Carlos Oberti 2004

Genome-Wide Linkage Scan Identifies a Novel Genetic Locus onChromosome 5p13 for Neonatal Atrial Fibrillation Associated WithSudden Death and Variable Cardiomyopathy

Carlos Oberti, MD, MS*, Lejin Wang, MD*, Lin Li, PhD, Jiamei Dong, MD, Shaoqi Rao, PhD,Wei Du, BS, and Qing Wang, PhD, MBAFrom the Center for Molecular Genetics, Department of Molecular Cardiology, Lerner ResearchInstitute (C.O., L.W., L.L., J.D., S.R., W.D., Q.W.) and the Center for Cardiovascular Genetics,Department of Cardiovascular Medicine (C.O., L.W., L.L., J.D., S.R., W.D., Q.W.), The ClevelandClinic Foundation; the Department of Molecular Medicine, Cleveland Clinic Lerner College ofMedicine of Case Western Reserve University (L.W., L.L., J.D., S.R., Q.W.), and the Departmentof Biological, Geological, and Environmental Sciences, Cleveland State University (W.D., Q.W.),Cleveland, Ohio; the Department of Cardiology, Ospedale Italiano Umberto I, Montevideo, Uruguay(C.O.); and Huazhong University of Science and Technology Human Genome Research Center,Wuhan, Hubei, P.R. China (Q.W.).

AbstractBackground—Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, andpatients with AF have a significantly increased risk for ischemic stroke. Approximately 15% of allstrokes are caused by AF. The molecular basis and underlying mechanisms and pathophysiology ofAF remain largely unknown.

Methods and Results—We have identified a large AF family with an autosomal recessiveinheritance pattern. The AF in the family manifests with early onset at the fetal stage and is associatedwith neonatal sudden death and, in some cases, ventricular tachyarrhythmias and waxing and waningcardiomyopathy. Genome-wide linkage analysis was performed for 36 family members andgenerated a 2-point logarithm of the odds (LOD) score of 3.05 for marker D5S455. The maximummultipoint LOD score of 4.10 was obtained for 4 markers: D5S426, D5S493, D5S455, andD5S1998. Heterozygous carriers have significant prolongation of P-wave duration on ECGscompared with noncarriers (107 versus 85 ms on average; P = 0.000012), but no differences betweenthese 2 groups were detected for the PR interval, QRS complex, ST-segment duration, T-waveduration, QTc, and R-R interval (P > 0.05).

Conclusions—Our findings demonstrate that AF can be inherited as an autosomal recessive traitand define a novel genetic locus for AF on chromosome 5p13 (arAF1). A genetic link between AFand prolonged P-wave duration was identified. This study provides a framework for the ultimatecloning of the arAF1 gene, which will increase the understanding of the fundamental molecularmechanisms of atrial fibrillation.

Keywordsarrhythmia; genetics; atrium; fibrillation; death; sudden

Correspondence to Dr Qing Wang, Center for Molecular Genetics, ND4-38, The Cleveland Clinic Foundation, 9500 Euclid Ave,Cleveland, OH 44195. E-mail [email protected].*The first 2 authors contributed equally to this work.The online-only Data Supplement, which contains a table and a figure, can be found with this article at http://www.circulationaha.org.

NIH Public AccessAuthor ManuscriptCirculation. Author manuscript; available in PMC 2006 October 23.

Published in final edited form as:Circulation. 2004 December 21; 110(25): 3753–3759.

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A trial fibrillation is “a supraventricular tachyarrhythmia characterized by uncoordinated atrialactivation with consequent deterioration of atrial mechanical function.”1 It is the most commonsustained cardiac rhythm disturbance, and its prevalence increases as the population ages.2Atrial fibrillation affects 2.2 million Americans, and each year 160 000 new cases arediagnosed.3 It is associated with significant morbidity and mortality.4,5 An estimated 70 000strokes each year in the United States are caused by atrial fibrillation.4

Atrial fibrillation can be associated with heart diseases, including coronary artery disease,congestive heart failure, hypertension, congenital heart disease, diabetes, rheumatic or othersignificant valvular heart disease, significant left ventricular systolic dysfunction, andhyperthyroidism.2,6 However, it can also occur in many patients without any other detectablecardiac and systemic diseases, known as “lone atrial fibrillation” (20% to 50% of the patientswith atrial fibrillation).1,6

Electrophysiological studies have demonstrated that ectopic foci of repetitive atrial activity,usually arising from the pulmonary veins, may initiate atrial fibrillation.7 Furthermore, atrialfibrillation can cause changes in cellular electrophysiology (electrical remodeling; altered geneexpression of the L-type calcium channel, potassium channels ITo, IK1, IKAch, etc).8 Atrialfibrillation is also associated with adaptive and maladaptive changes in tissue and cellulararchitecture (structural remodeling).9

Atrial fibrillation can occur in families, suggesting a genetic propensity for atrial fibrillationin some patients. Two genetic loci for autosomal dominant atrial fibrillation have been mappedto chromosomes 10q22–2410 and 6q14–1611; however, the specific genes have not yet beenidentified. A gain-of-function mutation (S140G) in the cardiac potassium channel geneKCNQ1 (KvLQT1) on chromosome 11p15.5, the type-1 long-QT syndrome (LQTS) gene,12was found to be associated with autosomal dominant atrial fibrillation in a Chinese family.13 However, it is important to note that more than 50% of the affected members (9 of 16) inthe family are also affected with LQTS, which is caused by loss-of-function or dominant-negative mutations in KCNQ1. The molecular basis of the majority of atrial fibrillation cases,however, remains unknown.

In this study, we describe the identification of a form of atrial fibrillation that is inherited inan autosomal recessive manner. We then used genome-wide linkage scan to map the geneticlocus for this type of atrial fibrillation (arAF1) and performed genotype-phenotype correlationstudies for heterozygous carriers and noncarriers in the family.

MethodsStudy Subjects and Phenotyping

The autosomal recessive atrial fibrillation family was identified in Uruguay, South America,and subsequently recruited to our laboratory for genetic analysis. Informed consent wasobtained from the participants or their guardians in accordance with standards established bythe Cleveland Clinic Foundation Institutional Review Boards on Human Subjects.

Phenotyping was performed on the basis of clinical history, physical examinations, and thedata from resting 12-lead ECG. Echocardiography was also performed for a few selected familymembers. The diagnosis was based on the criteria by the ACC/AHA/ESC Joint Committee ofExperts on the Management of Patients with Atrial Fibrillation, which include the followingtypical ECG features. (1) Discrete P waves are absent or difficult to count. (2) Rapid oscillationsor fibrillatory waves (f waves) that vary in size, shape, and timing are present. (3) An atrialrate (frequency of f waves) of 150 to 300 bpm is seen or is difficult to detect at high atrial rates.(4) There is an irregular, frequently rapid, ventricular rate because of constant stimulation from

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the atria, 100 to 200 bpm; irregularly irregular rhythm; or inconsistent R-R interval.1 QTc wasmeasured as the QT interval corrected for heart rate.14,15 P-wave duration was measured fromlead II.

Isolation of Genomic DNA and GenotypingGenomic DNA was prepared from whole blood with the DNA Isolation Kit for MammalianBlood (Roche Diagnostic Co).

A genome-wide scan was performed using 398 polymerase chain reaction–based short tandemrepeat polymorphic markers from chromosomes 1 to 22 (ABI Prism Linkage Mapping SetMD10) as described previously.16 These markers span the human genome by every 10 cM.Amplification of each short tandem repeat was performed by polymerase chain reaction usingthe PE 9700 PCR System with the standard procedure as instructed by the manufacturer (ABI).Genotyping was performed using an ABI 3100 Genetic Analyzer. Genotyping data were scoredusing the GeneMapper 2 software (ABI).

Fine mapping was performed using additional markers identified at the Genethon and Centerfor Medical Genetics, Marshfield Medical Research Foundation, databases.

Linkage AnalysisPhenotyping data, genotyping data, and pedigree information were introduced into a computer,and pairwise linkage analysis was performed using the Fastlink software package.17 Two-point linkage analysis was performed assuming an autosomal recessive pattern of inheritance,a disease-allele frequency of 0.001, and penetrance of 0 for carriers and noncarriers and 0.99for homozygous affected individuals. The logarithm of the odds (LOD) scores were calculatedusing the actual allele frequencies of each marker that were calculated from the genotypingdata of 38 independent individuals from Uruguay (Data Supplement Table). Gene frequencywas assumed to be equal between males and females. Multipoint linkage analysis wasperformed using the Simwalk2 program,18 with the fine mapping data formatted with theMega2 (version 2.5) program.19 The parameters used for multipoint linkage analysis areidentical to those for 2-point linkage analysis. The distance (cM) between markers was basedon the data from the Center for Medical Genetics, Marshfield Medical Research Foundation,database.

ResultsIdentification of Autosomal Recessive Atrial Fibrillation

We identified a large consanguineous atrial fibrillation family from Uruguay, South Americawith 57 family members (32 males and 25 females; ages from 3 months to 93 years) in 5 livinggenerations (Figure 1). Seven consanguineous marriages occurred in the family. The ECG fromproband V:11 and patient VI:2 showed the typical features of atrial fibrillation: absent ordifficult-to-count P waves, a fast atrial rate, and inconsistent R-R intervals (Data SupplementFigure). QTc (the QT interval corrected for heart rate) is 0.42 and 0.40 second for the probandV:11 and affected member VI:2, respectively. LQTS was not detected in any members fromthis family (Table 1). No abnormalities were found in other organ systems. The atrialfibrillation in the family appeared to be chronic atrial fibrillation. Interestingly, multiple suddendeaths occurred in the family: the proband (V:11) died at the age of 15 months, and affectedmembers V:9, V:10, and VI:1 also died suddenly at the ages of 3 months, 2 months, and 18months, respectively. The family history study also revealed 2 other claimed cases of suddendeath (IV:17, IV:18; Figure 1), but the exact cause of the death was not known. Patient VI:2died suddenly at the age of 1 year and 7 months after this article was submitted. Thus, autosomal



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recessive atrial fibrillation in this family appears to be associated with a particularly severeform of clinical outcome, early onset, and association with sudden death.

The proband (V:11) was delivered by caesarean section at week 36 of pregnancy because offetal tachycardia, with a heart rate of 250 bpm and atrial fibrillation/flutter. Supraventriculartachyarrhythmias continued after the delivery. The first echocardiogram at the age of 2 daysshowed marked dilatation of both atria and an ejection fraction of 52%, and amiodarone (10mg/d) was prescribed. At the age of 1 month, an electrophysiological study was performed anddetected atrial fibrillation/flutter. Because of the mild response to amiodarone and the familyhistory of sudden death, linear ablation on the left atrium was attempted but was unsuccessful.After application of a Furman catheter, ablation of the atrioventricular node was performed,setting a ventricular rate of 100 bpm. Two days later, a permanent pacemaker was implantedbecause of the high frequency of sudden death in the family. At the age of 2 months, only mildatrial dilatation was observed, and the ejection fraction was borderline normal, even thoughno anticongestive therapy was exercised. The ejection fraction was 52% at the age of 4 months.The patient, however, died suddenly at the age of 15 months.

For patient VI:2, a recent echocardiogram at the age of 15 months was normal.Echocardiography did not detect structural heart abnormalities in her mother and grandmother(data not shown), the obligate carriers. This patient was on medical treatment with digoxin(0.07 mg twice a day) and propafenone (30 mg/d), which apparently maintained the patient insinus rhythm with a heart rate of 125 bpm. The patient, however, died recently at the age of 1year and 7 months.

Affected member V:9 was born with supraventricular tachycardia. He was delivered bycaesarean section at week 37 of pregnancy because of fetal tachycardia. An ECG at the age of24 days showed atrial flutter, with a heart rate of 200 bpm. No structural heart abnormalitieswere detected by echocardiography at the age of 24 days, but a later echocardiogram showeddilatation of the left ventricle and left atrium and a decrease of contractility (ejection fractionof 43%).

Affected member V:10 was born with atrial tachycardia, and echocardiography did not detectstructural heart disease.

Affected member VI:1 was born with atrial tachycardia, and electrical cardioversion wasperformed at the age of 20 days. At the age of 1 month, an echocardiogram showed normalventricular and atrial sizes.

The atrial fibrillation in the family is inherited with an autosomal recessive pattern, becauseall parents have normal phenotype, and occurrence of atrial fibrillation is associated withconsanguineous marriages (Figure 1). These results suggest that atrial fibrillation can beinherited as an autosomal recessive trait.

Identification of a Novel Genetic Locus for Autosomal Recessive Atrial Fibrillation byGenome-Wide Linkage Analysis

A genome-wide scan was performed for 36 family members from kindred arAF1 (Figure 1)using markers that span the human genome by every 10 cM. Analysis of the pairwise LODscores identified significant linkage to one marker, D5S455, on chromosome 5p13 (Table 2;Figure 1). A peak LOD score of 3.05 was obtained for D5S455 at a recombination fraction of0 with the actual marker allele frequencies (Data Supplement Table) specific to the Uruguaypopulation. The LOD scores were also calculated with the commonly used allele frequenciesof 1/n, where n is the number of alleles observed, and the peak LOD score for D5S455 increasedto 3.44 (Table 2).



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Additional markers, including D5S1506, D5S493, D5S426, D5S1998, D5S1964, andD5S1490 (Figure 1), were used for fine mapping. The peak multipoint LOD score of 4.10 wasobtained for a region spanned by 4 markers D5S493, D5S426, D5S455, and D5S1998 (Figure2). The chromosomal segment from markers D5S426 to D5S1964 was homozygous for bothaffected patients with atrial fibrillation (V:11 and VI:2 in Figure 1). Markers D5S1506 andD5S1490 were positioned outside of the homozygous chromosomal segment at the telomericand centromeric directions, respectively. These results indicate that the autosomal recessiveatrial fibrillation gene (arAF1) is located between markers D5S1506 and D5S1490, a regionof 7.76 cM in length (Figure 3).

P-Wave Duration Is Significantly Prolonged in Heterozygous Carriers in the arAF1 FamilyGenotypic analysis identified 24 family members in kindred arAF1 as heterozygous carrierswho share one disease haplotype (Figure 1). ECGs were recorded from 19 carriers and 5noncarriers (Table 1). None of the heterozygous carriers are affected with atrial fibrillation.Detailed ECG parameters for the 19 carriers and 5 noncarriers were measured and analyzed(Table 1 and Figure 4). No significant differences between the carriers and noncarriers weredetected for the PR interval, QRS complex, ST-segment duration, T-wave duration, QT intervaland QTc (412 ms for carriers versus 416 ms for noncarriers), and R-R interval (P > 0.05)(Figure 4). By contrast, a highly significant difference was observed for the P-wave durationbetween the carriers and noncarriers (107 ms for carriers versus 85 ms for noncarriers, P =0.0000122) (Figure 4).

DiscussionOur study demonstrates that atrial fibrillation can be inherited as an autosomal recessive trait.Furthermore, we have mapped the autosomal recessive atrial fibrillation gene to chromosome5p13 (arAF1). The identification of the autosomal recessive form of atrial fibrillation may havean implication for our understanding of the more common sporadic atrial fibrillation cases.The majority of atrial fibrillation cases are considered to be sporadic. Although major mutationsin the genes for inherited atrial fibrillation would be anticipated to be responsible for monogenicforms of atrial fibrillation (such as in the family studied here), more common variants or single-nucleotide polymorphisms could be responsible for “sporadic” forms of atrial fibrillation inadults. After the autosomal recessive atrial fibrillation gene is identified, it will be interestingto perform mutation analysis or single-nucleotide polymorphism identification in sporadiccases to validate the above hypothesis.

The atrial fibrillation in the family studied here appeared to be very severe, because it ismanifested by early, fetal or infantile, age at onset and is associated with sudden death.Remarkably, the heterozygous carriers did not show signs of atrial fibrillation. These featuresare consistent with the recessive mode of inheritance, as in other diseases, including LQTS.Patients with autosomal recessive LQTS have a much more severe clinical outcome than thosewith the autosomal dominant form of LQTS.20–23

Ventricular tachyarrhythmia was detected at the fetal stage in 2 of the 5 affected individuals,proband V:11 and affected member V:9. Although ventricular tachyarrhythmia has beendemonstrated in atrial fibrillation patients,24,25 it is still interesting to detect ventriculartachyarrhythmia at the fetal stage in the arAF1 family. This is consistent with the earlierargument that the autosomal recessive form of atrial fibrillation in the arAF1 family isassociated with a severe outcome.

It has been reported that atrial fibrillation with a rapid ventricular response may lead to anonischemic cardiomyopathy.25 In the arAF1 family studied here, dilatation of both atria andan ejection fraction of 52% were found at the age of 2 days in proband V:11, but these



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disappeared or minimized later in his life. The patient was under medication with amiodarone(10 mg/d) and had a pacemaker implanted, but it is unknown whether these therapies led tothe later improvements on the echocardiographic parameters. Dilatation of left atrium and leftventricle and a low ejection fraction of 43% were found for affected member V:9. No structuralabnormalities were detected in other affected members, including VI:2, V:10, and VI:1. It isunknown whether atrial and ventricular dilatation in 2 of the 5 affected members in the arAF1family are caused by atrial fibrillation or whether they are accompanying features of theautosomal recessive atrial fibrillation in this family.

Although the heterozygous carriers in the arAF1 family studied here did not manifest the typicalECG features of atrial fibrillation, their P-wave duration, on average (107 ms), was abnormal(normal P-wave duration, 80 to 100 ms) and significantly longer than the value of 85 ms fromthe noncarriers (P = 0.000012, Figure 4). Thus, this study identifies a genetic link betweenprolongation of P-wave duration and atrial fibrillation. On the basis of this interestingobservation, we can speculate that prolonged P-wave duration may be a precursor to atrialfibrillation in this family, although it remains to be determined whether prolonged P-waveduration is a predictor for common sporadic or lone atrial fibrillation. Because the P wave onthe standard ECG corresponds to the intra-atrial conduction times,26 prolonged P-waveduration may be an indication of abnormal atrial conduction. Thus, atrial fibrillation can be anatrial conduction disorder. It is interesting to note that a parallel comparison can be made foratrial fibrillation and LQTS, because LQTS is characterized by prolongation of QTc andabnormalities in ventricular conduction.21 The P-wave durations for patients VI:1 and VI:2were 80 ms and 60 ms, respectively, on ECGs at sinus rhythm; however, caution should beexercised in interpreting these readings, because the ECGs were recorded while atrialfibrillation in the patients was cardioverted electrically and controlled pharmacologically.

It is anticipated that, like ventricular fibrillation, atrial fibrillation may be caused by mutationsin ion channel genes. The arAF1 gene is defined within a 7.76-cM region, and the only channelgene identified at this locus is SLC1A3, which encodes glial high-affinity, Na+-dependentglutamate transporter. Sequence analysis of all exons, including exonintron boundaries, ofSLC1A3 failed to identify any mutation in our arAF family. Furthermore, no cardiac ion channelgenes were mapped to the 2 autosomal dominant atrial fibrillation loci on chromosomes 10and 6. Continued mutational analysis with other candidate genes at the arAF1 locus shouldlead to the identification of the specific atrial fibrillation gene at this locus. Identification ofan atrial fibrillation gene is likely to greatly further our understanding of the molecularmechanism underlying the pathogenesis of atrial fibrillation, the most common cardiacarrhythmia.

Acknowledgements

This study was supported by an American Heart Association Established Investigator award; Chinese Ministry ofScience and Technology National High Technology 863 Project No. 2002BA711A07; and NIH grants R01-HL-65630and R01-HL-66251 (all to Dr Wang). We thank T. Parrado for help with the Fastlink and other linkage softwarepackages; B. Cerutti, S. Gelos, J.L. Montenegro, and R. Canessa at the Ospedale Italiano Umberto I and HospitalPereira Rosell and M. Chung and A. Natale at the Cleveland Clinic Foundation for help with clinical examinationsand ECG reviewing; A. Barbela for help with echocardiograms; and R. de la Fuente and other members of the Wanglaboratory for technical help and discussion.

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Figure 1.Mapping of a novel genetic locus for autosomal recessive atrial fibrillation (arAF1). Pedigreestructure of a family with autosomal recessive atrial fibrillation is shown. Genome-widelinkage analysis was performed with 398 polymorphic markers that span entire human genomeby an average interval of 10 cM. Results of genotypic analysis are shown for markersD5S1506, D5S426, D5S493, D5S455, D5S1998, D5S1964, and D5S1490 below eachindividual. Affected individuals are shown as filled circles (females) and squares (males).Normal individuals are shown as empty symbols, and deceased individuals are indicated byslashes. Proband is indicated by an arrow. Obligate carriers by genotyping are denoted withblack dots in symbols. Disease haplotype is denoted with a filled vertical bar, and normalhaplotypes are indicated by open vertical bars. ECG analysis of normal family members andobligate heterozygous carriers did not reveal any clinical features of atrial fibrillation. Sevenconsanguineous marriages are indicated by =.



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Figure 2.Multipoint LOD score analysis of atrial fibrillation (arAF1). Genotypes at D5S1506,D5S426, D5S493, D5S455, D5S1998, D5S1964, and D5S1490 were used to determinemultipoint LOD scores at chromosome 5p13. D5S1506 is arbitrarily plotted at 0 cM at abscissa.Other microsatellite markers near arAF1 locus are indicated along abscissa from telomere tocentromere. Multipoint LOD scores are plotted on ordinate.



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Figure 3.Ideogram of chromosome 5 with Geimsa banding patterns and localization of arAF1 locus.Genetic map with chromosome 5p13 markers and location of putative arAF1 gene is shownon right.



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Figure 4.ECG characterization of heterozygous carriers and noncarriers. A 2-tailed probability valuewas obtained by use of a Student’s t test with assumption of unequal variances and nullhypothesis of no difference between mean values. Error bar represents SD. Only significantdifference between carriers and noncarriers was identified for duration of P wave (P =0.0000122). P wave indicates P-wave duration recorded from lead II (normal range for P-waveduration is from 80 ms to 100 ms); PR, length of PR interval; QRS, duration of QRS complex;ST, length of ST segment; T, T-wave duration; QT, length of QT interval; RR, length of RRinterval; QTc, QT corrected for heart rate.



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TABLE 2Pairwise LOD Scores for Chromosome 5p13 Markers at the arAF1 Locus

Recombination Fraction

Marker 0.00 0.05 0.10 0.20 0.30 0.40

D5S1506 −0.02 0.25 0.58 0.63 0.48 0.25D5S493 0.22 0.08 −0.03 −0.17 −0.19 −0.13D5S426 2.10 1.72 1.40 0.95 0.63 0.32D5S455 3.05 2.61 2.17 1.39 0.84 0.43D5S1998 2.23 1.98 1.74 1.26 0.81 0.39D5S1964 1.91 1.77 1.60 1.20 0.78 0.37DS51490 −3.24 −0.58 −0.11 0.20 0.24 0.16

LOD scores were calculated by use of the FASTLINK linkage program assuming autosomal recessive inheritance, 99% penetrance, and the diseasefrequency of 0.001. Allele frequencies for LOD score calculation are actual frequencies in the Uruguay population derived from genotyping data of 38independent individuals (76 chromosomes) (supplementary Table 1). LOD scores varied little with the estimated disease frequencies: the LOD score forD5S455 increased to 3.06 at a disease frequency of 0.0001 and 0.00001. The LOD scores were also calculated using the allele frequencies of 1/n (wheren is the number of alleles observed), which increased the LOD scores for marker D5S455 to 3.440, 3.448, and 3.450 at a disease frequency of 0.001,0.0001, and 0.00001, respectively.


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Carlos Oberti 2004