a syndrome manifesting with

prolonged QT interval and syncopal spells


17

associated with deafness and a family

pattern that suggests autosomal recessive

inheritance. A few years later, it became

apparent that patients with normal hearing

could also suffer from these typical cardiac

features, and that this form of the disease

is inherited as an autosomal dominant trait

(Ward 1964; Romano 1965).

Clinical features

Congenital long QT syndrome

(LQTS) is characterized by a delayed

ventricular repolarization which leads to

prolongation of the QT interval on the

electrocardiogram (ECG) (Chien 1999)

(Figure 5). Other clinical features include

T-wave abnormalities, syncopal spells,

and torsade de pointes (twisting of the

points) ventricular arrhythmias (Figure 5).

These arrhythmias are typically induced by

physical exercise or emotional stress, and

can be fatal. The affected individuals show

no structural abnormalities of the heart

muscle.

Estimates of the prevalence of

autosomal dominant LQTS (Romano-Ward

syndrome, RWS, OMIM 192500) range

from 1:5 000 to 1:10 000. The autosomal

recessive form of LQTS (Jervell and Lange-

Nielsen syndrome, JLNS, OMIM 220400),

in which arrhythmias are associated with

congenital deafness, is extremely rare

(Ackerman 1998). Since many of the

individuals affected with dominant LQTS

are asymptomatic, this syndrome is likely

to remain undiagnosed. The comprehensive

set of diagnostic criteria for LQTS in use

since 1993 takes into account the QT

interval corrected for heart rate (QTc),

morphology of the T-wave, and patient’s

clinical and family history (Schwartz et al.

1993). Heart-rate-corrected QT interval is

calculated from the equation QTc = QT/

RR1/2 (sec), where RR denotes the heart rate

Figure 5. ECG strips (lead 2) demonstrating a) normal QT interval, b) prolonged QT interval, c) abnormal T-wave, and d) torsade de pointes ventricular arrhythmia.

a b

c d

18


in beats per minute (Bazett 1920).

Mortality estimates as high as 50% per

ten years have been presented for LQTS

(Moss et al. 1991; Ackerman 1998). This

is probably an overestimate reflecting the

fact that more severely affected individuals

were identified in the early years of LQTS

studies.

Molecular genetics of LQTS

LQTS can be divided into several subtypes

based on the mode of inheritance and

the genetic defect underlying the disease

(Table 1; see also for complete gene

names). Estimates are that a third (Wichter

et al. 2002) or even half (Moss 2003) of the

LQTS-causing mutations have been found

to date. Since there are many families

in whom the phenotype does not show

linkage to any of the known disease loci,

it is probable that unknown loci exist even

yet.

LQT1 (OMIM 192500) Mutations of

the KCNQ1 gene result in the LQT1 subtype

of the long QT syndrome and account for

approximately 42% of genetically defined

LQTS (Splawski et al. 2000). More than

one hundred mutations have been identified

in this gene (see the Internet database

Gene connection for the heart). KCNQ1 is

expressed in the heart, ear, kidney, lung, and

placenta (Wang et al. 1996a; Neyroud et al.

1997). In Finland, the founder mutation

G589S is detected in 30% of Finnish LQTS

patients (Piippo et al. 2001b; unpublished).

This mutation is located in the C-terminus

of the protein and has a clinically mild

phenotype. In functional studies, the

G589S mutant subunits formed functional

channels, but showed reduced currents

(Piippo et al. 2001b).

LQT2 (OMIM 152427) This subtype

results from mutations in the HERG gene

which is abundantly expressed in the heart

and brain (Wymore et al. 1997). HERG

(also called KCNH2, for potassium voltage-

gated channel, subfamily H, member 2) is

composed of 15 exons (Itoh et al. 1998)

and was originally described as the human

counterpart of the Drosophila ether a-go-go

related gene (Warmke and Ganetzky 1994).

Heterozygous mutations in the HERG gene

lead to the RW syndrome (Curran et al.

1995), but some homozygous patients have

also been described with a severe cardiac

phenotype but without associated hearing

loss (Hoorntje et al. 1999; this study). With

more than 130 HERG mutations identified

(Internet database Gene connection for the

heart), estimates are that 45% of inherited

LQTS is caused by mutations in this gene

(Splawski et al. 2000). In Finland, however,

mutations in the KCNQ1 gene appear to be

a more frequent cause of LQTS than are

mutations in the HERG gene (Piippo et al.

2001b; unpublished).

LQT3 (OMIM 603830) The SCN5A

gene consists of 28 exons and encodes

a voltage-gated cardiac sodium channel

(Wang et al. 1996b). The expression of

this gene is evident solely in cardiac tissue.


19

Mutations in SCN5A appear to be a relatively

rare cause of LQTS, representing only 8%

of the molecularly defined cases (Splawski

et al. 2000). In the Finnish population, only

one mutation has been described to date

(Piippo et al. 2001a). This V1667I mutation

causes a relatively mild form of LQT3, but

when halofantrine, an antimalarial drug,

was administered, the mutation carriers

experienced ventricular tachycardia and a

considerable prolongation of QTc (Piippo

et al. 2001a).

LQT4 (OMIM 600919) In a single

large French kindred the phenotype of

LQTS and sinus node dysfunction was

mapped to chromosome 4q25-27 (Schott et

al. 1995). Recently, in the affected family

members the ankyrin B (AnkB) gene was

shown to harbor a mutation (Mohler et al.

2003). AnkB is a 45-exon gene expressed in

most cell types and coding for a membrane

adaptor targeting numerous proteins to cell

membrane (Mohler et al. 2002). Mutations

in AnkB probably constitute an extremely

rare cause of LQTS, since there have been no

other reports of LQTS families’ phenotypes

showing linkage to this chromosomal

region. This gene has not been studied in

the Finnish LQTS population.

LQT5 (OMIM 176261) and LQT6

(OMIM 603796) are caused by mutations

in the KCNE1 and in the KCNE2 genes.

These genes, located only 79 kb apart,

are transcribed from oppositely oriented

frames. The gene product of KCNE2 is

a 123 amino-acid auxiliary subunit for

potassium channel KCNQ1, while KCNE3

codes for a 103 amino-acid protein which

possibly modulates the HERG channel

(Abbott et al. 1999). Heterozygous

mutations in the KCNE1 and in the KCNE2

genes lead to the RW type of LQTS

(Splawski et al. 1997; Abbott et al. 1999).

Mutations in the KCNE1 and in the KCNE2

genes account for 3% and 2% of inherited

LQTS, respectively (Splawski et al. 2000).

In Finland, no mutations have been detected

in these genes to date.

LQT7 (OMIM 170390) Recently,

mutations in the KCNJ2 gene were found in

patients affected with Andersen syndrome

(Tristani-Firouzi et al. 2002), a rare disorder

characterized by prolonged QT interval,

cardiac arrhythmias, periodic paralysis, and

dysmorphic features. In addition, mutations

were also detected in patients with LQTS

and periodic paralysis (Ai et al. 2002).

The KCNJ2 gene consists of 2 exons and

has a mRNA of 1.3 kb in size. It encodes

an inwardly rectifying potassium channel

comprising two transmembrane domains

and a pore region (Raab-Graham et al.

1994). Variable expression and penetrance

may account for the polymorphic clinical

phenotype resulting from KCNJ2 mutations.

Since many of the KCNJ2 mutation carriers

present with prolonged QT interval, this

gene has been labeled as the LQT7 gene

(Tristani-Firouzi et al. 2002).

JLNS In Jervell and Lange-Nielsen

20


syndrome, the arrhythmic symptoms

are accompanied by congenital deafness

(Jervell and Lange-Nielsen 1957). In

general, the hearing defect is inherited as

an autosomal recessive trait and the cardiac

phenotype as an autosomal dominant trait.

Heterozygous mutation carriers are usually

clinically unaffected or have borderline

prolonged ECGs. Mutations in genes

coding for potassium channel subunits

underlying the IKs

current, namely KCNQ1

(Neyroud et al. 1997) and KCNE1 (Tyson

et al. 1997), lead to the JNLS phenotype.

The rationale behind mutations in the same

genes resulting in both RWS and JLNS is

that KCNQ1 and KCNE1 are expressed

abundantly also in the inner ear. They are

involved in production of endolymph,

the potassium-rich fluid of the inner ear

(Neyroud et al. 1997). Complete loss of the

channel function destroys the endolymph

ion homeostasis, resulting in abnormal hair

cell function and deafness.

Although heterozygous KCNQ1

mutations usually lead to the Romano-

Ward syndrome, and homozygous or

compound heterozygous mutations result

in the Jervell and Lange-Nielsen syndrome,

some mutations differ from this pattern. In

particular, the A300T missense mutation in

the KCNQ1 gene has been reported to result

in a severe cardiac phenotype but normal

hearing in the homozygous offspring of

heterozygous parents with normal hearing

and no cardiac symptoms (Priori et al.

1998). In JLNS families, some heterozygous

carriers have clearly prolonged QTc intervals

and could therefore be classified as having

RW syndrome (Duggal et al. 1998; Piippo

et al. 2001b). These findings imply that no

straightforward classification of recessive

and dominant LQTS can be made.

Molecular pathogenic mechanisms of LQTS-causing mutations

Mutations in several different genes result

in the LQTS phenotype. At least four

distinct mechanisms underlie the delayed

repolarization that leads to prolonged QT

interval: 1) Defects in the delayed rectifier

potassium currents IKs

and IKr

(LQT1,

LQT2, LQT5, LQT6) result in reduced

outflow of potassium ions, thus lengthening

the repolarization phase (Shalaby et al.

1997; Splawski et al. 2000). 2) LQT3-

associated SCN5A mutants produce an

increased sodium current by resistance

to inactivation (Tan et al. 2003), while

3) KCNJ2 mutations in LQT7 result in

reduction of the inward potassium current

IK1

(Ai et al. 2002). 4) The gene that causes

LQT4 codes for the membrane-targeting

protein ankyrin B, and accordingly the

resultant pathogenic mechanism differs

from that the other subtypes (Mohler et

al. 2003). The only mutation described in

this gene has a loss-of-function phenotype

and thus results in the disrupted cellular

organization of the sodium pump, Na+/Ca2+

exchanger, and inositol-1,4,5-trisphosphate

receptors.


21

Several mechanisms have been

described by which the mutations in ion

channel genes lead to the LQTS phenotype.

In general, mutations in potassium channel

subunits show a loss-of-function phenotype

resulting in reduced potassium currents

(Roden 2001), whereas the LQTS-causing

mutations of the SCN5A gene have a gain-

of-function effect: increasing the flow of

sodium ions to the cell (Dumaine et al.

1996; Tan et al. 2003).

The dominant negative effect–in

which, when heterotetramers are formed,

the mutant channel subunit suppresses the

activity of wild-type subunits–is probably

the most common mechanism leading

Table 1. Summary of primarily electric arrhythmogenic disorders.

Disease Locus Key clinical features Gene Protein

LQT1 11p15.5 ECG abnormalities, syncope KCNQ1 K+ channel α subunit

LQT2 7q35-36 ECG abnormalities, syncope HERG K+ channel α subunit

LQT3 3p21-23 ECG abnormalities, syncope SCN5A Na+ channel α subunit

LQT4 4q25-27 ECG abnormalities, sinus node dysfunction

AnkB Adaptor protein

LQT5 21q22.1-22.2 ECG abnormalities, syncope KCNE1 K+ channel β subunit

LQT6 21q22.1-22.2 ECG abnormalities, syncope KCNE2 K+ channel β subunit

LQT7 17q23 Prolonged QT, periodic paralysis KCNJ2 K+ channel α subunit

JLNS1 11p15.5 Congenital deafness, cardiac manifestations

KCNQ1 K+ channel α subunit

JLNS2 21q22.1-22.2 Congenital deafness, cardiac manifestations

KCNE1 K+ channel β subunit

CPVT 1q42-43 Syncope, sudden death RYR2 Ca2+ release channel

CPVT 1p13.3-11 Syncope, sudden death CASQ2 Ca2+ binding protein

BS 3p21-23 ECG abnormalities, RBBB SCN5A Na+ channel α subunit

PCCD 3p21-23 Conduction disturbances SCN5A Na+ channel α subunit

PCCD 19q13.2-13.3 Conduction disturbances ? ?

WPW 7q36 Preexcitation PRKAG2 AMP-activated kinase γ subunit

AF 6q14-q16 Atrial arrhythmia ? ?

AF 10q22-q24 Atrial arrhythmia ? ?

AF 11p15.5 Atrial arrhythmia KCNQ1 K+ channel α subunit

LQT, long QT syndrome; KCNQ1, gene for potassium voltage-gated channel, KQT-like subfamily, member 1; HERG, human ether a-go-go-related gene; SCN5A, gene for sodium channel, voltage-gated, type V, alpha; AnkB, gene for ankyrin B; KCNE1, gene for potassium voltage-gated channel, Isk-related family, member 1; KCNE2, gene for potassium voltage-gated channel, Isk-related family, member 2; KCNJ2, gene for potassium inwardly rectifying channel, subfamily J, member 2; JLNS, Jervell and Lange-Nielsen syndrome; CPVT, catecholaminergic polymorphic ventricular tachycardia; RYR2, gene for ryanodine receptor, type 2; CASQ2, gene for calsequestrin 2; BS, Brugada syndrome; PCCD, progressive cardiac conduction defect; WPW, Wolf-Parkinson-White syndrome; PRKAG2, gene for protein kinase, AMP-activated, gamma 2 non-catalytic subunit; AF, atrial fibrillation; RBBB, right bundle branch block;

22


to reduction in the IKs

and IKr

currents

(Shalaby et al. 1997; Nakajima et al. 1998;

Splawski et al. 2000). Some mutants do

not form functional channels and therefore

do not exhibit a dominant-negative effect

(Sanguinetti et al. 1996a). The mutant

protein can also be misfolded, subsequently

recognized by the cellular machinery, and

targeted to degradation pathways, thus

never reaching the plasma membrane.

This type of trafficking defect results from

some KCNQ1 (Roden 2001) and HERG

mutations (Huang et al. 2001).

Genotype-phenotype correlation in LQTS

Most of the mutations underlying the

LQTS phenotype have been detected in

KCNQ1, HERG, and SCN5A genes, while

mutations in other known LQTS genes

appear to be rare (Internet database Gene

connection for the heart). But does the

locus of the causative mutation have any

effect on patient phenotype? Indeed, it

appears that the clinical course of LQTS

can at least in part be predicted based on the

underlying gene. A recent study evaluating

647 genotyped patients was aimed at risk

stratification in LQTS (Priori et al. 2003).

Its results were that cumulative survival

rate is higher among the patients carrying

a mutation in the KCNQ1 gene than among

those with a mutation in the HERG or

SCN5A gene. The same study shows that

a significant portion (36%) of KCNQ1

mutation carriers have a normal QTc,

whereas in the SCN5A group, this figure is

only 10%. The lethality of cardiac events is

higher in the SCN5A group than in the other

two groups (Zareba et al. 2003).

Gene-specific triggers have also been

identified. Individuals carrying a mutation

in the KCNQ1 gene have been shown

to experience most of their symptoms

(62%) during physical exercise, with only

3% of their arrhythmic events occurring

during sleep (Schwartz et al. 2001). With

HERG and SCN5A genes, the situation

differs: patients in these groups are more

likely to experience cardiac events during

rest or sleep. An auditory stimulus often

triggers cardiac events in the HERG group

(Jongbloed et al. 1999; Schwartz et al.

2001), but swimming is a frequent trigger

among KCNQ1-mutation carriers (Schwartz

et al. 2001). Beta-blocker therapy is more

effective among LQT1 patients than among

LQT2 and LQT3 patients (Schwartz et al.

2001).

Genetic and phenotypic heterogeneity of LQTS

Since an increasing number of genes (to

date, seven) and hundreds of mutations

underlying different LQTS phenotypes

have been detected (Table 1), it has become

clear that a single genetic defect does not

completely determine the phenotype of

each patient. Variable phenotypes in related

individuals with the same mutation suggests

the influence of environmental factors

and the presence of modifying genetic

effects (Priori et al. 1999a; Moss 2003).


23

At least in the LQT1 and LQT2 subgroups,

gender and age affect probability of cardiac

events (Zareba et al. 2003). The reduced

penetrance and variable expressivity

associated with mutations in the different

LQTS genes remain to be explained.

2.1.2 Catecholaminergic polymorphic ventricular tachycardia

Catecholaminergic polymorphic ventricular

tachycardia (CPVT, OMIM 604772), which

is also denoted FPVT (familial polymorphic

ventricular tachycardia), is characterized by

episodes of bidirectional and polymorphic

ventricular tachycardias (Leenhardt et al.

1995) (Figure 6). Physical or emotional

stress typically triggers the symptoms, with

no myocardial abnormalities observed. QT

intervals in affected subjects are normal

or borderline prolonged. The onset of

the disease is in adolescence or young

adulthood. Mortality from this syndrome

is high, reaching as high as 31% by the age

of 30 if left untreated (Swan et al. 1999).

Treatment with beta-adrenergic blockers

appears to be an effective therapy (Fisher et

al. 1999), but occasionally an implantable

cardiac defibrillator is necessary (Swan et

al. 1999). Table 2 shows the main clinical

features of CPVT.

In the initial study, 30% of patients had

a familial history of syncope or sudden

death (Leenhardt et al. 1995). Since then,

both autosomal dominant and recessive

modes of inheritance have been reported.

Our group mapped the dominant form of

the disease to chromosome 1q42-43 (Swan

et al. 1999), while the recessive form was

localized in the short arm of the same

chromosome (Lahat et al. 2001a). Recently,

the dominant form was shown to result from

mutations in the cardiac ryanodine receptor

gene (this study; Priori et al. 2001), while

Figure 6. Electrocardiogram (leads V1-V5) during exercise stress test of a 46-year-old male, demonstrating ventricular complexes (black arrows).

V1

V2

V3

V4

V5

24


mutations in the cardiac calsequestrin gene

were reported to underlie the recessive form

of CPVT (Lahat et al. 2001b) (Table 1).

2.1.3 Other inherited electrical arrhythmias

Brugada’s syndrome (BS, idiopathic

ventricular fibrillation, OMIM 601144)

This is an ubiquitous disease with a

prevalence of 1 in 2 000 in the general

population (Antzelevitch et al. 2003).

It is, however, more prevalent in South

Asia (Vatta et al. 2002), where it is called

sudden unexplained nocturnal death. For

some unknown reason, it more often affects

males. Symptoms include ventricular

fibrillation and a heightened risk of cardiac

sudden death. The ECG shows an elevated

ST segment and shortened action potentials.

BS can be caused by loss-of-function

mutations of the SCN5A gene (Chen et al.

1998), while the LQT3 phenotype results

from gain-of-function mutations in the

same gene, but many BS patients lack

SCN5A mutations. Another locus has been

mapped to chromosome 3p22-25 (Weiss

et al. 2002) near the SCN5A gene, but no

defective gene has yet been identified.

Progressive cardiac conduction

defect (PCCD, OMIM 113900) is

characterized by progressive slowing of

the electrical conduction through the His-

Purkinje system, manifesting as widening

of the QRS complexes. This may lead to

atrioventricular block and sudden death.

SCN5A missense mutations underlie PCCD

(Schott et al. 1999), and also underlie the

LQT3 and Brugada syndrome. Compound

heterozygosity for two particular SCN5A

mutations results in severe disturbances

and degenerative changes in the conduction

system, whereas the heterozygous carriers

of these same mutations were asymptomatic

(Bezzina et al. 2003). A second PCCD locus

was localized to chromosome 19q13.2-13.3

(Brink et al. 1995), but the actual disease-

causing gene remains unknown.

Wolff-Parkinson-White syndrome

(WPW, OMIM 194200) is characterized

by pre-excitation of the ventricles by

accessory atrioventricular fibers (Al-Khatib

Table 2. Summary of symptoms and findings in CPVT (Modified from Swan and Laitinen 2002).

Symptoms Exercise- or stress-related syncopal spells or sudden death

Resting ECG Usually normal findingsMinor QT interval prolongation possible

Electrophysiologic study Programmed electrical stimulation rarely induces ventricular tachycardiaIn some patients isoproterenol infusion induces ventricular arrhythmias

Exercise stress test and ambulatory ECG

Ventricular premature complexes, ventricular bigeminy, salvoes of polymorphic ventricular premature complexes, or non-sustained polymorphic ventricular tarchycardia during physical effort

Cardiac imaging studies Usually normal findingsLocalized right ventricular aneurysms or dyskinetic areas in some patients


25

and Pritchett 1999). Electrocardiographic

findings include short PR interval and

prolonged QRS. The prevalence of WPW

has been 1.5 to 3.1 per 1 000 individuals

in Western countries (Gollob et al. 2001).

Most cases are sporadic, but also familial

occurrence with autosomal dominant

inheritance has been reported. Clinical

manifestations of WPW are diverse,

since the symptoms of WPW can be

accompanied by atrial septal defect, mitral

valve prolapse (Al-Khatib and Pritchett

1999), dilated cardiomyopathy (Grunig

et al. 1998), and many other conditions.

Approximately 10% of patients affected

have hypertrophic cardiomyopathy (HCM),

and the defective gene underlying WPW

has been localized to 7q3 with linkage

analysis in families affected with WPW

or HCM or both (MacRae et al. 1995).

Haplotype analysis in WPW families with

autosomal dominant inheritance refined

the linkage area in chromosome 7q34-36,

and a mutation in the gene coding for the

gamma-2 regulatory subunit of the AMP-

activated protein kinase (PRKAG2) was

subsequently detected in two families

(Gollob et al. 2001). The PRKAG2 protein

modulates glucose uptake and glycolysis,

and mutations in PRKAG2 gene lead to

increased glycogen storage (Arad et al.

2003).

Atrial fibrillation (AF), a common

cardiac arrhythmia associated with

significant morbidity and mortality, is

characterized by rapid and irregular

activation of the atrium (Go et al. 2001).

The prevalence ranges from 0.1% in adults

younger than 55 to 9% in persons aged

80 or older. AF can be associated with a

number of genetic disorders, including

hypertrophic and dilated cardiomyopathy

and long QT syndrome type 4. Although

AF is usually secondary to other conditions

such as hyperthyroidism, hypertension, or

left ventricular dysfunction, a substantial

minority of patients have “lone AF” without

accompanying metabolic or cardiovascular

abnormalities, and may thus possess a

hereditary predisposition to this disease

phenotype.

Autosomal dominantly inherited AF

(OMIM 607554) has been localized to three

distinct loci. Three families showed linkage

to 10q22-24 (Brugada et al. 1997) and one

family to 6q14-16 (Ellinor et al. 2003),

but the defective gene at both of these loci

remains unknown. A genome-wide scan in

a four-generation family with autosomal

dominant hereditary AF localized the

defective gene to chromosome 11p15.5

(Chen et al. 2003). Mutation screening of

the positional candidate genes revealed a

missense mutation in the KCNQ1 gene.

In functional studies, channels harboring

this S140G mutation showed a gain-of-

function phenotype (Chen et al. 1998). As

mentioned above, LQTS-causing mutations

in the same gene result in a loss-of-function

phenotype.

26


2.2 ARRHYTHMOGENIC DISORDERS

ASSOCIATED WITH STRUCTURAL

ABNORMALITIES

2.2.1 Hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM,

OMIM 192600) is primarily a disorder of

the myocardium which causes distinctive

anatomic and histologic features and can

result in severe clinical manifestations

(Chien 1999). Left ventricular wall

thickening diminishes the chamber

volumes. The myocyte arrangement is

distorted, and the proportion of fibroblasts

is increased, reducing the elasticity of the

chamber walls and decreasing contractility.

Clinical features include dyspnea, chest

pain, atrial and ventricular arrhythmias,

heart failure, and elevated risk of sudden

death (Arad et al. 2002). The symptoms

usually begin at a later age, but age of onset

and degree of symptoms vary significantly

(Arad et al. 2002; Towbin and Bowles

2002). In the general population, HCM has

a prevalence of 1 in 500 (Maron et al. 1995).

It is estimated that more than half the HCM

cases are familial, most often inherited as

an autosomal dominant trait.

It was long thought that HCM was

purely a disease of the sarcomere, since

mutations in genes encoding contractile

proteins cause the disease (Seidman and

Seidman 2001). Recently, research has

shown that mutations in other genes also

can lead to the HCM phenotype (Blair et

al. 2001; Arad et al. 2002). The unifying

feature of the consequences of the mutations

seems to be inefficient utilization of ATP,

leading to the increased energy cost of

force production (Crilley et al. 2003). This

is in line with the late onset of the disease:

when the cell grows older, cellular energy

production decreases, and thus a subtle

defect in one of the components needed for

muscle contraction begins to take its toll.

To date, more than ten genes have been

linked to the HCM phenotype (Table 3).

Mutations in some of these are associated

with extracardiac symptoms, which renders

the classification of a phenotype such

as HCM difficult. Mutations of cardiac

myosin-binding protein C and β-myosin

heavy-chain genes appear to be the most

frequent cause of inherited HCM (Richard

et al. 2003). Together, mutations in these

two genes account for 82% of all HCM

mutations identified. In Finland, mutations

of the gene coding for cardiac myosin-

binding protein C account for 38% of

familial HCM (Jääskelainen et al. 2002),

whereas the β-myosin heavy chain gene

does not constitute a significant cause of

HCM (Jääskelainen et al. 1998).

In addition to mutations in nuclear

genes, mutations in mitochondrial DNA

or nuclear encoded mitochondrial proteins

also result in hypertrophic and dilated

cardiomyopathy. These mutations include

deletions and point mutations in transfer

RNA genes (Suomalainen et al. 1992;


27

Shi et al. 1999). The phenotype of the

mitochondrial mutations often include

symptoms of other organs (Naviaux 2000;

Wallace 2000).

2.2.2 Dilated cardiomyopathy

Ventricular dilatation and diminished

contraction characterize the phenotype of

dilated cardiomyopathy (DCM, OMIM

115200). This disease has a prevalence

of 1:2 500 and causes heart failure,

serious arrhythmias, and thromboembolic

events. The clinical course of DCM may

be progressive, and roughly half of the

affected individuals die within 5 years

of diagnosis without transplantation

(Towbin and Bowles 2002). Symptoms

include dyspnea, palpitations, and easy

fatigue (Grunig et al. 1998). The onset

of symptoms is most commonly between

the ages of 18 and 50, but children of all

ages and the elderly can also be affected

(Towbin and Bowles 2002). Approximately

25 to 30% of DCM is inherited (Grunig

et al. 1998) as an autosomal recessive, X-

linked or mitochondrial trait with reduced

penetrance. Cardiac symptoms are often

accompanied by symptoms in other organs

(Table 3).

More than 15 loci for DCM have

been identified (Table 3), but only a small

number of causative genes have been found,

and apparently most of the disease-causing

mutations remain unknown. Substantial

phenotypic and genetic variety suggests that

yet more disease loci are likely to appear.

Most DCM genes code for cytoskeletal,

contractile, or inner-nuclear membrane

proteins, while the function of the

disease-causing gene tafazzin is unknown

(D’Adamo et al. 1997). Many of the genes

mutated in HCM also harbor mutations

leading to the DCM phenotype, but the

mutations differ (Watkins 2003). There

appears to be a wide range of molecular

pathogenic mechanisms underlying the

DCM phenotype, a general feature being

disruption of myocyte architecture or

viability (Watkins 2003).

Intriguingly, two DCM loci, 6q12-16

(Sylvius et al. 2001) and 10q21-23 (Bowles

et al. 1996), overlap with the loci found to

associate with atrial fibrillation (Brugada

et al. 1997; Ellinor et al. 2003). As also

the clinical phenotypes of these disorders

overlap, it is reasonable to hypothesize that

these diseases could share predisposing

genes.

2.2.3 Arrhythmogenic right ventricular dysplasia

The major clinical features of

arrhythmogenic right ventricular

dysplasia (ARVD, OMIM 107970) are

fibrofatty replacement of right ventricular

myocardium and life-threatening ventricular

arrhythmias. The incidence and prevalence

of ARVD are unknown. However, it is

unusually common in northeastern Italy

(prevalence 1:1 000) (Rampazzo et al.

1994), accounting for more than 20% of

sudden deaths among young Italian athletes

28


(Corrado et al. 1998). ARVD is typically

inherited as an autosomal dominant trait

with variable penetrance. Naxos disease,

an autosomal recessive form of this

trait, is associated with woolly hair and

palmoplantar keratoderma (McKoy et al.

2000).

Seven different loci for ARVD have

been identified (Table 3). It is of particular

interest that one subtype of this disease,

ARVD2, was mapped to chromosome

1q42-43 (Rampazzo et al. 1995), and

affected patients had mutations in the

gene coding for ryanodine receptor type

2 (RYR2) (Tiso et al. 2001). The defective

genes in Naxos disease (McKoy et al. 2000)

and in ARVD8 (Rampazzo et al. 2002)

have been identified for plakoglobin and

desmoplakin, respectively.

As most of the ARVD genes are still

unknown, the molecular pathogenic

mechanism underlying these phenotypes

cannot be established. Some of the ARVD2

mutations in the RYR2 gene are reported to

result in reduced binding of the regulatory

protein FKBP12.6 (calstabin 2) (Tiso et

al. 2002). Plakoglobin and desmoplakin

encode for proteins involved in cell-cell

and cell-adhesion complexes (McKoy et al.

2000) (Rampazzo et al. 2002). Most likely,

mutations in these genes result in decreased

integrity of cell-cell junctions which leads

Table 3. Arrhythmogenic disorders associated with structural abnormalities.

Disease Locus Additional phenotype Defective protein Reference

HCM 1q32 None Cardiac troponin T (Thierfelder et al. 1994)

2q24.3 None Titin (Satoh et al. 1999)

3p21 Skeletal myopathy Essential myosin light chain (Poetter et al. 1996)

7q36 Pre-excitation (WPW) AMP-activated kinase, γ subunit

(Blair et al. 2001)

10q24 Lactic acidosis, seizures Cytochrome C oxidase assembly protein COX 15

(Antonicka et al. 2003)

11p11 None Cardiac myosin binding protein-C

(Watkins et al. 1995)

11p15.1 None Muscle LIM protein (Geier et al. 2003)

12q23-21 Skeletal myopathy Regulatory myosin light chain

(Poetter et al. 1996)

14q12 None Cardiac β myosin heavy chain

(Geisterfer-Lowrance et al. 1990)

14q12 None Cardiac α myosin lightchain (Niimura et al. 2002)

15q14 None Cardiac actin (Mogensen et al. 1999)

15q22 None α tropomyosin (Thierfelder et al. 1994)

19q13 None Cardiac troponin I (Kimura et al. 1997)

20q13.3 None Myosin light-chain kinase (Davis et al. 2001)


29

DCM 1q21.2 Conduction disease Lamin A/C (Fatkin et al. 1999)

1q32 None Cardiac troponin T (Kamisago et al. 2000)

2q14-22 Conduction disease ? (Jung et al. 1999)

2q24.3 None Titin (Gerull et al. 2002)

2q35 Skeletal myopathy Desmin (Li et al. 1999)

3p25-22 Conduction disease ? (Olson and Keating 1996)

4q12 None β sarcoglycan (Barresi et al. 2000)

5q33 None δ sarcoglycan (Tsubata et al. 2000)

6q12-16 None ? (Sylvius et al. 2001)

6q22.1 Heart failure Phospholamban (Schmitt et al. 2003)

6q23 Skeletal myopathy, conduction defect

? (Messina et al. 1997)

6q23-24 Sensorineural hearing loss

? (Schonberger et al. 2000)

9q13-22 None ? (Krajinovic et al. 1995)

10q21-23 Mitral valve prolapse ? (Bowles et al. 1996)

11q15.1 None Muscle LIM protein (Knoll et al. 2002)

14q11 None Cardiac β myosin heavy chain

(Kamisago et al. 2000)

15q14 None Cardiac actin (Olson et al. 1998)

15q2 None α tropomyosin (Olson et al. 2001)

17q12 None T-cap (Knoll et al. 2002)

Xp21 Skeletal myopathy Dystrophin (Muntoni et al. 1993)

Xq28 Short stature, neutropenia

Tafazzin (D’Adamo et al. 1997)

ARVD 1q42-43 None Ryanodine receptor 2 (Tiso et al. 2001)

2q32 None ? (Rampazzo et al. 1997)

3p23 None ? (Ahmad et al. 1998)

6q24 None Desmoplakin (Rampazzo et al. 2002)

10p14-12 None ? (Li et al. 2000a)

14q12-22 None ? (Severini et al. 1996)

14q23-24 None ? (Rampazzo et al. 1994)

Naxos disease

17q21 Palmoplantar keratoderma

Plakoglobin (McKoy et al. 2000)

HCM, hypertrophic cardiomyopathy; WPW, Wolf-Parkinson-White syndrome; DCM, dilated cardiomyopathy; ARVD, arrhythmogenic right ventricular dysplasia.In addition, mitochondrial inheritance of HCM and DCM has been reported.

30


to disruption of the cellular architecture in

the tissue.

3 ION CHANNEL DISEASES

Ion channel diseases (”channelopathies”)

represent a relatively new field of inherited

diseases, although more than 400 different

ion channels exist (Gargus 2003). Voltage-

gated ion channels generating electrical

signals in cells share several structural

similarities but differ in their ion selectivity

and tissue specificity (Terlau and Stuhmer

1998). As described below, mutations in

essentially the same ion channels produce

distinct but yet predictable phenotypes in

different tissues. A typical feature of ion

channel diseases, phenotypic heterogeneity,

can also be observed: a wide range of

phenotypes can be produced with allelic

mutations in ion channel genes (Barchi

1998).

3.1 RYANODINE RECEPTOR DEFECTS–

EXAMPLES OF DISEASES OF

CALCIUM METABOLISM

Except for the cardiac RYR2 channel,

there exist two other isoforms of ryanodine

receptors: RYR1 is expressed mainly

in the skeletal muscle, whereas RYR3

has a wide expression pattern, although

originally identified in the brain. A high

level of structural similarity exists between

different RYRs (Tunwell et al. 1996).

RYR1 and RYR2 especially are very

homologous, sharing 87% of amino acids

in their putative transmembrane segments

and showing an overall identity of 66%

(Otsu et al. 1990). Differences between

the amino acid sequences are reflected in

the structure of the proteins (Sharma et al.

1998) and are probably responsible for the

functional differences–for example, their

differing sensitivities to Ca2+ activation/

inactivation and to Mg2+ inhibition.

Mutations in the RYR1 gene cause

malignant hyperthermia (MH) and central

core disease (CCD) (see McCarthy et al.

2000 for review). MH is a pharmacogenetic

disorder of skeletal muscle. In susceptible

individuals, administration of halogenated

anesthetics will trigger a possibly fatal

clinical MH episode characterized by

hypermetabolism, muscle rigidity, rapid

increase in body temperature, lactic and

respiratory acidosis, and tachycardia

(MacLennan 2000). CCD exhibits major

clinical variability, but common symptoms

include hypotonia and proximal muscle

weakness. Histological examination

shows amorphous central regions (cores)

in type 1 muscle fibers with a relative lack

or complete absence of mitochondria in

these core areas. The loss of mitochondria

is probably caused by continuous high

cytoplasmic Ca2+ concentration. Since

mitochondria participate in removal of

the excess calcium, this leads to Ca2+

overload in the mitochondria and their

subsequent destruction (Missiaen et al.

2000). CCD is closely related to MH,


31

and CCD patients are normally treated as

MH-susceptible. Mutant RYR1 channels

display an increased sensitivity to caffeine

and a significantly reduced calcium release

(Monnier et al. 2000). This supports the

hypothesis of channels releasing Ca2+

inadequately, which leads to depletion of

the Ca2+ stores.

All hitherto identified RYR1 mutations

are clustered in three distinct regions of the

gene in exons 2 to 18, 39 to 46, and 98 to 105

(McCarthy et al. 2000), corresponding to the

cytoplasmic N-terminal portion, the central

portion encompassing the FKBP12.6-

binding region and the C-terminal portion

comprising the transmembrane domains.

Interestingly, the recently identified RYR2

mutations also seem to cluster in exactly

the same areas (this study; Priori et al. 2001;

Tiso et al. 2001; Bauce et al. 2002; Marks

et al. 2002; Priori et al. 2002). Intuitively,

two plausible explanations exist: either

mutations elsewhere in the channel protein

are not well tolerated, or these regions play

a particularly functional role.

Mutations in the RYR1 and RYR2 genes

result in phenotypes with serious clinical

manifestations. Intriguingly, no reports

of any neurological symptoms among

patients with the RYR2 mutations have

appeared even though RYR2 is the major

ryanodine receptor isoform expressed in

the brain (McPherson and Campbell 1993).

The gene coding for RYR3 has a wide

expression pattern and is yet to be linked to

any clinical phenotype. This renders RYR3

an extremely interesting candidate gene for

multisystemic neuromuscular disorders.

3.2 DISEASES OF POTASSIUM AND

SODIUM SIGNALING

The ion channel genes underlying LQTS

phenotypes belong to three gene families.

The KCNQ family comprises four genes,

all linked to clinical phenotypes. Mutations

in the genes coding for the muscle isoforms

KCNQ2 and KCNQ3 underlie benign

familial neonatal convulsions–a generalized

transient epilepsy of the newborn (Hubner

and Jentsch 2002). These two channel

subunits assemble to form heteromeric

channels. The KCNQ4 gene is expressed in

the outer hair cells of the inner ear (Cooper

and Jan 1999). These cells’ function is

increased sensitivity of sound perception.

Mutations in the KCNQ4 gene result in

autosomal dominantly inherited childhood-

onset deafness (Kubisch et al. 1999).

The LQTS-causing genes KCNE1 and

KCNE2 code for small auxiliary subunits

of voltage-gated potassium channels and

belong to the KCNE gene family. The third

member of this family is KCNE3, which is

abundantly expressed in skeletal muscle.

A single missense mutation in the KCNE3

gene is associated both with hypokalemic

and hyperkalemic periodic paralysis

(Abbott et al. 2001).

The sodium channel gene SCN5A

harbors mutations resulting in various

32


arrhythmic diseases including LQTS,

Brugada syndrome, and progressive cardiac

conduction defect. Other members of the

SCN family have also been characterized.

Mutations in the SCN1A and SCN2A genes

underlie the phenotype of generalized

epilepsy with febrile seizures in childhood

and sometimes also afebrile seizures in

adulthood (Escayg et al. 2000; Sugawara

et al. 2001; Hubner and Jentsch 2002).

The SCN4A gene underlies a number of

neuromuscular entities: paramyotonia

congenita, potassium-aggravated myotonia,

hypo- and hyperkalemic periodic paralysis,

and malignant hyperthermia (Gargus

2003).

4 MOLECULAR DIAGNOSIS IN INHERITED ARRHYTHMIC DISEASES

Identification of individuals at risk for

an arrhythmia or sudden cardiac death

is clinically important, but, variable

phenotype and age of onset as well as

reduced penetrance impede the clinical

diagnosis in many arrhythmic disorders.

Increased knowledge of the genetic basis

of inherited arrhythmias has supplied the

means for accurate detection of patients at

risk. Molecular genetic testing can provide

diagnostic and prognostic information

that complements clinical examination

in assessment for the diagnosis. When a

genetic defect causing a familial disease

is identified, the pathophysiological

substrate is conclusively determined, and

unequivocal identification of gene carriers

becomes possible (Priori 1997).

4.1 Indications and limitations in genetic screening

In the diagnosis or management of patients

when exact diagnosis is hindered by reduced

penetrance or variable phenotype, or when

the clinical course varies markedly by

genotype or mutation type, DNA analysis

is particularily likely to be helpful (Vincent

2001). A major question for future research

is related to the screening of individuals

for mutations and polymorphisms that

may predispose to induction of arrhythmia

(Behr and McKenna 2003). The feasibility

of genetic screening is, however, of concern

(Vincent 2001; Marian and Roberts

2003). Optimally, to permit unequivoical

diagnosis of all affected individuals with a

certain phenotype, all the genes and their

mutations causing the disease should be

known. Unfortunately, this is not the case

for any inherited disease.

A recent paper reports the screening of

nine hypertrophic cardiomyopathy-causing

genes in 197 unrelated probands (Richard

et al. 2003). The disease-causing mutation

was identified in 63% of the patients, and

62% of the mutations were novel. This

situation resembles that of other arrhythmic

disorders, including LQTS (Chiang and

Roden 2000; Richard et al. 2003), and

emphasizes the need for comprehensive

screening of the complete sequence of

known causal genes in each individual,


33

rather than screening restricted to known

mutations or particular parts of genes

(Marian and Roberts 2003; Richard et al.

2003).

Due to the variable phenotype and

penetrance of LQTS, genetic diagnosis

is likely to become particularly useful.

The corrected QT interval (QTc) is one of

the major clinical criteria of LQTS, but a

marked overlap exists in the QTc intervals

of affected and unaffected individuals

(Vincent et al. 1992; Saarinen et al. 1998;

Chiang and Roden 2000). Mutation

carriers with normal or borderline QTc can

be at risk for syncope and sudden death

(Piippo et al. 2001b), especially when

predisposed to QT-prolonging drugs (Priori

et al. 1999b). Identification of these non-

penetrant individuals with genetic analysis

is therefore important.

4.2 Different strategies in molecular diagnosis

Linkage analysis (Terwilliger and Ott 1994)

can be used for DNA diagnosis if the locus

for the disease has already been determined

(Eng and Vijg 1997). This method is

applicable only when DNA samples at least

of the parents and unaffected siblings of

the patient in interest are available (Priori

1997).

As faster and more cost-effective

mutation screening methods have

developed, the DNA samples of

ungenotyped probands can be screened

for mutations in multiple known disease

genes. However, mutation screening is

still time- and money consuming, and in

most laboratories, a complete screening

of several genes cannot be provided as a

routine diagnostic method. In addition,

some probands may have mutations in

unknown genes, or detection of a specific

mutation of a particular gene may fail, due

to limitations in analytic techniques (Li et

al. 2000b; Vincent 2001). If the mutation

is however known in a family, family

members can undergo mutation detection-

based DNA diagnosis.

In Finland, the peculiar population

history of Finns has provided a unique

opportunity to utilize founder mutations

in molecular diagnosis of LQTS. Because

the founder mutation KCNQ1-Fin accounts

for approximately 30% of all LQTS

cases, a single low-cost test performed on

individuals suspected to be affected with

LQTS identifies a considerable portion of

the disease carriers (Piippo et al. 2001b).

Since the syndrome is relatively rare,

population-level screening would most

likely be fruitless.

34

AIMS

AIMS

The aim of this study was to clarify the genetic background of two severe ventricular arrhythmic diseases: catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome (LQTS), with special emphasis on elucidation of the molecular pathogenesis of CPVT, as no information was available on the causative gene, its mutations, or their functional consequences. Due to its central role in regulation of cardiac repolarization, HERG was considered a particularly important LQTS gene, and no information existed on possible Finnish-type HERG mutations at the start of the present study.

The specific aims were:

1. Fine-map the CPVT locus on chromosome 1q42-43.

2. Identify the CPVT gene and characterize the disease-causing mutations.

3. Study the functional properties of the genetic defects causing CPVT.

4. Survey the HERG gene for mutations and polymorphisms in Finnish LQTS patients. Develop diagnostic tests for routine detection of the gene mutations predisposing to life-threatening arrhythmias.

PATIENTS AND METHODS

35


1 PATIENTS AND CONTROLS

At the commencement of this study,

two Finnish families affected with


tachycardia (CPVT) had been identified and

the disease locus mapped to chromosome

1q42-43 (Swan et al. 1999). The phenotypic

characteristics of CPVT included exercise-

induced ventricular premature complexes

during a stress test. In addition, the patients

had experienced polymorphic ventricular

tachycardias, and many of the families

had a history of unexplained sudden

death. No structural or histopathological

abnormalities of the myocardium existed.

In Study I, two additional Finnish families

suspected to carry the same disease were

studied by haplotype analysis to confirm

the diagnosis and to delineate the disease

locus. These four families comprised study

group 1, which underwent screening of the

complete coding region of the RYR2 gene.

Study group 2 consisted of 12 new probands

whose DNA had not been analyzed

previously. In this group, we analyzed

approximately half of the coding region of

the RYR2 gene. While the screening of the

RYR2 gene was in progress in study group

2, clinical studies revealed seven new

Finnish families (group 3) affected with

CPVT. In Study III, the samples of groups 2

and 3 (n = 19) were screened for mutations

in the CASQ2 gene. The two amino acid

polymorphisms that were identified in the

CASQ2 gene were also determined in a

cohort of 185 unrelated probands with long

QT syndrome (LQTS), in which previous

molecular studies had failed to detect

disease-causing mutations.

In Study V, 40 Finnish probands

with congenital LQTS were screened

for mutations in the HERG gene, which

had been previously shown to underlie

LQTS. The patients fulfilled at least two

of the following diagnostic criteria: 1)

prolonged heart-rate-corrected QT interval

(> 440 ms), 2) presence of syncopal

spells or documented torsade de pointes

tachycardia or both, and 3) family history

of LQTS or sudden death. In the same

study, LQTS patients (n = 81) carrying

the KCNQ1-Fin mutation (Piippo et al.

2001b) were genotyped for HERG K897T

polymorphism. Study IV concentrated on a

severely affected LQTS proband who was

also included in Study V.

When a mutation was detected in

a proband, all his or her relatives and

an appropriate number of control DNA

samples were also studied for the mutation.

The control cohort comprised 300 Finnish

blood donors from the Helsinki area. The

36


sex ratio of this cohort was 1:1, and the

cohort contained only adults. Informed

consent was obtained from the study

subjects, and the Ethics Review Committee

of the Department of Medicine, University

of Helsinki, approved this study.

2 MOLECULAR GENETIC STUDIES

The methods are described in more detail in

the original publications I to V.

2.1 DNA EXTRACTION AND POLYMERASE

CHAIN REACTION

Genomic DNA was extracted from

peripheral lymphocytes, either by the

standard phenol extraction method

(Blin and Stafford 1976) or the salting-

out method with the PureGene DNA

Purification Kit (Gentra, Minneapolis,

MN). The polymerase chain reactions

(PCR) (Mullis et al. 1986) were performed

under varying conditions and with intronic

primers, whenever possible.

2.2 MUTATION SCREENING

Sequencing PCR-amplified DNA fragments

were purified and directly sequenced with

BigDye Terminator (Applied Biosystems,

Foster City, CA) chemistry based on the

use of di-deoxynucleotides in controlled

termination of the elongation reaction

(Sanger et al. 1977). The fragments were

analyzed with the ABI377 Prism automated

sequencer (Applied Biosystems).

Denaturing high-performance

liquid chromatography (dHPLC) An

application of the heteroduplex formation

method, dHPLC, was used in mutation

screening in Studies I and III. The

optimal melting curves for PCR-amplified

DNA fragments were calculated with

Wavemaker software (Transgenomics,

Omaha, NE), and fragments were run with

the DNA-sep column and Wave 3500A

Nucleic Acid Fragment Analysis system

(Transgenomics).

2.3 MICROSATELLITE ANALYSIS

Several databases of different genome

centers (including the Sanger Centre, the

Whitehead Institute, and The Genetic

Location Database) were searched

for multiallelic, highly polymorphic

microsatellite markers located in the

areas of interest. Primer sequences came

from The Genome Database. Genomic

fragments were amplified with primers

containing a fluorescent label in the 5’

primer. After pooling, these fragments were

run on ABI377, and results were analyzed

with GenScan and Genotyper computer

programs (Applied Biosystems). See

Internet references for detailed addresses of

the genome centers.

2.4 LINKAGE AND HAPLOTYPE ANALYSIS

Pairwise linkage analyses were performed

with the MLINK and ILINK options of the


37

FASTLINK v 4.1P package. Haplotypes,

which were used to study the relatedness

of the families carrying the HERG-Fin

mutation and fine mapping of the CPVT

locus, were constructed manually.

2.5 BIOCOMPUTING

Several sequence databases, computer

programs, and search tools were utilized

in this study (see Internet references for

details). Of these, the following were

particularly valuable: the BLAST2 computer

program served for the determination of the

genomic organization of CPVT candidate

genes coding for the G-protein γ4 subunit

(GNG4, 2 coding exons), the type-3

muscarinic receptor (CHRM3, 1 exon),

and the cardiac ryanodine receptor (RYR2,

105 exons). PCR primers were designed

with the Primer3-program. The Clustal W-

alignment program was used in comparing

different homologs of the proteins of

interest. Evolutionary conservation of

amino acids altered by these mutations was

deduced based on these alignments.

2.6 SPECIFIC DETECTION METHODS FOR

DNA ALTERATIONS

Upon identification of each nucleotide

alteration, a specific detection method was

set up (Table 4). This method was then

utilized in the screening of relatives of the

mutation/polymorphism carriers and of the

DNA samples of at least 100 healthy control

individuals. The majority of these methods

were based on restriction endonuclease

digestion, since many mutations alter the

recognition site of a certain enzyme. When

a mutation did not result in a change of

restriction pattern, an artificial recognition

site was generated by primer-induced

restriction analysis (PIRA) (Haliassos et

al. 1989). Solid-phase minisequencing

(Syvänen et al. 1990) and heteroduplex

analysis (White et al. 1992) both were used

in detection of single-nucleotide alterations,

while two mutations were detected with

direct sequencing.

3 FUNCTIONAL ANALYSES

Site-directed mutagenesis was utilized

in creating constructs for the functional

studies. RYR2 mutations P2328S, Q4201R,

and V4653F were separately introduced

to partial RYR2 cDNA in the pBS vector

with the Chameleon double-stranded,

site-directed mutagenesis kit (Stratagene,

La Jolla, CA). The fragments containing

the desired mutations were subsequently

subcloned to full-length RYR2 cDNA in the

pCMV5 vector. The L552S HERG mutation

was introduced to HERG cDNA in the

pcDNA3 plasmid with the QuickChange

site-directed mutagenesis kit (Stratagene).

Each of the mutant RYR2 cDNAs was

co-transfected to HEK-293 cells with

FKBP1B cDNA coding for the regulatory

protein FKBP12.6. These transfections

were performed by the Ca2+ phosphate

precipitation method. Microsomes

38


containing recombinant RYR2 channels

were isolated and in vitro phosphorylated

with the protein kinase A catalytic

subunit. Single-channel recordings were

performed under voltage-clamp conditions

(Gaburjakova et al. 2001). Data were

analyzed with Fetchan software (Axon

Instruments, Union City, CA).

Transfections of HERG-L552S mutants

to COS7 cells were made with Effectene

Transfection Reagent (Qiagen, Valencia,

CA). Whole-cell membrane currents were

measured with the EPC-9 amplifier and

Pulse/Pulsefit software (HEKA, Lambrecht,

Germany).

4 STATISTICAL ANALYSES

Fisher’s exact and the χ2 test served for

comparison of categorical variables.

Analysis of variance (ANOVA) taking

relatedness into account was used in Study

V, which compared the QTc intervals of

Table 4. Specific detection methods to detect mutations and polymorphisms identified in Studies I and III-V.

Gene Alteration Detection Allele2

RYR2 Mutation V2306I TaaI mutant

P2328S HaeIII wild-type

Q4201R PvuII wild-type

V4653F NcoI (PIRA1) wild-type

P4902L HaeIII mutant

R4959Q BspI wild-type

Polymorphism G1885E sequencing -

Q2958R BsrGI (PIRA1) wild-type

13682+67 C>T NlaIII mutant

HERG Mutation R176W SmaI mutant

P451L AluI mutant

L552S NcoI (PIRA1) mutant

Y569H RsaI mutant

G584S AluI mutant

G601S SfiI wild-type

T613M sequencing -

453delC sequencing -

1631delAG heteroduplex-analysis -

Polymorphism K897T BstUI (PIRA1) mutant

CASQ2 Polymorphism T66A Hin6I mutant

V76M minisequencing -

1PIRA, primer-induced restriction analysis used in the detection2denotes whether the wild-type or mutant allele is cleaved by the restriction enzyme


39

KCNQ1-Fin carriers with different HERG

K897T genotypes. Unpaired Student’s t-

test was used in comparing the means of

in vitro experimental results. A P-value

of < 0.05 was considered statistically

significant.

40

RESULTS

Figure 7. Pedigrees of families affected with CPVT. Linkage and haplotype analyses as well as screening of the whole coding region of the RYR2 gene were performed in families 1-4. In families 5-7, approximately half of the RYR2 gene and the complete coding region of the CASQ2 gene were studied.

RESULTS

1 IDENTIFICATION OF THE GENE CAUSING DOMINANT CPVT

1.1 FINE-MAPPING THE CPVT LOCUS (I)

When this study was initiated, two families

(Figure 7, families 1 and 2) affected with

autosomal dominantly inherited CPVT

had been identified, and a genome-wide

scan had assigned the disease locus to

chromosome 1q42-43 (Swan et al. 1999).

In the present study, linkage analysis

in two novel CPVT families revealed

maximum pairwise lod scores of 2.43

(family 3) and 1.06 (family 4) at marker

D1S2670, thus suggesting linkage to the

locus identified earlier. The combined lod

score value of all the families was 8.23 at

the same marker. Haplotype analysis in

RESULTS

41

1.3 IDENTIFICATION OF RYR2 MUTATIONS

IN CPVT PATIENTS (I, III)

As the next step in search of the gene

underlying CPVT, the candidate gene

ryanodine receptor type 2 (RYR2) was

screened for mutations in the probands of

families 1 to 4 (I). Genetic analysis of the

complete coding region (105 exons) of RYR2

revealed three nucleotide substitutions.

These alterations were predicted to result

in amino acid changes P2328S, Q4201R,

and V4653F (Figure 9A), in families 1, 4,

and 3. Several individuals in each of the

families were shown to carry the respective

mutation (Table 5). No causative mutation

was detected in family 2, in which a LOD

score of 2.07 was demonstrated at marker

D1S2670 (Swan et al. 1999).

In Study III, 12 new CPVT probands

were enrolled for analysis of the RYR2

gene. The screening was concentrated

in regions of the RYR2 gene proposed

in Study I to be pathophysiologically

significant and shown to harbor mutations.

Figure 8. Summary of informative haplotypes delineating the disease locus in chromosome 1q42-43. Box: disease locus. Families shared no common haplotype.

all the four CPVT families showed that

the families shared no common haplotype.

However, recombinations in families 1 and

3 delineated the disease locus as between

markers D1S235 and D1S2670 (Figure 8).

This narrowed the linkage region from 9

centiMorgans (cM) (Swan et al. 1999) to

approximately 1.5 cM, which, according to

the Ensembl Genome Browser, corresponds

to approximately 2.5 megabases.

1.2 SCREENING OF CANDIDATE GENES (I)

The narrowed linkage area contained

several genes. GNG4 (Kalyanaraman et

al. 1998), coding for a G-protein subunit γ,

and CHMR3 (Shi et al. 1999), coding for

a muscarinic receptor ion channel, were

selected for mutation screening based on

their position and function. The samples

from the four original probands were

screened for mutations in these genes by

direct sequencing. No sequence alterations

were detected compatible with a pathogenic

role.

42

RESULTS

Three novel point mutations were revealed

(Figure 9A): V2306I, P4902L, and

R4959Q. The missense mutation V2306I

was detected in the affected son of the

proband, but not in her parents. Paternity

was confirmed by haplotype analysis.

P4902L and Q4959Q were present only

in the respective probands. All the six

RYR2 gene alterations described in Studies

I and III co-segregated with the disease

phenotype in the corresponding families,

but were absent from control individuals.

In addition to the six mutations, two amino

acid polymorphisms were detected (Table

9, Discussion).

1.4 PHENOTYPIC CHARACTERISTICS OF

RYR2 MUTATIONS (I, II)

Some of the phenotypic features of RYR2

mutation carriers are depicted in Table 5.

In general, most of the mutation carriers

had experienced syncopes associated with

physical or emotional stress. On exercise

testing, when heart rates exceeded an

individual threshold, isolated unifocal and

multifocal ventricular premature complexes

appeared. Penetrance of the mutations was

not complete, as two carriers of the P2328S

mutation were asymptomatic. Most patients

had experienced their first symptoms

around the age of 10 years, but age of onset

ranged from 6 to 40.

Figure 9a) A schematic view of RYR2 protein containing all mutations described until 2/2004. Mutations described in this study (●), mutations described by Priori et al (Priori et al. 2001; Priori et al. 2002) ( ), Tiso et al (Tiso et al. 2001), and Bauce et al (Bauce et al. 2002) (□). The mutations with bold font result in CPVT phenotype. Mutations with normal font have been described as associating with ARVD2, or the associated phenotype is ambiguous. b) Approximate location of all the reported CASQ2 mutations by Lahat et al (□) (Lahat et al. 2001b) and Postma et al ( ) (Postma et al. 2002). The polymorphisms (●). Location of the predicted Ca2+ binding region is indicated.

Cytoplasm

SR lumen

b

P2328S

R4959Q

E4950KN4104K

Q4201R

N4895D

I4867M

A4860G

R4497CV4771I

V4653F

V76M

T66A

R33X62delA

532+1G>A

D307H

Ca2+ bindingregion

S2246L V2306IE2311D

N2386IY2392C

L3778F

G3946S

R2474ST2504M

P4902L

L433PR420WR176Q

aNH2

NH2

COOH

COOH

RESULTS

43

Table 5. Clinical characteristics of RYR2 mutation carriers (mean, or mean ± SD values).

V2306I P2328S Q4201R V4653F P4902L R4959Q

Family no. 5 1 4 3 6 7

Carriers (n) 2 16 3 10 1 1

Age, yr 14 ± 13 29 ± 14 56 ± 16 45 ± 20 56 60

Resting heart rate, bpm

47 71 ± 12 57 78 ± 16 41 72

Threshold heart rate, bpm

105 129 ± 17 NA 133 ± 20 100 100

QTc, ms 445 430 ± 20 448 440 ± 50 380 438

Sudden deaths (n) at age < 35 yr in family

0 4 4 5 0 0

SD, standard deviation; NA, not available.

Threshold rates at which nonsustained

ventricular arrhythmias occurred during

the exercise test did not significantly differ

between families 1 and 3 (Table 5). Other

families were too small to permit any

genotype-phenotype correlations (Figure

7).

1.5 FUNCTIONAL PROPERTIES OF

MUTANT RYR2 CHANNELS (II)

The mutant RYR2 channels, when

compared to the wild-type channels,

showed several functional abnormalities.

When phosphorylated, the mutant

P2328S, Q4201R, and V4653F channels

showed a significant increase in open

probability, gating frequency, and mean

open time values. Long-lasting open states,

not characteristic of wild-type RYR2

channels, were also observable. Unlike

phosphorylated wild-type channels, the

phosphorylated homotetrameric mutant

RYR2 channels showed resistance to

inhibition by incremental concentrations

of Mg2+, the physiologic inhibitor of RYR2

channels. As magnesium concentration

increased, the activity of wild-type channels

became completely inhibited, whereas the

mutant channels showed a significantly

higher activity and required higher Mg2+

concentrations for inhibition.

Since CPVT is inherited as an autosomal

dominant trait, it was pertinent to co-

express the wild-type RYR2 cDNA with

mutant RYR2 cDNAs. Open probability,

gating frequency, and resistance to

inhibition by Mg2+ resembled those of the

homotetrameric mutant channels. In short,

after PKA phosphorylation, the hetero-

and homotetrameric mutant channels

display a gain-of-function abnormality.

This is consistent with a “leaky” channel

phenotype and a defect in regulation by the

physiologic inhibitor Mg2+.

Recently, the 1,4-bentzothiazepine

derivative (JTV519) was shown to inhibit

44

RESULTS

progression of heart failure in a dog model,

and it was proposed that this may involve

regulation of RYR2 activity (Yano et al.

2003). Accordingly, the effect of JTV519

on the activity of PKA phosphorylated

P2328S mutant channels was also

examined. JTV519 treatment resulted in

normalization of channel activity and re-

binding of FKBP12.6. This indicates that

this experimental drug may rescue the gain-

of-function defect in P2328S channels.

2 SCREENING OF THE CASQ2 GENE IN THE FINNISH CPVT PROBANDS (III)

Since mutations of calsequestrin type

2 (CASQ2) were shown to result in the

recessive (Lahat et al. 2001b), and possibly

also the dominant form of CPVT (Postma

et al. 2002), the gene appeared to be a

reasonable candidate in the Finnish CPVT

families. Screening of the complete coding

region of the CASQ2 gene (11 exons)

revealed a sample from a 42-year-old CPVT

proband to be heterozygous for two amino

acid changes, T66A and V76M (Figure

9B). Subcloning of the corresponding

PCR product into a plasmid and its

subsequent sequencing demonstrated that

both aberrations were present in the same

allele. The affected son and mother of the

proband, as well as his unaffected daughter

were heterozygous for both T66A and

V76M. Seven other CPVT probands were

heterozygous for T66A, but V76M was

detected in none of the 18 other probands

studied. Both of these alterations were also

detected in the control cohort. No other

significant alterations were identified.

Comparison in LQTS probands and

Table 6. Genotype and allele frequencies for the two CASQ2 polymorphisms.

CPVT indexes (n = 19) LQTS (n = 177) Blood donors (n = 265)

T66A Genotypes TT 57.8% 57.1% 51.3%

AT 42.1% 34.5% 40.4%

AA 0% 8.5% 8.3%

Alleles Allele T 78.9% 74.3% 71.5%

Allele A 21.2% 25.7% 28.5%

V76M Genotypes VV 94.7% 94.6% 94.8%

VM 5.3% 5.4% 5.2%

MM 0% 0% 0%

Alleles Allele V 97.4% 97.3% 97.4%

Allele M 2.6% 2.7% 2.6%

All differences statistically non-significant

RESULTS

45

blood donors of the frequencies of the

two polymorphisms alone (Table 6)

and the resulting haplotypes showed no

significant differences in allele or genotype

frequencies.

3 SURVEY OF THE HERG GENE IN FINNISH LQTS PATIENTS

3.1 IDENTIFICATION OF PRIVATE HERG

MUTATIONS AND THE HERG-FIN

FOUNDER MUTATION (IV, V)

Direct sequencing of the complete coding

region of the HERG gene in 39 LQTS

probands revealed a total of nine mutations

and one amino acid polymorphism (Figure

10). Eight of these mutations, two deletions

and six point mutations, were each found

in one family alone (Table 7). Nucleotide

change T1655C, predicted to result in

substitution of leucine 552 by serine

(L552S), was detected in the heterozygous

state in four families. In addition, one

pedigree consisted of two LQTS families,

both having its own separate proband.

In this family, the parents of the nuclear

family were heterozygous for the L552S

mutation, while their two daughters were

homozygous for L552S.

All nine mutations co-segregated in the

families with the disease phenotype and

were absent from DNA samples of at least

100 blood donors.

In order to construct disease-associated

haplotypes, microsatellite markers adjacent

to the HERG gene locus were analyzed in

all six families shown to carry the HERG

L552S mutation. A common haplotype

was present in all affected probands,

thus providing evidence for a founder

mutation. The affected grandparents of the

homozygous individuals originated from

the same commune in Lapland, whereas the

ancestors of the other families were from

eastern Finland.

3.2 PHENOTYPIC CHARACTERISTICS OF

HERG MUTATION CARRIERS (IV, V)

Most HERG mutation carriers showed

a marked prolongation of QT interval

Extracellular

Intracellular

T613M

del453CL552S

Y569H

1631delAG

K897T

T601SG584S

R176W

P451L

NH2

COOH

Figure 10. The approximate location of HERG mutations (●) and polymorphism (□). Finnish founder mutation L552S underlined.

46

RESULTS

compared to that of non-carriers (Table 7),

but phenotypic variation even amongst the

carriers of a single mutation was evident.

In the case of the P451L mutation, none

of the carriers had QTc exceeding 440 ms.

However, the proband and her affected

sister had experienced syncopal spells,

suggesting that this is a disease-causing

mutation. Of the 45 carriers with the

private mutations other than L552S, 16

were symptomatic.

In the one family with the homozygous

L552S carriers, the unrelated heterozygous

parents were asymptomatic, while both

their homozygous daughters were severely

affected; one of them died at age 4. The

mean QTc of the heterozygous L552S

carriers was significantly higher than that of

the family members without the mutation.

In the absence of any drug treatment, the

QTc values of the homozygous L552S

carriers were 677 ms and 513 ms.

Of the 35 L552S carriers 25 (71%) were

asymptomatic. Of the ten symptomatic

mutation carriers, nine were women. Of

symptomatic patients, 90% experienced

symptoms while at rest. Symptomatic

mutation carriers had a mean QTc of 500 ±

59 ms, whereas the asymptomatic carriers

had 452 ± 34 ms and noncarriers 412 ±

23 ms. These differences were highly

significant; in ANOVA analysis the p value

between all groups was < 0.001. The p value

between symptomatic and asymptomatic

carriers was < 0.001.

3.3 FUNCTIONAL PROPERTIES OF THE

HERG-L552S MUTATION IN VITRO (IV)

When the HERG protein containing the

L552S mutation was transiently expressed

in COS7 cells, it produced functional

channels, with robust currents similar in

Table 7. Main clinical features of families affected with HERG gene mutations (mean, or mean ± SD values).

453delC R176W P451L 1631delAG L552S1 Y569H G584S G601S T613M

Carriers (n) 19 6 4 1 35 1 10 3 1

Non-carriers (n)

32 10 10 1 43 4 8 12 11

Age, y

carriers 33 ± 21 39 ± 23 34 ± 23 52 35 ± 18 16 42 ± 20 32 ± 15 28 ± 20

non-carriers

45 ± 18 41 ± 17 38 ± 25 76 36 ± 16 34 ± 13 36 ± 24 42 ± 25 28 ± 20

QTc

carriers 470 ± 42 463 ± 42 421 ± 16 488 466 ± 47 512 465 ± 21 531 ± 45 536

non-carriers

419 ± 20 408 ± 23 411 ± 30 461 412 ± 23 423 ± 14 417 ± 23 418 ± 27 413 ± 26

Symptomatic patients (n)

4 2 2 1 10 1 4 1 1

1Only individuals heterozygous for L552S mutation included.

RESULTS

47

amplitude to those generated by the HERG-

wt construct. However, L552S displayed a

marked increase in the rate of activation and

deactivation. Such an increased deactivation

rate may result in lower channel activity at

the end of the repolarization phase.

3.4 COMMON AMINO ACID

POLYMORPHISM IN THE HERG

GENE (V)

In addition to several mutations, screening

of the HERG gene revealed a common

nucleotide alteration, A2690C, predicted

to result in the amino acid polymorphism

K897T. Population frequencies, as

determined in a sample cohort of healthy

blood donors, were 0.84 for allele A and

0.16 for allele C.

To study the possible phenotypic effects

of K897T polymorphism, 170 KCNQ1-Fin

carriers from 28 families were screened for

this polymorphism. KCNQ1-Fin carriers

with the HERG T897T genotype had a

slightly lower mean QTc value than did

individuals with the K897K and K897T

genotypes, and gender-specific analysis

indicated that this difference was present

in women only. In ANOVA analysis

taking family relatedness into account,

this difference was no longer significant,

however.

48

DISCUSSION

DISCUSSION

Long QT syndrome (LQTS) and


tachycardia (CPVT) share common

features: syncopal spells, absence of

structural myocardial aberrations, a usually

autosomal dominant pattern of inheritance,

and risk of sudden death in youth. Initially,

CPVT was considered an incomplete

(”forme fruste”) subtype of LQTS, because

triggering factors and patient age resemble

those described in LQTS, in the absence of

an unequivocal QT interval prolongation.

However, meticulous cardiological and

genetic analyses have revealed that

these diseases differ both clinically and

molecularly.

1 IDENTIFICATION OF GENES UNDERLYING CPVT

1.1 MUTATIONS OF THE RYANODINE

RECEPTOR GENE IN DOMINANT CPVT

This study aimed at identification of the

disease-causing gene of catecholaminergic

polymorphic ventricular tachycardia

(CPVT). First, the location of the disease

gene was delineated by construction of a

dense panel of microsatellite markers for

the chromosomal region in 1q42-43. These

markers were then utilized in haplotype

analysis, which led to the notable reduction

in linkage area. This facilitated the selection

of candidate genes for further analysis. As

screening of the candidate genes TWIK-1

(Swan et al. 1999), GNG4, and CHMR3

revealed no significant alterations, the

gene coding for ryanodine receptor type 2

(RYR2) remained as the primary candidate.

Since the RYR2 gene is one of the largest

known human genes (cDNA of 15.5 kb,

105 exons), the screening was initiated with

an indirect method (dHPLC). However, it

was subsequently considered necessary

to sequence the whole coding region of

the RYR2 gene in all the four probands

enrolled in the study. In Study I, RYR2

mutations were identified in three of the

four probands.

Simultaneously, RYR2 mutations were

reported in Italian families affected with

CPVT (Priori et al. 2001). Interestingly,

Italian probands affected with ARVD2, a

variant form of ARVD previously mapped

to the same chromosomal region (Rampazzo

et al. 1995), also carried mutations in RYR2

gene (Tiso et al. 2001).

It is unclear whether CPVT and ARVD2

represent two distinct diseases or a single

one with variable expression. ARVD2

patients often present with fibro-fatty

replacement of the myocardium and discrete

myocardial atrophy, but considerable

DISCUSSION

49

H.sapiens RYR2 ---LSVDDAKLQGAGEEEAKGGKRPKEGLLQMKLPEPVKLQMCLLLQYLCDCQVRHRIEA 1924O.cuniculusR YR2 ---PSVEDTKLEGAGEEEAKVGKRPKEGLLQMKLPEPVKLQMCLLLQYLCDCQVRHRIEA 1924M.musculus RYR2 ---ISIEDAKLE--GEEEAKGGKRPKEGLLQMKLPEPVKLQMCLLLQYLCDCQVRHRIEA 1923H.sapiens RYR3 ---TQVEEKAVE---AGEKAGKEAPVKGLLQTRLPESVKLQMCELLSYLCDCELQHRVEA 1825H.sapiens RYR1 ETAQEKEDEEKEEEEAAEGEKEEGLEEGLLQMKLPESVKLQMCHLLEYFCDQELQHRVES 1957

H.sapiens RYR2 LVSKGYPDIGWNPVEGERYLDFLRFAVFCNGESVEENANVVVRLLIRRPECFGPALRGEG 2339O.cuniculus RYR2 LVSKGYPDIGWNPVEGERYLDFLRFAVFCNGESVEENANVVVRLLIRRPECFGPALRGEG 2340M.musculus RYR2 LVSKGYPDIGWNPVEGERYLDFLRFAVFCNGESVEENANVVVRLLIRRPECFGPALRGEG 2339H.sapiens RYR3 LLAKGYPDVGWNPIEGERYLSFLRFAVFVNSESVEENASVVVKLLIRRPECFGPALRGEG 2236H.sapiens RYR1 LVAKGYPDIGWNPCGGERYLDFLRFAVFVNGESVEENANVVVRLLIRKPECFGPALRGEG 2372

H.sapiensRYR2 YILEFDGGSRG-KGEHFPYEQEIKFFAKVVLPLIDQYFKNHRLYFLSAASRPLCSGGHAS 2997O.cuniculusRYR2 YILEFDGGSRS-KGEHFPYEQEIKFFAKVVLPLIDQYFKNHRLYFLSAASRPLCSGGHAS 2998M.musculusRYR2 YILEFDGGSRS-KGEHFPYEQEIKFFAKVVLPLIDQYFKNHRLYFLSAASRPLCTGGHAS 2997H.sapiensRYR3 FIAHLEAIVSSGKTEKSPRDQEIKFFAKVLLPLVDQYFTSHCLYFLSSPLKPLSSSGYAS 2893H.sapiensRYR1 FIAHLEAVVSSGRVEKSPHEQEIKFFAKILLPLINQYFTNHCLYFLSTPAKVLGSGGHAS 3032

H.sapiens RYR2 VNEGGEKEKMELFVNFCEDTIFEMQLAAQISESDLNERSANKEESEK-----ERPEEQGP 4231O.cuniculus RYR2 VNEGGEKEKMELFVNFCEDTIFEMQLAAQISESDLNERSANKEESEK-----ERPEEQGP 4232M.musculus RYR2 VNEGGEKEKMELFVNFCEDTIFEMQLAAQISESDLNERLANKEESEK-----ERPEEQAP 4231H.sapiens RYR3 VNEGGEQEKMELFVNFCEDTIFEMQLASQISESDSADRPEEEEEDEDSSYVLEIAGEEEE 4132H.sapiens RYR1 VNEGGEAEKMELFVSFCEDTIFEMQIAAQISEPEGEPETDEDEGAGAAEAGAEGAEEGAA 4280

H.sapiens RYR2 QPSEDDIKGQWDRLVINTQSFPNNYWDKFVKRKVMDKYGEFYGRDRISELLGMDKAALDF 4679O.cuniculus RYR2 QPSEDDIKGQWDRLVINTQSFPNNYWDKFVKRKVMDKYGEFYGRDRISELLGMDKAALDF 4681M.musculus RYR2 QPSEDDIKGQWDRLVINTQSFPNNYWDKFVKRKVMDKYGEFYGRDRISELLGMDKAALDF 4679H.sapiens RYR3 QPSEDDIKGQWDRLVINTPSFPNNYWDKFVKRKVINKYGDLYGAERIAELLGLDKNALDF 4584H.sapiens RYR1 QPEDDDVKGQWDRLVLNTPSFPSNYWDKFVKRKVLDKHGDIYGRERIAELLGMDLATLEI 4751

H.sapiens RYR2 AIIQGLIIDAFGELRDQQEQVKEDMETKCFICGIGNDYFDTVPHGFETHTLQEHNLANYL 4919O.cuniculus RYR2 AIIQGLIIDAFGELRDQQEQVKEDMETKCFICGIGNDYFDTVPHGFETHTLQEHNLANYL 4921M.musculus RYR2 AIIQGLIIDAFGELRDQQEQVKEDMETKCFICGIGNDYFDTVPHGFETHTLQEHNLANYL 4919H.sapiens RYR3 AIIQGLIIDAFGELRDQQEQVREDMETKCFICGIGNDYFDTTPHGFETHTLQEHNLANYL 4822H.sapiens RYR1 AIIQGLIIDAFGELRDQQEQVKEDMETKCFICGIGSDYFDTTPHGFETHTLEEHNLANYM 4990

H.sapiens RYR2 FFLMYLINKDETEHTGQESYVWKMYQERCWEFFPAGDCFRKQYEDQLN 4967O.cuniculus RYR2 FFLMYLINKDETEHTGQESYVWKMYQERCWEFFPAGDCFRKQYEDQLN 4969M.musculus RYR2 FFLMYLINKDETEHTGQESYVWKMYQERCWEFFPAGDCFRKQYEDQLN 4967H.sapiens RYR3 FFLMYLINKDETEHTGQESYVWKMYQERCWDFFPAGDCFRKQYEDQLG 4870H.sapiens RYR1 FFLMYLINKDETEHTGQESYVWKMYQERCWDFFPAGDCFRKQYEDQLS 5038

V2306I P2328S

Q4201R

V4653F

P4902L

R4959Q

G1885E

Q2958R

Figure 11. Alignment of the human RYR2 protein with RYR2 proteins from other species and human ryanodine receptor isoforms. Only relevant parts of the proteins are included. Mutations and polymorphisms (italics) identified during the present study in bold with a gray background in the amino acid sequence.

50

DISCUSSION

interfamilial variability exists (Rampazzo

et al. 1995). In the Finnish CPVT patients,

the left and right ventricular dimensions as

well as systolic and diastolic functions were

normal, with the exception of two cases in

family 4, who showed minor local bulging

of the right outflow tract. There appears to

be no evident correlation between location

of mutation and resulting phenotype.

In Study III, the samples from 12

CPVT probands were screened for RYR2

mutations. Three point mutations were

identified, each present in a distinct family.

In the Finnish CPVT population, of the

16 probands studied, six (38%) showed

RYR2 mutations. Because nine of the ten

remaining families were insufficiently large

to accommodate linkage studies, it was

impossible to judge whether a mutation

located in the regulatory regions or introns

of the RYR2 gene, or another totally different

gene, underlie the CPVT phenotype in

these families. In one family (Family 2,

Figure 7), linkage was established to the

same chromosomal region, but sequencing

of the entire coding region of the RYR2

gene, as well as the other candidate genes

GNG4, CHMR3, and TWIK-1, revealed no

significant alterations.

1.2 FUNCTIONAL AND PHENOTYPIC

PROPERTIES OF RYR2 MUTATIONS

Each of the RYR2 mutations detected in this

study alters an evolutionarily conserved

amino acid residue (Figure 11). With the

exception of the original families in our

linkage study (Swan et al. 1999; Study

I), most of the RYR2 mutations identified

until now have occurred in small pedigrees

(Priori et al. 2001; Priori et al. 2002). This

may reflect reduced survival and unrealized

reproduction of the mutation carriers. Since

the identification of individuals affected

with CPVT is difficult, and sudden deaths

occur frequently among affected subjects

if they receive inadequate treatment, this

explanation is plausible.

After the detection of mutations in the

RYR2 gene, several groups have studied

their functional consequences in order

to clarify the pathological mechanism

underlying the clinical phenotypes. The

results of these studies have been somewhat

contradictory.

In the first study published on this

topic, the mutation R4496C was introduced

in the mouse RYR2, equivalent to the

human CPVT-causing mutation R4497C

(Jiang et al. 2002). In ryanodine-binding

studies, single-channel recordings and

single-cell Ca2+ imaging experiments, the

mutant displayed enhanced basal activity,

further demonstrated by replacement of

the arginine with glutamate, a negatively

charged amino acid. Thus, a positive charge

at this particular position appeared to be

important for stabilizing the closed state

of the channel at low Ca2+ concentrations.

These authors suggest that the increased

activity of RYR2 lowers the critical SR

DISCUSSION

51

Ca2+ concentration at which spontaneous

calcium release occurs, and that this is the

mechanism by which the enhanced basal

activity leads to ventricular arrhythmia.

As RYR2 mutations appear to underlie

both entirely electric disorders (CPVT)

and disorders associated with structural

abnormalities (ARVD2), the functional

differences between these mutations were

studied in yeast two-hybrid experiments

(Tiso et al. 2002). These mutations are

located in the central area of the channel

encompassing the binding region for the

regulatory protein FKBP12.6 (Marx et

al. 2000). The results of Tiso et al (2002)

suggest that some of the phenotypic

variability may be explained by the

differing effects that these mutations

exert on FKBP12.6 binding. According

to their results, CPVT mutations seemed

to increase and ARVD2 mutations to

reduce FKBP12.6 binding to RYR2. Since

binding of FKBP12.6 increases the open

probability of RYR2 channels, they suggest

that CPVT mutations do not significantly

alter the cytoplasmic calcium levels while

ARVD2 associated mutations increase

RYR2-mediated calcium release in to the

cytoplasm.

Quite opposing results come from a

recent study addressing the functional

properties of three mutations underlying

CPVT (Wehrens et al. 2003). Mutations

generated in the human RYR2 gene were

expressed in mammalian a cell line, after

which, the channels were used in single-

channel recordings. All three mutants

displayed reduced affinity to FKBP12.6,

and, after PKA phosphorylation, increased

channel activity compared to that of wild-

type channels. PKA phosphorylation of

the channels occurs in vivo during exercise

(Marks 2000), in line with the phenotype of

affected patients showing symptoms only

during physical or emotional stress.

The mechanism proposed by

Wehrens et al (2003) is challenged by

a study in which the CPVT-associated

RYR2 mutations S2246L, N4104K,

and R4497C were expressed in HL-1

cardiomyocytes (George et al. 2003). At

rest, the phenotypic characteristics of

the cells expressing the mutant channels

were indistinguishable from the wild type.

When the RYR2 channels were activated

by mechanistically different RYR agonists

(caffeine and 4-chloro-m-cresol) or by

beta-adrenergic stimulation, Ca2+ release

was augmented significantly more through

the mutant channels than through the wild

type channels. The RYR2-FKBP12.6

complex was severely disrupted after beta-

adrenergic stimulation, but no difference

appeared in the FKBP12.6 dissociation rate

between the mutant and wild type channels

(George et al. 2003). These results indicate

that the defect in Ca2+ release is FKBP12.6

independent.

Our functional experiments performed

with the RYR2 channels containing P2328S,

52

DISCUSSION

Q4201R, and V4653F mutations were

consistent, with a pathogenic mechanism

similar to that suggested by Wehrens et al

(2003). We observed the same phenotype

of “leaking” channels in all RYR2 channels

studied, and thus the mutations probably

resulted in global destabilization of the

large channel complex rather than specific

disruptions of various functional domains.

In addition, our studies demonstrate that

the mutated channels have a decreased

sensitivity to inhibition by Mg2+, an

important endogenous channel modulator.

Our results with the experimental

drug JTV519 demonstrate a molecular

mechanism where JTV519 enhances

the affinity of RYR2 for FKBP12.6 and

rescues the gain-of-function phenotype

of the mutant channels. These results thus

provide a basis for targeted drug treatment

of exercise-induced cardiac arrhythmias.

In conclusion, several studies have

focused on revealing the molecular

pathogenic mechanisms of the specific

RYR2 mutations underlying the

phenotypes of CPVT and ARVD2. Results

of these studies, however, do not agree. It is

therefore necessary to obtain more detailed

data on the structure-function aspects of

RYR2 channels in order to understand

how the mutations result in augmented

intracellular Ca2+ release.

Very recent findings suggest that

mutations in the RYR2 gene may also

underlie the phenotypes of hypertrophic

and dilated cardiomyopathy (Cui et al.

2003; Furino et al. 2003), which underlines

the importance of the ryanodine receptor

in cardiac myocytes. Different mutations

appear to have very different outcomes,

implying that alterations in the RYR2 gene

may result in numerous cardiac phenotypes

showing both electric and structural

characteristics. One can hypothesize that

this may reflect the differential functional

effects of the mutations: some mutations

may result in transient elevation of

cytoplasmic calcium concentration and

thus lead to an electric disorder like CPVT.

Other mutations may cause more prolonged

changes in calcium homeostasis, resulting

in structural abnormalities of the cardiac

muscle, as in hypertrophic and dilated

cardiomyopathy.

1.3 CASQ2–THE SECOND GENE FOR

CPVT

Soon after the identification of RYR2

mutations in CPVT patients, the autosomal

recessive form of CPVT was characterized

and the disease gene localized to

chromosome 1p13-21 (Lahat et al. 2001a).

All the affected individuals belonged to

the same Bedouin tribe, and their parents

were often cousins. The phenotype of the

affected individuals was severe, with high

mortality. Average age of onset was 7.8 ±

4 years, a figure markedly lower than in the

dominant form (Swan and Laitinen 2002).

Subsequent analysis of candidate genes

DISCUSSION

53

located in the 1p13-21 region revealed a

single homozygous missense mutation in

the CASQ2 gene in all the affected patients

(Lahat et al. 2001b). Heterozygous mutation

carriers were clinically unaffected. The

CASQ2 gene encodes a protein located in

the sarcoplasmic reticulum and functioning

as a calcium buffer. The CASQ2 protein

is associated physically with RYR2

through interaction with junctin and triadin

(Zhang et al. 1997). Of the three additional

mutations in the CASQ2 gene have been

reported (Postma et al. 2002), two were

found to segregate in an autosomal

recessive manner. The third mutation,

however, showed a possible autosomal

dominant mode of inheritance.

Genetic analysis of the complete coding

region of the CASQ2 gene in 19 Finnish

CPVT probands disclosed two amino

acid polymorphisms but no apparently

causative mutations. This indicates that

this gene plays a minor if any role in the

molecular pathogenesis of CPVT in the

Finnish patients. The two novel amino

acid polymorphisms detected, T66A and

V76M, were relatively common in a cohort

of healthy blood donors, and between the

control and patient groups their allele,

genotype or haplotype frequencies did not

differ. Therefore, they most likely represent

innocent genetic polymorphisms.

2 HERG ALTERATIONS IN LQTS FAMILIES

2.1 POINT MUTATIONS AND SMALL

DELETIONS

In this study, the complete coding region of

the HERG gene was screened for mutations

by direct sequencing. Nine mutations,

seven missense mutations and two small

deletions, were detectable in families

affected with LQTS (Table 7). Three of

the point mutations (Y569H, G601S,

and T613M) had occurred de novo in the

proband or in a parent of the proband, and

two of these (G601S and T613M) also

occur in other populations (Akimoto et al.

1998; Jongbloed et al. 1999). This region,

encoding for the pore-forming portion of

the protein, may thus represent a mutational

“hot spot” in the HERG gene.

The clinical phenotype of those

individuals affected by the private HERG

gene mutations described in this study

shows marked variation. For example,

none of the carriers of the P451L mutation

had a prolonged QTc interval, while the

individuals carrying the 453delC mutation

had a mean QTc of 470 ± 42 ms. More

than a third of the mutation carriers were

symptomatic. As for the HERG-Fin

founder mutation, the mean QTc interval

of the symptomatic heterozygous mutation

carriers was significantly longer than

that of the asymptomatic carriers and

noncarriers. The spectrum of the phenotype

54

DISCUSSION

of the heterozygous HERG-Fin carriers was

wide, however, ranging from asymptomatic

patients to those who had experienced

recurrent syncopal spells.

Each of the HERG point mutations

identified alters an amino acid conserved in

evolution, a fact revealed when the amino

acid sequence of the HERG ion channel

was compared with those of homologous

channels from other species (Figure 12). For

example, the HERG-Fin (L552S) mutation

substitutes a leucine that is conserved not

only in mammals but also in Drosophila

melanogaster.

In functional studies, the Finnish

founder mutation L552S displayed a

marked increase in both activation and

inactivation rates, when compared to wild-

H.sapiens NEDGAVIMFILNFEVVMEKDMVGSPAHDTNHRGPPTSWLAPGRAKTFRLKLPALLALTAR 176O.cuniculus NEDGAVIMFILNFEVVMEKDMVGSPARDTNHRGPPTSWLAPGRAKTFRLKLPALLALTAR 159R.norvegicus NEDGAVIMFILNFEVVMEKDMVGSPAHDTNHRGPSTSWLASGRAKTFRLKLPALLALTAR 176M.musculus NEDGAVIMFILNFEVVMEKDMVGSPAHDTNHRGPSTSWLASGRAKTFRLKLPALLALTAR 176D.melanogaster QTQETPLWLLLQVAPIRNE----------------------------------------- 139

H.sapiens LLLVIYTAVFTPYSAAFLLKETEEGPPATECGYACQPLAVVDLIVDIMFIVDILINFRTT 474O.cuniculus LLLVIYTAVFTPYSAAFLLKETEEGPPAPECGYACQPLAVVDLIVDIMFIVDILINFRTT 459R.norvegicus LLLVIYTAVFTPYSAAFLLKETEDGSQAPDCGYACQPLAVVDLLVDIMFIVDILINFRTT 476M.musculus LLLVIYTAVFTPYSAAFLLKETEDGSQAPDCGYACQPLTVVDLIVDIMFIVDILINFRTT 476D.melanogaster LCLTFYTAIMVPYNVAFKNKTSEDVS-----------LLVVDSIVDVIFFIDIVLNFHTT 292

H.sapiens RLVRVARKLDRYSEYGAAVLFLLMCTFALIAHWLACIWYAIGNMEQPHMDSRIGWLHNLG 590O.cuniculus RLVRVARKLDRYSEYGAAVLLLLMCTFALIAHWLACIWYAIGNMEQPHMDSRIGWLHNLG 575R.norvegicus RLVRVARKLDRYSEYGAAVLFLLMCTFALIAHWLACIWYAIGNMEQPHMDSHIGWLHNLG 592M.musculus RLVRVARKLDRYSEYGAAVLFLLMCTFALIAHWLACIWYAIGNMEQPHMDSHIGWLHNLG 592D.melanogaster RLGRVVRKLDRYLEYGAAMLILLLCFYMLVAHWLACIWYSIG-RSDADNGIQYSWLWKLA 411

H.sapiens DQIGKPYNSSG--------LGGPSIKDKYVTALYFTFSSLTSVGFGNVSPNTNSEKIFSI 642O.cuniculus DQMGKPYNSSG--------LGGPSIKDKYVTGLYFTFSSLTSVGFGNVSPNTNSEKIFSI 627R.norvegicus DQIGKPYNSSG--------LGGPSIKDKYVTALYFTFSSLTSVGFGNVSPNTNSEKIFSI 644M.musculus DQIGKPYNSSG--------LGGPSIKDKYVTALYFTFSSLTSVGFGNVSPNTNSEKIFSI 644D.melanogaster NVTQSPYSYIWSNDTGPELVNGPSRKSMYVTALYFTMTCMTSVGFGNVAAETDNEKVFTI 471

H.sapiens RQ--------------RKRKLSFRRRTDKDTEQPGEVSALG--PGRAGAG--PSSRGRPG 924O.cuniculus RQ--------------RKRKLSFRRRTDKDTEQPGEVSALG--PGRAGAG--PSSRGRPG 909R.norvegicus RQ--------------RKRKLSFRRRTDKDTEQPGEVSALGQGPARVGPG--PSCRGQPG 928M.musculus RQ--------------RKRKLSFRRRTDKDTEQPGEVSALGQGPARVGPG--PSCRGQPG 928D.melanogaster RRKNEPQLPQNQDHLVRKIFSKFRRTPQVQAGSKELVGGSGQSDVEKGDGEVERTKVLPK 771

R176W

P451L

L552S Y569H G584S

G601S T613M

K897T

Figure 12. Amino acid alterations predicted to result from the HERG point mutations and polymorphism (italics) detected compared with homologous proteins from four different species. Only relevant parts of the amino acid sequence are illustrated. Mutations and polymorphism are in bold and a with gray background.

DISCUSSION

55

type channels. Increase in deactivation rate

is expected to reduce conductance during

terminal action potential repolarization

and therefore to prolong the repolarization

phase.

The functional properties of the HERG

channel with the missense mutation G601S

have been studied in HEK-293 cells and

in Xenopus oocytes (Furutani et al. 1999).

This mutant showed defective protein

trafficking, with reduced current amplitude,

but did not suppress the function of the

wild-type subunit in a dominant negative

manner. The T613M mutation has also been

functionally characterized (Huang et al.

2001). When expressed alone in Xenopus

oocytes, the mutant channel did not

produce any detectable current. When the

mutant channel subunit was co-expressed

with the wild-type subunit, a dominant

negative effect and defective trafficking to

the cell membrane could be observed.

These three examples: increased

deactivation rate, defective trafficking to

the cell membrane, and dominant negative

effect, illustrate the diverse mechanisms by

which the mutations can lead to the LQTS

phenotype. This could in part explain the

variability in clinical phenotype between

various mutations.

Recently, an inherited short QT interval

was described in two families (Gaita et al.

2003). The patients exhibited a QTc interval

of < 300 ms, and the families shared a

history of sudden deaths. Two distinct

point mutations, both resulting in the same

amino acid change N588K, were identified

in the HERG gene (Brugada et al. 2004).

The pathological mechanism by which

this mutation leads to the shortening of

QT interval, a totally different phenotype

than in other HERG mutations, is a gain-

of-function abnormality (Brugada et al.

2004).

2.2 PHENOTYPIC EFFECTS OF K897T

POLYMORPHISM

The effect of the K897T polymorphism

on myocardial repolarization, tested in

a random middle-aged population (226

men/187 women) (Pietilä et al. 2002),

indicates that the K897T polymorphism

appears to have a gender-related effect on

certain components of QT interval. Women

homozygous for the wild-type allele had

shorter total ventricular repolarization

(QTcmax

) and longer QTc measured at

lead V2 than did women carrying the

polymorphism.

A recent study further explored the

phenotypic effects of this polymorphism

both in vitro and in vivo (Paavonen et al.

2003). When expressed in HEK-293 and

COS cells, the channel containing the 897T

allele showed reduced surface expression

and increased inactivation, which were

proposed to result in reduced macroscopic

currents. The effect of this polymorphism

was also investigated in a cohort of 261

KCNQ1-Fin carriers who underwent an

56

DISCUSSION

exercise stress test. No difference in QTc

interval was observable between the groups

when all patients were included in the

analysis or when men and women were

considered separately. However, with only

the patients who had prolonged QT interval

(QTc > 440 ms) at rest (n = 39) included,

a difference in QT interval dynamics

occurred. During exercise, the patients

with the K897T and T897T genotypes had

longer QT intervals than did those with the

K897K genotype (Paavonen et al. 2003).

Despite many attempts to delineate

the physiopathological significance of the

HERG K897T polymorphism, it appears

that further studies are needed to elucidate

its exact phenotypic effects.

3 IMPLICATIONS FOR MOLECULAR DIAGNOSIS IN THE VENTRICULAR ARRHYTHMIA DISEASES CPVT AND LQTS

CPVT has an almost complete penetrance

and a high mortality rate (Lahat et al.

2001a; Swan and Laitinen 2002). Two

genes have recently been identified that

underlie this life-threatening disease (this

study; Lahat et al. 2001b). In LQTS, caused

by one of at least seven different genes

(Moss 2003), the penetrance varies, and

many mutation carriers are asymptomatic

(Priori et al. 1999b; Piippo et al. 2001b).

In both LQTS and CPVT, identification of

a genetic defect underlying the disease has

practical consequences. Early molecular

diagnosis permits effective advice and drug

treatment and can therefore prevent sudden

death (Priori 1997; Moss et al. 2000).

Exact molecular diagnosis in LQTS

is of paramount importance. It identifies

asymptomatic mutation carriers who may

be at risk for arrhythmias (Priori et al.

1999b), so that accurate and personalized

information about gene-specific risks such

as sports or medication can be provided.

For example, swimming is a particular risk

factor in LQT1 type of LQTS (Schwartz et

al. 2001), whereas sudden loud noises such

as alarm clocks should be avoided by the

LQT2 type (Wilde et al. 1999; this study).

The penetrance of LQTS mutations

can be very low. For example, our study

demonstrates that more than 70% of the

carriers of the HERG L552S mutation are

asymptomatic. Genetic testing in families

affected with LQTS mutations may

therefore seem questionable. However,

subtle genetic defects may predispose to

drug-induced arrhythmias (Priori et al.

1999b). This suggests that even though

the mutation may seem benign, it can

predispose to serious arrhythmias if a QT

interval-prolonging drug is administered to

the patient.

In CPVT, onset of symptoms is

usually in adolescence (Swan and Laitinen

2002). Only two Finnish mutation

carriers were non-penetrant at the age

of 15 years or older without manifesting

ventricular arrhythmias. Since the

DISCUSSION

57

Table 8. Present status for occurrence of the mutations in RYR2 and HERG genes identified in this study.

Gene Mutation Coding effect Location in the channel Families (n) Carriers (n)Non-carriers (n)

RYR2 V2306I G6916A cytoplasmic 1 2 12

P2328S C6928T cytoplasmic 1 17 30

Q4201R A12602G transmembrane regions 1 3 22

V4653F G13957T transmembrane regions 1 10 26

P4902L C14705T transmembrane regions 1 1 36

R4959Q G14876A transmembrane regions 1 1 3

HERG 453delC frameshift truncated protein 1 36 38

R176W C526T cytoplasmic 1 7 11

P451L C1352T S2 1 5 11

L552S T1655C S5 12 91 131

1631delAG frameshift truncated protein 1 1 1

Y569H T1705C S5 1 1 4

G584S G1750A S5-pore 1 16 19

G601S G1801A S5-pore 1 3 12

T613M C1838T pore 1 1 12

26 194 368

first symptom of CPVT can be lethal,

presymptomatic diagnosis is of utmost

importance. Established mutation carriers

are adequately monitored and advised to

avoid competitive sports and to take beta-

adrenergic-blocking drugs. Beta-blocking

therapy seems to be effective in preventing

malignant arrhythmias, as there have

been no sudden cardiac deaths among the

Finnish CPVT patients since the patients

were recruited to this study. In one patient,

a cardiac defibrillator has been implanted

due to non-responsiveness to drug therapy.

Since the six mutations in RYR2 and

nine mutations in HERG described here

were identified, several more carriers

of these particular mutations have been

molecularly diagnosed. Table 8 summarizes

the situation in February 2004. Of note is

the number of HERG-Fin carriers: a total

of 12 families carry this mutation. In all,

the KCNQ1-Fin (Piippo et al. 2001b)

and HERG-Fin mutations account for

approximately 64% of the molecularly

defined LQTS cases in Finland. Simple

PCR-based assays are routinely used in our

laboratory to screen these two mutations in

all new LQTS probands, and when positive,

in their family members as well.

58

DISCUSSION

4 CONTINUING SEARCH FOR ARRHYTHMIA GENES

Over the last decade, a large amount of

information of genes predisposing to

inherited arrhythmia diseases has been

provided through meticulous clinical and

genetic studies. The recent completion of

a draft sequence of the human genome has

revolutionized the process of disease gene

identification, as availability of the genomic

sequence enables exact localization of

microsatellite markers and candidate genes

as well as computational identification

of previously unknown genes. Many

LQTS probands still lack identification

of a mutation in any of the seven known

LQTS genes. Furthermore, only 38% of

the Finnish CPVT probands had a mutation

in the RYR2 gene and none in the CASQ2

gene. Inevitably, more arrhythmia genes are

likely to be identified, and many hitherto

insufficiently studied ion channel genes are

good candidates.

Indeed, there are several promising

candidate genes encoding for ion channels.

The gene encoding the skeletal muscle

L-type calcium channel (CACNA1S)

harbors mutations in families affected

with malignant hyperthermia (Monnier et

al. 1997), thus establishing a second MH

locus. Mutations leading to hypokalemic

periodic paralysis have also been identified

in the same gene (Ptacek et al. 1994). But,

the cardiac L-type calcium channel gene

(CACNA1C) has as yet been linked to no

clinical phenotype (Gargus 2003). A similar

situation exists for the tissue isoforms

of the sarco/endoplasmic reticulum Ca2+

ATPase (SERCA). Mutations in the skeletal

muscle isoform SERCA1, encoded by

ATP2A1 gene, result in Brody’s disease, a

skeletal myopathy (MacLennan 2000). The

isoform SERCA2, encoded by the ATP2A2

gene, has two splice variants: SERCA2b is

expressed in the skin and harbors mutations

leading to Darier’s disease, a skin disorder

characterized by warty papules and plaques

in seborrheic areas, and distinctive nail

abnormalities (Sakuntabhai et al. 1999),

while the cardiac isoform SERCA2a has

yet to reveal a pathogenic allele.

While these genes represent obvious

candidates for the arrhythmic diseases,

recent studies have shown that even

some less likely candidates should not be

ignored. The LQT4 gene turned out to be

a membrane adaptor protein (Mohler et

al. 2003). Hypertrophic cardiomyopathy

results also from mutations in genes other

than sarcomere protein-coding (Blair

et al. 2001). Careful clinical evaluation

and phenotyping of affected patients and

their family members is necessary for

identification of new inherited disease

entities. In addition, alternative approaches

to gene identification will most likely be

needed, as diseases with reduced penetrance

and of variable phenotype are difficult to

study with conventional linkage analyses

requiring large pedigrees. Accordingly,

DISCUSSION

59

homology analysis revealed a previously

unidentified potassium ion channel gene

(KCNE3) to underlie one form of periodic

paralysis (Abbott et al. 2001).

In conclusion, several disease genes

for arrhythmic disorders still await

identification. The characterization of these

genes and their mutations will provide

a molecular diagnosis for an increasing

number of individuals. Even more

importantly, identification of the novel

disease genes will enable the investigation

and clarification of molecular pathogenic

mechanisms underlying the disease

phenotypes, and thus provide a basis for

new therapeutic interventions.

60

CONCLUSIONS

CONCLUSIONS

Inherited arrhythmic syndromes involving

no structural abnormalities of the heart

muscle are rare but clinically important

entities. This study investigated the genetic

background of two of these diseases,


tachycardia (CPVT) and long QT syndrome

(LQTS). At the commencement of this

study, the genetic locus of CPVT had been

mapped to chromosome 1q42-43, and

several genes underlying LQTS had been

described. The main results and conclusions

of the present study are as follows:

• The location of the gene causing CPVT

was narrowed to a region of 1.5 cM.

Several functionally relevant positional

candidate genes were screened for

mutations. Subsequently, mutations

were identified in the gene coding for

ryanodine receptor type 2 (RYR2), a

calcium-release channel located in the

sarcoplasmic reticulum. This study

revealed the genetic background of six

Finnish CPVT families.

• In functional studies, the mutant

RYR2 channels displayed increased

open probability. This is in line with

the hypothesis that the channels

inappropriately leak calcium into the

cytoplasm. In addition, the mutant

channels displayed insensitivity to the

physiological inhibitor magnesium.

Treatment with the experimental

drug JTV519 normalized the channel

function. These data may have

relevance to patient treatment in the

future.

• While this study was in progress, the

gene underlying recessively inherited

CPVT was identified as calsequestrin

2 (CASQ2). This gene was screened

for mutations in Finnish CPVT

probands, and two novel amino acid

polymorphisms were detected but no

pathogenic mutations. Thus far, CASQ2

does not appear to be a major cause of

Finnish CPVT.

• A known LQTS-causing gene,

HERG, was screened for mutations

in 40 Finnish LQTS probands.

Nine mutations, including two

small deletions and seven missense

mutations, were detected in this gene

coding for a delayed rectifier potassium

channel. One of the point mutations

(L552S) represented a Finnish founder

mutation and was therefore designated

HERG-Fin. This mutation accounts

for approximately 10% of molecularly

defined Finnish LQTS cases.

• The first common amino acid

polymorphism in the HERG gene

CONCLUSIONS

61

was also identified. For a study of

the phenotypic effects of K897T

polymorphism, 170 individuals

carrying a Finnish founder mutation

KCNQ1-Fin underwent screening

for this polymorphism. A genotype-

phenotype relationship was suggested,

but additional studies are required for

confirmation of this result.

• The functional properties of the HERG-

Fin mutation were studied with a patch-

clamp technique. The mutant channel

showed an increased deactivation rate

that may result in decreased channel

activity at the end of the repolarization

phase.

• The results of this study are useful in

the molecular diagnosis in each of the

affected LQTS families. As a result of

this study, all Finnish LQTS probands

are routinely screened for the presence

of the HERG-Fin mutation. Molecular

diagnosis is of major importance

as it also identifies asymptomatic

individuals who may be at risk for

drug-induced arrhythmias.

• In CPVT, the first symptom may be

lethal, and beta-adrenergic blocking

therapy has been effective in preventing

malignant arrhythmias. DNA-based

diagnosis in the CPVT families

therefore provides an important asset

in identifying affected individuals

presymptomatically. No sudden cardiac

deaths have occurred in our Finnish

CPVT families since the time that the

patients were recruited to this study.

• Identification of the mutations

underlying CPVT and LQTS has

enabled investigation of the functional

defects that lead to arrhythmia

induction.

62

ACKNOWLEDGEMENTS

ACKNOWLEDGEMENTS

The current study was carried out during

the years 1998-2003 at the laboratory of

Professor Kimmo Kontula at the Department

of Medicine, and Research Program

in Molecular Medicine, Biomedicum

Helsinki, University of Helsinki. I wish to

express my sincere gratitude to all those

people who have contributed to this work.

Especially, I wish to thank:

Professor Kimmo Kontula, my

supervisor, whose enthusiasm and

encouragement have been crucial

throughout this work. Despite his

overwhelmingly hectic schedule, Kimmo

has always found time for discussion and

has given the larger perspective to this

project. I also wish to thank Kimmo for

providing me the financial opportunity

to visit co-workers abroad and to attend

scientific conferences.

Professor Reijo Tilvis, the head of

the Institute of Clinical Medicine, and

Professor Kimmo Kontula, the head of the

Department of Medicine, for providing

excellent research facilities.

Docent Katarina Pelin and Docent Anu

Wartiovaara for carefully reviewing this

thesis and for valuable comments. Carol

Norris for hilarious English lessons and for

revising the language of my thesis.

Docent Lauri Toivonen, Docent Matti

Viitasalo, and Dr Heikki Swan from the

Department of Cardiology for pleasant

collaboration. Especially, my sincere

thanks go to Heikki who has always been

ready to help with open questions. Minna

Härkönen and Hanna Ranne for expert

technical assistance.

Professor Andrew R. Marks and all the

members of his laboratory at the Center

for Molecular Cardiology, University of

Columbia, New York, Dietrich A. Stephan,

PhD, from the Research Center of Genetic

Medicine, Children’s National Medical

Center, Washington D.C., and Docent

Michael Pasternack and his research group,

Institute of Biotechnology, University of

Helsinki for valuable collaboration.

Heidi Fodstad and Kirsi Piippo, my

colleague “rytmihäiriötytöt”, for being

such wonderful persons to work with.

You have been there to help with practical

laboratory problems and to discuss issues

ranging from the meaning of genetic

studies of arrhythmias to possible career

option as tomato farmers. You have been

great company during congress trips, and

most of all, you have always been great

friends. For Kirsi I am most grateful for

supervision and guidance during the early

parts of this work.

Saara Nyqvist, Tuula Soppela, Sirpa

ACKNOWLEDGEMENTS

63

Vartiovuoren Tytöt for being an important

factor in balancing work with other things

in life. Satku for being such a great friend

and for doing the lay out of this thesis. Mia

for drawing most of the figures and Ville

for the design and execution of the thesis

cover.

My family for continuous love and

support. Especially I wish to thank my

parents who have always trusted in me and

my decisions.

My dear life-companion Pasi. You have

been an irreplaceable source of support,

encouraged me when things have been hard

and celebrated with me at the moments of

success.

All the patients and families who have

participated in this study.

This study was financially supported

by the Biomedicum Helsinki Foundation,

the Finnish Cultural Foundation, the Ida

Montin Foundation, the Maud Kuistila

Foundation, the Research Funds of the

University of Helsinki, the Sigrid Juselius

Foundation and the Special State Share of

the Helsinki University Central Hospital.

Espoo, February 2004

Stick and Susanna Tverin for expert

technical assistance and for being ever so

helpful. Tuula Hannila-Handelberg, Maarit

Lappalainen, Kristian Paavonen, and Kaisa

Valli-Jaakola for your help and company. I

wish you the best of luck in finishing your

thesis projects. All the present and former

members of the lab: Alpo, Camilla, Helena,

Ilse, Jaana, Jukka, Laura, Paulina, Sam,

Sami, Tarja, and Timo.

Raija Selivuo for her help with practical

matters.

Docent Maija Wessman for referring

me to Kimmo’s group when I was a second

year genetics student. I haven’t regretted

my decision to join the arrhythmia study!

Maija, Mari Kaunisto and Riitta Sallinen

for relaxing coffee breaks and helpful

discussions.

All the people who worked in the

basement labs of the Meilahti Hospital and

currently work in the Molecular Medicine

labs in the Biomedicum for creating an

enjoyable, lively, and nice atmosphere.

Eija Hämäläinen and Susanna Mehtälä for

cheerful company, help, and advice. What

is left of our old “bunker” team: Heidi,

Kaisa, Kirsi, Mari, and Riitta for numerous

non-scientific discussions, evenings out,

and for sharing the ups and downs of lab

work and writing.

All my friends. The Marjorie gang,

which has held on from the very first year

of our studies in biology, for numerous

unforgettable moments. My scouting troop

64

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2 INTERNET REFERENCES

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