ORIGINAL ARTICLE
Molecular genetic analysis
of
six Dutch
families with atrial fibrillation
M.M. Entius, A. Groenewegen, A. Pronk, J.J van der Smagt, P. Loh, R.N. Hauer, R. Derksen, I.C. van Gelder, D.J.A. Lok, P.A. Doevendans
Background. Atrial fibrillation (AF), the most common cardiac arrhythmia, is characterised by rapid and irregular contraction of the atrium. The risk of AF increases with age and AF increases the risk of various heart disorders, stroke and mortality. AF can occur in a sporadic or familial form. The underlying mechanism leading to AF is not well known but genetic analysis can increase our insight into the molecular pathways in AF. Detailed information on the molecular mechanisms of a disorder increase options for diagnosis and treatnent. Recently, a gain-of-function mutation in exon 1 of the KCNQ1 gene located on chromosome 11 was identified in a large Chinese AF family. KCNQ1 associates with KCNE1 or KCNE2 (both located on chromosome 21) to form cardiac potassium dcannels. Subsequent analysis ofChinese families showed a KCNE2 mutaton in two Hmilies.
Other genetic studies show linkage to chromosome 6 and 10, indicating genetic heterogeneity. A number of studies have shown that altered expression ofthe atrial connexin40 protein is a risk factor forAF. Connexin genes encode gap-junction proteins that are important in cardiac conduction and for normal wave propagation. Ob*jectives/metbods. In this study we analysed the role of KCNQ1, KCNE1 coding region and Cx4O promoter region in six Dutch AF families by sequence analysis. Conclusion. No mutations were found in these genes. The absence of mutations indicates genetic heterogeneity in familial AF; however, fiurther research is needed. Candidate genes are being sequenced, linkage analysis in a large family will be performed and additional AF families will be collected. (NedHeartJ2005;13:269-73.) Key words: familial atrial fibrillation, genetic analysis
M.M. Entius PA. Doevendans Department of Cardiology, Heart Lung Centre Utrecht Interuniversity Cardiology Institute of the Netherlands, Utrecht A. Gromeiwegen Department of Medical Physiology, Utrecht University Medical Centre, Utrecht A. Pronk Department of Cardiology, Heart Lung Centre Utrecht J.J. van der Smagt Division of Medical Genetics, Utrecht University Medical Centre, Utrecht P. Loh R.N. Hauer R. Dk sen Department of Cardiology, Heart Lung Centre Utrecht I.C. van Gelder Department of Cardiology, Groningen University Hospital,
Correspondence to: M.M. Entius Department of Cardiology, Heart Lung Centre Utrecht, PO Box 85500, 3508 GA Utrecht E-mail: [email protected]
A trial fibrillation (AF) is the most common cardiac arrhythmia characterised by rapid and erratic activation of the atria due to multiple wavelets of excitation that propagate through the atrial myocardium causing an irregular ventricular rhythm. AF can occur in a paroxysmal, persistent or permanent form. Diagnosis ofAF is based on clinical history and examination and confirmed by a 12-lead electrocardiogram (ECG). AF can become persistent over time and can cause several cardiac disorders including heart failure, stroke and mortality. The risk of AF also increases with age: about 0.5% ofthe population aged 50 years suffer from AF to up to 10% ofthe people over 80 years. In addition, many forms of cardiac disorders and environmental factors can contribute to the aetiology and complicate diagnosis." 2 Individuals without a heart disorder who suffer from AF are known as 'lone' or idiopathic AF patients, who can be seen at a relatively young age. The underlying mechanisms leading to AF are not yet clear but most likely involve pathways
Netherlands Heart Journal, Volume 13, Number 7/8, August 2005
269
Groningen D.J.A. Lok
Department of Cardiology, Deventer Hospital, Deventer
Molecular genetic analysis of six Dutch families with atrial fibrillation
concerned with cardiac conduction or molecular architecture in the atria. A detailed understanding of the molecular background of a disorder facilitates insight into the mechanisms leading to the disease, which can be used for the development of an effective treatment or an early detection method.3 Some lone AF patients suffer from a familial form of AF (FAF).4 FAF is inherited in an autosomal dominant fashion at high penetrance. Molecular genetic analysis ofhuman hereditary diseases including cardiac diseases has led to the identification of several mutated genes underlying the disease. The genes that are related to cardiac disorders can either be structural or nonstructural and they can be dassified on the basis of their function. They encode ion channel proteins, cytoskeletal proteins or sarcomeric proteins. Mutations in genes connected to nonstructural cardiac arrhythmias all encode ion channels, these genes are also known as channelopathy genes.5 Inward currents of positively charged ions (Na+, Ca+) depolarise the cell membrane and outward current (K+) provides repolarisation. Socalled 'afterdepolarisation' can lead to abnormal focal activity. The heart requires an accurate interaction of ion channels, ion currents, gap junctions and structural proteins for a normal function. An additional mechanism leading to AF is the occurrence of a reentry circuit. Monogenetic disorders, in which a single gene mutation leads to a mutant phenotype, are attractive study models due to the relatively straightforward genotype-phenotype correlation. Most disorders, however, are more complex to correlate as they involve genetic and environmental factors.6-8 Recent genetic studies on AF in Chinese families shed light on the molecular background of the disorder. Mutations in KCNQ1 and KCNE2 were found in these AF families.9"10 KCNQI is located on chromosome l lpl5.5 and encodes a pore-forming asubunit of the cardiac potassium I(I,) channel. The identified mutation in KCNQI leads to the missense mutation S14OG in affected members. Electrophysiological analysis showed a gain-of-function effect on KCNQ1/KCNEI and KCNQ1/KCNE2 potassium channels. Loss-of-function mutations in KCNQ1 have previously been reported for long-QT syndrome (LQT)."1 A subsequent study of the same Chinese group in 28 AF kindreds led to the identification ofthe same gain-of-function mutation in KCNE2 in two AF families (C79T).10 No mutations were found in HERG, KNCE1, KCNE3, KCNE4, KCNE5 and KCNJ2. Loss-of-function mutations in KCNE1 and KCNE2 have been found in patients with LQT5 and LQT6 syndromes, respectively.'2"13 In earlier studies two single nucleotide polymorphisms (SNPs) were identified in the coding region of KNCEl. These SNPs can influence the expression or activity of KNCEl. At amino acid position 38 the A-to-G polymorphism alters the original serine (S) amino acid to glycine (G)14 and the G-to-A poly270
p25 p24 p23
p22 p21.3
p15 p14 p13
p21.1
p12
p12
p1t
q12 q12 q14 q15
ql
q16
q21
q22 AFgoen
q22
qq2 AFgm
q23
q24 q23 q25 q24 q25
q26
q26 q27
Chromoso
6
Chronosome 10
Figure 1. Drawing of chromosomes 6 and 10 on basis of Giemsa staining. Loci thatlink tofamilialAF are indicated by 'AFgene'
morphism at amino acid position 85 changes aspartic acid (D) to asparagine (N).15 An increased frequency of the 38G showed an association with AF.16
Two genetic studies on familial AF show linkage to chromosome 10q22 and chromosome 6q14-16 (figure 1 ),17,18 however no disease-causing gene has been identified in this region. On both chromosomes candidate genes have been sequenced for mutations, Cx62, 5HTlE and TRIP7 on chromosome 6 and DLG5 on chromosome 10.18 19 Other important factors for normal wave propagation across the heart are gap junctions. Gap junctions facilitate intercellular coupling.20 In the heart three types of gap-junction proteins are found: connexin40 (Cx40), Cx43 and Cx45. The mammalian heart shows a regionally differently expressed pattern for the molecules. Cx4O is predominantly expressed in the atrium. Animal studies on AF show an altered Cx40 expression and distribution.2' In addition, heterogeneous distribution of Cx40 was found in human AF tissue.22 Structural changes in atrial myocytes in sustainedAF correlated with Cx40 distribution. Recent
Netherlands Heart Journal, Volume 13, Number 7/8, August 2005
Molecular genetic analysis of six Dutch families with atrial fibillation
Table 1. Clinical data on AF families. Family 1 2 3 4 5 6 *
# Rhythm Size family affected
16 3 3 12 16 22
2 2 1 5 3 9
I
I
SR SR SR SR AF SR SR
Axis
PQ
QRS
0
59 -18 447 2 72 0
158 142 128 264 130 178
84 128 104 108 106 114 96
Remarks
431 469 410 417 392 388 457
Normal ECG RBBB Early repolarisation and slow R wave progression V1-V3 LAFB, sinus bradycardia LVH, negative T waves V3-V5 Early repolarisation and slow R wave progression V1-V3
Slow R wave progression V1-V3
indicates a nonfamilial individual.
studies show that two polymorphisms at nucleotides -44 (G-to-A) and +71 (A-to-G) are associated with arrhythnia susceptibility.23 In this study we analysed the possible role of KCNQ1, KCNE1 and Cx4O polymorphisms in six families with AF. Material, methods and clinical evaluation Patients Blood samples were collected from index patients of six Dutch families and one nonfamilial individual suspected of having FAF, who were diagnosed with AF (table 1). A diagnosis was made on the basis of 12lead ECGs. Informed consent was obtained from participating family members according to guidelines of the medical ethics committees of Maastricht University Hospital and Utrecht University Medical Centre. Standard 12-lead ECG recordings and blood for DNA extraction were obtained from all participating relatives. Gene analysis was performed on the index patient from each family. ECG analysis The PR, QRS, and QTc intervals were determined from ECGs obtained from the patients and their relatives. The ECGs were recorded in the supine position, and no medication was used. The data were statistically analysed by Student's t test for unpaired data. Genetic analysis of KCNQI, KCNEI and Cx40 DNA for genetic analyses was extracted from peripheral blood lymphocytes using standard protocols. Sequencing reactions were performed with primer sequences for KCNQ1. Sequences were obtained as described by Neyroud et al.24 Sequencing reactions for KNCE1 were performed using primer sequences for KCNE1 forward primer-GAGGTGTGCCTGGGA AGTTTGAG (accession number: AP000324: 41103181) and reverse primer-AGGCAGGATGTGTCC AGTFITTAG (accession number: AP000324: 27342456). Alternations in Cx40 upstream sequences were
#i
QTC
(msec) (msec)
Netherlands Heart Journal, Volume 13, Number 7/8, August 2005
performed as described before.23 Sequences were analysed by direct sequencing of PCR products generated with forward primer 5'-TGAGGACAA GGAC-AACAGGCAG-3' and reverse primer 5'CCTTCCTCTGGCTACT-TCATATC-3'. Results
The results of the clinical investigation of the six families and one nonfamilial individual are shown in table 1. In all families multiple persons are affected, four families are multigeneration families (figure 2). Onset age of AF was
Molecular genetic analysis
of
six Dutch
families with atrial fibrillation
M.M. Entius, A. Groenewegen, A. Pronk, J.J van der Smagt, P. Loh, R.N. Hauer, R. Derksen, I.C. van Gelder, D.J.A. Lok, P.A. Doevendans
Background. Atrial fibrillation (AF), the most common cardiac arrhythmia, is characterised by rapid and irregular contraction of the atrium. The risk of AF increases with age and AF increases the risk of various heart disorders, stroke and mortality. AF can occur in a sporadic or familial form. The underlying mechanism leading to AF is not well known but genetic analysis can increase our insight into the molecular pathways in AF. Detailed information on the molecular mechanisms of a disorder increase options for diagnosis and treatnent. Recently, a gain-of-function mutation in exon 1 of the KCNQ1 gene located on chromosome 11 was identified in a large Chinese AF family. KCNQ1 associates with KCNE1 or KCNE2 (both located on chromosome 21) to form cardiac potassium dcannels. Subsequent analysis ofChinese families showed a KCNE2 mutaton in two Hmilies.
Other genetic studies show linkage to chromosome 6 and 10, indicating genetic heterogeneity. A number of studies have shown that altered expression ofthe atrial connexin40 protein is a risk factor forAF. Connexin genes encode gap-junction proteins that are important in cardiac conduction and for normal wave propagation. Ob*jectives/metbods. In this study we analysed the role of KCNQ1, KCNE1 coding region and Cx4O promoter region in six Dutch AF families by sequence analysis. Conclusion. No mutations were found in these genes. The absence of mutations indicates genetic heterogeneity in familial AF; however, fiurther research is needed. Candidate genes are being sequenced, linkage analysis in a large family will be performed and additional AF families will be collected. (NedHeartJ2005;13:269-73.) Key words: familial atrial fibrillation, genetic analysis
M.M. Entius PA. Doevendans Department of Cardiology, Heart Lung Centre Utrecht Interuniversity Cardiology Institute of the Netherlands, Utrecht A. Gromeiwegen Department of Medical Physiology, Utrecht University Medical Centre, Utrecht A. Pronk Department of Cardiology, Heart Lung Centre Utrecht J.J. van der Smagt Division of Medical Genetics, Utrecht University Medical Centre, Utrecht P. Loh R.N. Hauer R. Dk sen Department of Cardiology, Heart Lung Centre Utrecht I.C. van Gelder Department of Cardiology, Groningen University Hospital,
Correspondence to: M.M. Entius Department of Cardiology, Heart Lung Centre Utrecht, PO Box 85500, 3508 GA Utrecht E-mail: [email protected]
A trial fibrillation (AF) is the most common cardiac arrhythmia characterised by rapid and erratic activation of the atria due to multiple wavelets of excitation that propagate through the atrial myocardium causing an irregular ventricular rhythm. AF can occur in a paroxysmal, persistent or permanent form. Diagnosis ofAF is based on clinical history and examination and confirmed by a 12-lead electrocardiogram (ECG). AF can become persistent over time and can cause several cardiac disorders including heart failure, stroke and mortality. The risk of AF also increases with age: about 0.5% ofthe population aged 50 years suffer from AF to up to 10% ofthe people over 80 years. In addition, many forms of cardiac disorders and environmental factors can contribute to the aetiology and complicate diagnosis." 2 Individuals without a heart disorder who suffer from AF are known as 'lone' or idiopathic AF patients, who can be seen at a relatively young age. The underlying mechanisms leading to AF are not yet clear but most likely involve pathways
Netherlands Heart Journal, Volume 13, Number 7/8, August 2005
269
Groningen D.J.A. Lok
Department of Cardiology, Deventer Hospital, Deventer
Molecular genetic analysis of six Dutch families with atrial fibrillation
concerned with cardiac conduction or molecular architecture in the atria. A detailed understanding of the molecular background of a disorder facilitates insight into the mechanisms leading to the disease, which can be used for the development of an effective treatment or an early detection method.3 Some lone AF patients suffer from a familial form of AF (FAF).4 FAF is inherited in an autosomal dominant fashion at high penetrance. Molecular genetic analysis ofhuman hereditary diseases including cardiac diseases has led to the identification of several mutated genes underlying the disease. The genes that are related to cardiac disorders can either be structural or nonstructural and they can be dassified on the basis of their function. They encode ion channel proteins, cytoskeletal proteins or sarcomeric proteins. Mutations in genes connected to nonstructural cardiac arrhythmias all encode ion channels, these genes are also known as channelopathy genes.5 Inward currents of positively charged ions (Na+, Ca+) depolarise the cell membrane and outward current (K+) provides repolarisation. Socalled 'afterdepolarisation' can lead to abnormal focal activity. The heart requires an accurate interaction of ion channels, ion currents, gap junctions and structural proteins for a normal function. An additional mechanism leading to AF is the occurrence of a reentry circuit. Monogenetic disorders, in which a single gene mutation leads to a mutant phenotype, are attractive study models due to the relatively straightforward genotype-phenotype correlation. Most disorders, however, are more complex to correlate as they involve genetic and environmental factors.6-8 Recent genetic studies on AF in Chinese families shed light on the molecular background of the disorder. Mutations in KCNQ1 and KCNE2 were found in these AF families.9"10 KCNQI is located on chromosome l lpl5.5 and encodes a pore-forming asubunit of the cardiac potassium I(I,) channel. The identified mutation in KCNQI leads to the missense mutation S14OG in affected members. Electrophysiological analysis showed a gain-of-function effect on KCNQ1/KCNEI and KCNQ1/KCNE2 potassium channels. Loss-of-function mutations in KCNQ1 have previously been reported for long-QT syndrome (LQT)."1 A subsequent study of the same Chinese group in 28 AF kindreds led to the identification ofthe same gain-of-function mutation in KCNE2 in two AF families (C79T).10 No mutations were found in HERG, KNCE1, KCNE3, KCNE4, KCNE5 and KCNJ2. Loss-of-function mutations in KCNE1 and KCNE2 have been found in patients with LQT5 and LQT6 syndromes, respectively.'2"13 In earlier studies two single nucleotide polymorphisms (SNPs) were identified in the coding region of KNCEl. These SNPs can influence the expression or activity of KNCEl. At amino acid position 38 the A-to-G polymorphism alters the original serine (S) amino acid to glycine (G)14 and the G-to-A poly270
p25 p24 p23
p22 p21.3
p15 p14 p13
p21.1
p12
p12
p1t
q12 q12 q14 q15
ql
q16
q21
q22 AFgoen
q22
qq2 AFgm
q23
q24 q23 q25 q24 q25
q26
q26 q27
Chromoso
6
Chronosome 10
Figure 1. Drawing of chromosomes 6 and 10 on basis of Giemsa staining. Loci thatlink tofamilialAF are indicated by 'AFgene'
morphism at amino acid position 85 changes aspartic acid (D) to asparagine (N).15 An increased frequency of the 38G showed an association with AF.16
Two genetic studies on familial AF show linkage to chromosome 10q22 and chromosome 6q14-16 (figure 1 ),17,18 however no disease-causing gene has been identified in this region. On both chromosomes candidate genes have been sequenced for mutations, Cx62, 5HTlE and TRIP7 on chromosome 6 and DLG5 on chromosome 10.18 19 Other important factors for normal wave propagation across the heart are gap junctions. Gap junctions facilitate intercellular coupling.20 In the heart three types of gap-junction proteins are found: connexin40 (Cx40), Cx43 and Cx45. The mammalian heart shows a regionally differently expressed pattern for the molecules. Cx4O is predominantly expressed in the atrium. Animal studies on AF show an altered Cx40 expression and distribution.2' In addition, heterogeneous distribution of Cx40 was found in human AF tissue.22 Structural changes in atrial myocytes in sustainedAF correlated with Cx40 distribution. Recent
Netherlands Heart Journal, Volume 13, Number 7/8, August 2005
Molecular genetic analysis of six Dutch families with atrial fibillation
Table 1. Clinical data on AF families. Family 1 2 3 4 5 6 *
# Rhythm Size family affected
16 3 3 12 16 22
2 2 1 5 3 9
I
I
SR SR SR SR AF SR SR
Axis
PQ
QRS
0
59 -18 447 2 72 0
158 142 128 264 130 178
84 128 104 108 106 114 96
Remarks
431 469 410 417 392 388 457
Normal ECG RBBB Early repolarisation and slow R wave progression V1-V3 LAFB, sinus bradycardia LVH, negative T waves V3-V5 Early repolarisation and slow R wave progression V1-V3
Slow R wave progression V1-V3
indicates a nonfamilial individual.
studies show that two polymorphisms at nucleotides -44 (G-to-A) and +71 (A-to-G) are associated with arrhythnia susceptibility.23 In this study we analysed the possible role of KCNQ1, KCNE1 and Cx4O polymorphisms in six families with AF. Material, methods and clinical evaluation Patients Blood samples were collected from index patients of six Dutch families and one nonfamilial individual suspected of having FAF, who were diagnosed with AF (table 1). A diagnosis was made on the basis of 12lead ECGs. Informed consent was obtained from participating family members according to guidelines of the medical ethics committees of Maastricht University Hospital and Utrecht University Medical Centre. Standard 12-lead ECG recordings and blood for DNA extraction were obtained from all participating relatives. Gene analysis was performed on the index patient from each family. ECG analysis The PR, QRS, and QTc intervals were determined from ECGs obtained from the patients and their relatives. The ECGs were recorded in the supine position, and no medication was used. The data were statistically analysed by Student's t test for unpaired data. Genetic analysis of KCNQI, KCNEI and Cx40 DNA for genetic analyses was extracted from peripheral blood lymphocytes using standard protocols. Sequencing reactions were performed with primer sequences for KCNQ1. Sequences were obtained as described by Neyroud et al.24 Sequencing reactions for KNCE1 were performed using primer sequences for KCNE1 forward primer-GAGGTGTGCCTGGGA AGTTTGAG (accession number: AP000324: 41103181) and reverse primer-AGGCAGGATGTGTCC AGTFITTAG (accession number: AP000324: 27342456). Alternations in Cx40 upstream sequences were
#i
QTC
(msec) (msec)
Netherlands Heart Journal, Volume 13, Number 7/8, August 2005
performed as described before.23 Sequences were analysed by direct sequencing of PCR products generated with forward primer 5'-TGAGGACAA GGAC-AACAGGCAG-3' and reverse primer 5'CCTTCCTCTGGCTACT-TCATATC-3'. Results
The results of the clinical investigation of the six families and one nonfamilial individual are shown in table 1. In all families multiple persons are affected, four families are multigeneration families (figure 2). Onset age of AF was
