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Investigation of Association between PFO Complicated by Cryptogenic Stroke and a Common Variant of the Cardiac Transcription Factor GATA4

  • Mahdi Moradi Marjaneh ,

    Contributed equally to this work with: Mahdi Moradi Marjaneh, Edwin P. Kirk, Maximilian G. Posch

    Affiliations The Victor Chang Cardiac Research Institute, Darlinghurst, Australia, Faculty of Medicine, University of New South Wales, Kensington, Australia

  • Edwin P. Kirk ,

    Contributed equally to this work with: Mahdi Moradi Marjaneh, Edwin P. Kirk, Maximilian G. Posch

    Affiliations Faculty of Medicine, University of New South Wales, Kensington, Australia, Department of Medical Genetics, Sydney Children's Hospital, Randwick, Australia

  • Maximilian G. Posch ,

    Contributed equally to this work with: Mahdi Moradi Marjaneh, Edwin P. Kirk, Maximilian G. Posch

    Affiliations The Experimental and Clinical Research Center (ECRC), Charité – Universitätsmedizin, Berlin, Germany, Deutsches Herzzentrum Berlin, Berlin, Germany

  • Cemil Ozcelik,

    Affiliation The Experimental and Clinical Research Center (ECRC), Charité – Universitätsmedizin, Berlin, Germany

  • Felix Berger,

    Affiliation Deutsches Herzzentrum Berlin, Berlin, Germany

  • Roland Hetzer,

    Affiliation Deutsches Herzzentrum Berlin, Berlin, Germany

  • Robyn Otway,

    Affiliation The Victor Chang Cardiac Research Institute, Darlinghurst, Australia

  • Tanya L. Butler,

    Affiliations Heart Centre for Children, The Children's Hospital at Westmead, Westmead, Australia, Sydney Medical School, University of Sydney, Sydney, Australia

  • Gillian M. Blue,

    Affiliation Heart Centre for Children, The Children's Hospital at Westmead, Westmead, Australia

  • Lyn R. Griffiths,

    Affiliation Genomics Research Centre, Griffith University, Gold Coast, Australia

  • Diane Fatkin,

    Affiliations The Victor Chang Cardiac Research Institute, Darlinghurst, Australia, Faculty of Medicine, University of New South Wales, Kensington, Australia, Cardiology Department, St Vincent's Hospital, Darlinghurst, Australia

  • Jeremy J. Martinson,

    Affiliation University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America

  • David S. Winlaw,

    Affiliations Heart Centre for Children, The Children's Hospital at Westmead, Westmead, Australia, Sydney Medical School, University of Sydney, Sydney, Australia

  • Michael P. Feneley,

    Affiliations The Victor Chang Cardiac Research Institute, Darlinghurst, Australia, Faculty of Medicine, University of New South Wales, Kensington, Australia, Cardiology Department, St Vincent's Hospital, Darlinghurst, Australia

  • Richard P. Harvey

    r.harvey@victorchang.edu.au

    Affiliations The Victor Chang Cardiac Research Institute, Darlinghurst, Australia, Faculty of Medicine, University of New South Wales, Kensington, Australia

Investigation of Association between PFO Complicated by Cryptogenic Stroke and a Common Variant of the Cardiac Transcription Factor GATA4

  • Mahdi Moradi Marjaneh, 
  • Edwin P. Kirk, 
  • Maximilian G. Posch, 
  • Cemil Ozcelik, 
  • Felix Berger, 
  • Roland Hetzer, 
  • Robyn Otway, 
  • Tanya L. Butler, 
  • Gillian M. Blue, 
  • Lyn R. Griffiths
PLOS
x

Abstract

Patent foramen ovale (PFO) is associated with clinical conditions including cryptogenic stroke, migraine and varicose veins. Data from studies in humans and mouse suggest that PFO and the secundum form of atrial septal defect (ASDII) exist in an anatomical continuum of septal dysmorphogenesis with a common genetic basis. Mutations in multiple members of the evolutionarily conserved cardiac transcription factor network, including GATA4, cause or predispose to ASDII and PFO. Here, we assessed whether the most prevalent variant of the GATA4 gene, S377G, was significantly associated with PFO or ASD. Our analysis of world indigenous populations showed that GATA4 S377G was largely Caucasian-specific, and so subjects were restricted to those of Caucasian descent. To select for patients with larger PFO, we limited our analysis to those with cryptogenic stroke in which PFO was a subsequent finding. In an initial study of Australian subjects, we observed a weak association between GATA4 S377G and PFO/Stroke relative to Caucasian controls in whom ASD and PFO had been excluded (OR = 2.16; p = 0.02). However, in a follow up study of German Caucasians no association was found with either PFO or ASD. Analysis of combined Australian and German data confirmed the lack of a significant association. Thus, the common GATA4 variant S377G is likely to be relatively benign in terms of its participation in CHD and PFO/Stroke.

Introduction

Stroke is the third leading cause of death [1] and the major cause of serious, long-standing disability in the United States [2] affecting about 800,000 individuals each year [3]. Among cases with ischemic stroke, 18–42% have no identifiable cause and are termed cryptogenic [4][7]. A well-recognised risk factor for cryptogenic stroke, particularly in young patients, is patent foramen ovale (PFO) [8], a common cardiac atrial septal variant in which the septum primum fails to fuse with the septum secundum after birth, leading to the potential for inter-atrial communication. Approximately 50% of stroke patients younger than 55 years have PFO [9][12], which is at least double the prevalence in the general population [13], [14]. A likely mechanism for involvement of PFO in stroke is “paradoxical embolism”, in which a venous thrombus crosses the atrial septum and enters the systemic circulation, although an abnormal septum may also be thrombogenic [8]. The severity of PFO in the normal population is variable, ranging from small pinhole openings to large tunnel-shaped corridors, and only ∼1% of PFO show an inter-atrial communication of ≥10 mm [14]. However, PFO in patients with stroke are generally large (>2 mm) [15], [16] and often associated with increased mobility or aneurysm of the fossa ovalis membrane, the remnant of embryonic septum primum [17], [18].

PFO is also associated with other conditions. A number of studies show a higher prevalence of PFO in migraineurs with aura [19], [20], [21] and a dramatic lessening of migraine attacks after PFO closure [22], [23], suggesting that PFO acts as a risk factor for migraine. However, not all studies found such an association [24]. Neurological decompression sickness is also more common in divers with PFO (and ASD) [25] and a recent study found that the prevalence of PFO was higher in patients with symptomatic varicose veins due to great saphenous incompetence [26].

There is no conclusive data on the underlying causes of PFO, although there is good evidence that genetic factors are involved [27][31]. The newly reported association between PFO and varicose veins [26], a common and highly hereditary vascular abnormality [32], also indicates genetic contribution to PFO.

ASD is a more severe atrial septal abnormality than PFO [33]. In the secundum form of ASD (ASDII), there is incomplete coverage of the fossa ovalis by the atrial septum primum, leading to persistent shunting of blood from left to right atrium. ASDII accounts for approximately 7% of all cases of congenital heart defect (CHD) [34], [35].

A link between ASD and PFO has not been formally established in humans, although the largest and most complex PFO can be difficult to distinguish from ASD and are often regarded as form fruste ASD. This relationship is also strongly suggested by family studies, including those bearing mutations in cardiac transcription factor genes. Mutations in TBX20, for example, are associated with defects in septation including ASD and PFO with permanent left-to-right shunt [27]. Heterozygous TBX20 mutant mice show an increased background prevalence of PFO and septal dysmorphogenesis, as well as a genetic predisposition to ASD [30]. Mutations in the homeodomain factor NKX2-5, a key transcriptional regulator of cardiac development, cause familial ASD in humans, and in mice lead to a high prevalence of PFO correlating with the severe end of a spectrum of atrial septal dysmorphogenesis [31], [36][42]. The prevalence of PFO in mice is also highly genetic background-dependent. Collectively, these data suggest that ASD and PFO exist in an anatomical continuum with a common genetic basis. Furthermore, the genetic underpinnings of PFO are likely to be highly heterogeneous. Indeed, analysis of quantitative trait loci (QTL) contributing to atrial septal defects or PFO in inbred strains of mice identified 13 significant or suggestive QTL [43].

GATA4 belongs to the conserved GATA family of zinc finger transcription factors which play an important role in mammalian cardiac lineage specification and morphogenesis [44]. Mutations in GATA4 are associated with CHD in humans, particularly ASDII [45]. Thus, it is apparent that perturbations in multiple members of the evolutionarily conserved cardiac transcription factor network cause or predispose to ASDII and PFO. The most prevalent variant of the GATA4 gene is an A>G non-synonymous transition at position c.1647 resulting in a serine to glycine change at amino acid 377 (GATA4 S377G) with a minor allele frequency of 0.11 [46]. Although this is a conservative amino acid change, the polymorphism is located within the C-terminal GATA4 domain which is highly conserved in vertebrate species and essential for GATA4 transcriptional activity [45], [47], [48]. In vitro functional analysis using the alpha myosin heavy chain promoter revealed that GATA4 S377G has a significantly enhanced transcriptional activity compared to wildtype GATA4 [49]. Although S377G showed no significant association with migraine [50], an association with CHD including ASD and PFO has not been characterized so far.

Here, we address whether there is a significant association between this GATA4 variant and ASD or PFO in patients unselected for family history. To focus on the more severe end of the PFO spectrum, PFO subjects comprised largely patients with cryptogenic stroke in whom PFO was subsequently found by echocardiography.

Materials and Methods

Global distribution of S377G

The distribution of S377G allele (dbSNP ref. 3729856) was determined in different indigenous populations [51] (Table 1) using Fluorescence Polarisation [52].

Ethics Statement

All human experiments in the Australian study were carried out under Human Research Ethics Committee approval from the South East Health Research Ethics Committee – Eastern Division, the St Vincent's Hospital Research Ethics Committee and the Children's Hospital at Westmead Research Ethics Committee.

The German study protocol was approved by the Institutional Review Board of the Charité-Universitätsmedizin Berlin, Germany.

Written informed consent was obtained from all participants (or parental/legal guardian on behalf of the children participating in the study).

Australian Study

Subjects were recruited from Sydney hospitals from 1982 to 2004 and those of Caucasian descent were selected for the study. Adult ASD and CHD patients were recruited mostly from St. Vincent's Hospital and St. Vincent's Private Hospital (SVHs), while children were from the Sydney Children's Hospital (SCH) and the Children's Hospital at Westmead (CHW). Most were recruited prospectively during outpatient services and were unselected for family history or particular cardiac anomalies. PFO and Stroke patients were recruited prospectively from St Vincent's Hospital and St Vincent's Private Hospital after referral to echocardiography services for a variety of indications including stroke or transient ischemic episode (TIA). Allocation to case (PFO with cryptogenic stroke; n = 58) and control (PFO without stroke/TIA, n = 29; stroke/TIA without PFO/ASD, n = 66) groups occurred retrospectively. Cases consisted of 58 individuals under investigation for cryptogenic stroke in whom PFO was demonstrated by transesophageal echocardiography (TEE). In control groups, PFO was either absent or an incidental finding, as also determined by TEE.

Clinical evaluation included medical history, 12-lead electrocardiography and transthoracic echocardiography and/or TEE with intravenous saline contrast injection during the strain and release phases of the Valsalva maneuvre. Two additional control groups were also assessed. TEE controls recruited from St. Vincent's Hospital (n = 113) had undergone TEE for a range of indications and were reported to have structurally normal hearts, in particular an intact atrial septum. The second group (population controls; n = 391) were Australian Caucasians in generally good health (no stroke or known heart disease) although unselected for septal status [50]. The PFO/Stroke group was restricted to patients with cryptogenic stroke. Since atrial fibrillation (AF) is a known cause of stroke, all stroke patients had Holter monitor evaluation unless there was prior evidence in their medical history for AF. We excluded all patients with AF from the PFO/Stroke group. Stroke/no PFO/ASD were also evaluated by Holter monitor as part of their clinical evaluation, although those with AF were included in the study.

Initially, GATA4 coding exons were amplified by polymerase chain reaction (PCR) from 100 ng of leukocyte DNA, purified with PCR Cleanup Plates (Millipore) and sequenced using Big Dye Terminator v3.1 kit (Applied Biosystems) and ABI PRISM® 3700 DNA Analyser. Subsequently, GATA4 S377 status was confirmed in all samples, including population and TEE controls, using a commercial SNP analysis platform (Genera Biosystems). Chi-square or the Fisher's exact test was used to detect differences between allele frequencies.

German study

A vast majority of individuals were of Caucasian origin and recruited from Berlin hospitals between 2005 and 2007. There were three Arab patients who were included in the study since the allele frequency of S377G is similar in both Arab and Caucasian populations (Table 1). Ninety-six and 95 patients had isolated ASDII and isolated PFO, respectively. ASDII probands represent a subgroup of a previously published CHD cohort included in genetic screening [53]. Most PFO probands (91%) had been admitted for interventional PFO closure after experiencing one or more thromboembolic events. All 191 cases were recruited at the Department of Pediatric Cardiology, Deutsches Herzzentrum Berlin, and Adult Cardiology Department at the Charité - Universitätsmedizin Berlin, Campus Virchow (CVK).

All patients were characterized by physical examination, 12-lead electrocardiography and TEE. Ninety-six patients attending the Department of Cardiology CVK were used as controls after exclusion of ASD and PFO by contrast-enhanced TEE after Valsalva maneuvre. All ASDII and PFO patients underwent 24 hour Holter monitor. AF was found in none of the PFO patients and all were designated as having cryptogenic stroke.

PCR fragments including the S377G variation were analysed by single stranded conformational polymorphism (SSCP). Differential migration patterns allowed a secure distinction of all three possible genotypes (AA, AG, GG) on 12% Polyacrylamide gels at 20°C. All three genotypes were confirmed in 12 samples by direct DNA sequencing. Discrete variables were expressed as counts or percentages and compared using chi-square test without Yates correction. Fisher's exact test was employed for association of counts of which one subgroup was smaller than four. Continuous variables were checked for Gaussian distribution and expressed as mean value ± standard deviation and were compared by use of unpaired, two sided t-test.

Results

Global distribution of GATA4 S377G

The global distribution of GATA4 S377G was determined by analysis of DNA from world indigenous populations [51]. European, Australian, American and Middle Eastern Caucasians had the highest allele frequency (11.3–20.2%), while Indians/Pakistanis had a lower frequency (7.7%) (Table 1). By contrast, Africans, East Asians and Pacific Islanders showed a very low frequency (0–0.4%). Allele frequency for all populations was in Hardy-Weinberg equilibrium. These data suggest a relatively recent and Caucasian origin for S377G, potentially in a single individual among Neolithic farming populations from the Near East before their expansion into Europe within the last 10,000 years [54]. Further comparisons of S377G frequency were restricted to Caucasian subjects.

Australian study

In Table 2, baseline demographics and relevant clinical data are tabulated. As ASD and PFO likely exist in an anatomical continuum with a common genetic basis, we also included ASD subjects (n = 129) which were a mixture of children and adults (mean age 21.6 yrs) and the majority had pure ASDII. Other CHD subjects (n = 109) which served as controls, were mostly children (mean age 3.9 yrs) and the majority had isolated ventricular septal defect (VSD), VSD with minor abnormalities, VSD with outflow tract defects including tetralogy of Fallot, pulmonary atresia, transpositions or double outlet right ventricle (DORV). Both ASD and Other CHD subjects were unselected for family history, although 26.6% and 11%, respectively, had a known family history of CHD.

Within PFO/stroke cases (n = 58), the mean age was 51.3 yrs, some 12–14 yr younger than in our three adult control groups in which septal status was known (PFO without Stroke, Stroke without PFO/ASD, and TEE Controls), suggesting over-representation of patients with premature stroke. No patient had severe atheroma or AF. Only a minority (10.7%) had a family history of CHD. No significant gender bias was evident in either cases or controls.

In all study populations, GATA4 S377G allele distribution was in Hardy-Weinberg equilibrium. Prevalence of heterozygosity and allele frequency varied somewhat, although not significantly, between case and control groups (Table 3). In healthy Population Controls, which were unselected for atrial septal status (n = 391), the allele frequency was 12.4%, in good agreement with other world Caucasian populations (Table 1). The frequency was not significantly different in other CHD subjects, those with PFO without Stroke or Stroke without PFO/ASD. However, allele frequency in our gold standard TEE Controls who did not have stroke and in whom ASD and PFO had been specifically excluded (n = 113), was lower at 9.3%. Although this fell short of significance when compared to Population Controls (p = 0.20), it would be the expected trend if S377G played a causative role in PFO at the severe end of the spectrum.

Allele frequency was also low in ASD subjects (9.3%), indicating no role for S377G in causation of ASDII. The differences between the ASD group and controls were not statistically significant. However, an excess of S377G was observed in cases with PFO and stroke, with an allele frequency of 18.1% (p = 0.02 comparing to the TEE control group).

German study

We also assessed S377G allele frequency independently in German Caucasians with ASD and PFO compared to the TEE controls. Basic demographics and clinical information from ASD and PFO subjects are shown in Table 4. The ASD group (n = 96) comprised children and adults (aged 2.9 yrs to 75.9 yrs) who were mostly female (70.5%). This female preponderance of ASD is consistent with a previous epidemiologic report [55]. PFO probands (n = 95) were all adults (mean age 52.9) who mostly had been admitted subsequent to neurologic events (91.2%). During TEE, atrial septal aneurysm (ASA) was detected in a relatively high number of ASDs and PFOs (37.4% and 40.7% respectively). It is known that ASA is often associated with other atrial septal defects, in particular ASDII and PFO, and is also a risk factor for stroke [56], [57], [58]. More than two thirds of ASD subjects had an ECG pattern of right bundle branch block (RBBB) and about 14% had atrioventricular block (AVB). In the PFO group, RBB was found in just 20% and one patient had AVB.

In the German Caucasians, the S377G allele frequency was not significantly different between ASDs, PFOs and TEE controls (range 12% to 13.5%), and it was close to the allele frequency of other world Caucasian populations (Table 5).

Pooled data

We continued recruitment of Australian Caucasians in the same St. Vincent's Hospital treatment population using identical inclusion criteria and patient classification as in the original study, but over two years were only able to recruit 32 new cases of PFO with neurologic event. Among them, only 8 had undergone PFO closure. This low number may be because of depletion of adults with severe PFO in the treatment population. However, in the same time frame, we collected an additional independent group of 134 TEE controls.

The power of both Australian and German studies was weakened by the low number of subjects. Given relative homogeneity of two studies, we pooled data from all subjects including the additional TEE controls, into one data set for analysis (Table 6). Subjects with PFO (n = 183) and ASD (n = 234) showed S377G allele frequencies of 13.9% and 10.7%, respectively, which were not significantly different compared to TEE controls (n = 340, S377G allele frequency = 11.9%).

Discussion

GATA transcription factors regulate several biological processes during embryogenesis including cardiac development. Of the six vertebrate GATA factors, GATA4, GATA5 and GATA6 are involved in cardiogenesis [59][62]. Several mutations in GATA4 have been previously described in patients with CHD, including 677delC and Arg283His which were reported in patients with complex heart malformations encompassing ASD, VSD, common atrioventricular canal, and PFO [63]. S377G is located within the conserved and essential C-terminal GATA4 domain and leads to an increase in transcriptional activity of GATA4 in vitro [49]. Given the causal role of GATA4 in CHD, this common allele may play a modifier role.

We initially screened for GATA4 S377G in three different sets of CHD patients including those with ASD, PFO and other forms of CHD, all of Australian Caucasian origin. We observed high allele frequency of S377G in subjects with PFO and stroke (18%) which was significantly higher than in TEE controls in whom ASD and PFO were excluded (p = 0.02). This finding suggested a role for S377G in the etiology of PFO complicated by stroke. However, several observations make this result less compelling. Around one-fourth of Caucasians unselected for septal status would be expected to have PFO [14]. If S377G was involved in causation of PFO we would expect its frequency in Population Controls to be higher than that in TEE Controls. Indeed, this was the case (12.4% vs 9.3%). However the difference was not statistically significant (p = 0.20). Furthermore, the difference in S377G frequency between patients with PFO and stroke, and population controls (18.1% vs 12.4%) was non-significant.

It is possible that an increased frequency of S377G in the patients with PFO and stroke is due to an independent effect of this variant on stroke risk. For example, GATA4 represses the transcriptional activity of apolipoprotein(a) gene [64], high levels of which are an independent risk factor for premature atherosclerosis and stroke [65], [66]. However, a direct association between S377G and stroke is unlikely because the allele frequency of S377G in the “stroke/no PFO/ASD” group (12.1%) was not different from that of TEE or population controls.

There was no association between S377G and PFO complicated by cryptogenic stroke when we analysed an independent group of German Caucasians including 95 subjects with PFO, most of whom had experienced a neurologic event. The S377G allele frequency was not significantly different in any comparison between ASDs, PFOs and TEE controls. Analysis of combined Australian and German data confirmed the lack of association.

In our initial screen of 162 subjects (including 43 PFO/stroke, 9 incidental PFO with no stroke and 110 ASD subjects), all coding exons of the GATA4 gene were sequenced. No GATA4 variants were identified in the PFO/stroke cohort. However, we found 4 non-synonymous variants, 3 in the ASD group (full gene deletion; E359del; A411V) and one in an incidental PFO subject (D425N). These changes were not detected by subsequent screening in 270 controls. A411V and D425N have been reported in a separate publication describing a larger screen [67]. While potentially deleterious, there were no obvious functional consequences of these mutations in transient transfection assays.

In summary, the study of independent and pooled populations of Australian and German Caucasians failed to show a significant association between S377G and PFO with stroke. We conclude that the common GATA4 variant S377G is relatively benign in terms of its participation in CHD and PFO/Stroke. However, it remains possible that common mutations in other cardiac transcription factors play a significant role in the pathogenesis of cryptogenic stroke through causation of PFO.

Acknowledgments

We would like to thank Haley Crotty, Olivia Baddeley, Janan Fornusek, and Katharina Schmitt for clinical data collection and Leticia Castro and Andreas Perrot for laboratory assistance.

Author Contributions

Conceived and designed the experiments: MMM EPK MGP LRG DF JJM DSW MPF RPH. Analyzed the data: MMM EPK MGP CO. Wrote the paper: MMM EPK MGP RPH. Critique of paper: CO FB RH RO TLB GMB LRG DF JJM DSW MPF. Collected data: MMM EPK MGP RO TLB GMB JJM. Research director: CO FB RH. Raised funding: CO FB RH RPH. Patient recruitment: FB LRG DF DSW MPF. Project coordinator: DSW MPH RPH. Clinical coordinator: TLB GMB.

References

  1. 1. Centers for Disease Control and Prevention (2007) WISQARS Leading Causes of Death Reports, 1999–2006. Centers for Disease Control and Prevention2007WISQARS Leading Causes of Death Reports, 1999–2006.Available: http://webappa.cdc.gov/sasweb/ncipc/leadcaus10.html via the Internet. Accessed 27 April 2010. Available: http://webappa.cdc.gov/sasweb/ncipc/leadcaus10.html via the Internet. Accessed 27 April 2010.
  2. 2. Centers for Disease Control and Prevention (2001) Morbidity and Mortality Weekly Report (MMWR), 2001. 50: 120–125.Centers for Disease Control and Prevention2001Morbidity and Mortality Weekly Report (MMWR), 2001.50120125
  3. 3. Lloyd-Jones D, Adams RJ, Brown TM, Carnethon M, Dai S, et al. (2010) Executive summary: heart disease and stroke statistics–2010 update: a report from the American Heart Association. Circulation 121(7): 948–954.D. Lloyd-JonesRJ AdamsTM BrownM. CarnethonS. Dai2010Executive summary: heart disease and stroke statistics–2010 update: a report from the American Heart Association.Circulation1217948954
  4. 4. Homma S, Sacco RL, Di Tullio MR, Sciacca RR, Mohr JP (2002) Effect of medical treatment in stroke patients with patent foramen ovale. Circulation 105: 2625–2631.S. HommaRL SaccoMR Di TullioRR SciaccaJP Mohr2002Effect of medical treatment in stroke patients with patent foramen ovale.Circulation10526252631
  5. 5. Musolino R, La Spina P, Granata A, Gallitto G, Leggiadro N, et al. (2003) Ischaemic stroke in young people: a prospective and long-term follow-up study. Cerebrovasc Dis 15(1–2): 121–128.R. MusolinoP. La SpinaA. GranataG. GallittoN. Leggiadro2003Ischaemic stroke in young people: a prospective and long-term follow-up study.Cerebrovasc Dis151–2121128
  6. 6. Bang OY, Lee PH, Joo SY, Lee JS, Joo IS, et al. (2003) Frequency and mechanism of stroke recurrence after cryptogenic stroke. Ann Neurol 54: 227–234.OY BangPH LeeSY JooJS LeeIS Joo2003Frequency and mechanism of stroke recurrence after cryptogenic stroke.Ann Neurol54227234
  7. 7. Sacco RL, Ellenberg JH, Mohr JP, Tatemichi TK, Hier DB, et al. (1989) Infarcts of undetermined cause: the NINCDS Stroke Data Bank. Ann Neurol 25(4): 382–390.RL SaccoJH EllenbergJP MohrTK TatemichiDB Hier1989Infarcts of undetermined cause: the NINCDS Stroke Data Bank.Ann Neurol254382390
  8. 8. Di Tullio MR, Homma S (2002) Mechanisms of cardioembolic stroke. Curr Cardiol Rep 4: 141–148.MR Di TullioS. Homma2002Mechanisms of cardioembolic stroke.Curr Cardiol Rep4141148
  9. 9. Lechat P, Mas JL, Lascault G, Loron P, Theard M, et al. (1988) Prevalence of patent foramen ovale in patients with stroke. N Engl J Med 318(18): 1148–1152.P. LechatJL MasG. LascaultP. LoronM. Theard1988Prevalence of patent foramen ovale in patients with stroke.N Engl J Med3181811481152
  10. 10. Di Tullio M, Sacco RL, Gopal A, Mohr JP, Homma S (1992) Patent foramen ovale as a risk factor for cryptogenic stroke. Ann Intern Med 117(6): 461–465.M. Di TullioRL SaccoA. GopalJP MohrS. Homma1992Patent foramen ovale as a risk factor for cryptogenic stroke.Ann Intern Med1176461465
  11. 11. Ranoux D, Cohen A, Cabanes L, Amarenco P, Bousser MG, et al. (1993) Patent foramen ovale: is stroke due to paradoxical embolism? Stroke 24(1): 31–34.D. RanouxA. CohenL. CabanesP. AmarencoMG Bousser1993Patent foramen ovale: is stroke due to paradoxical embolism?Stroke2413134
  12. 12. Lamy C, Giannesini C, Zuber M, Arquizan C, Meder JF, et al. (2002) Clinical and imaging findings in cryptogenic stroke patients with and without patent foramen ovale: the PFO-ASA Study. Atrial Septal Aneurysm. Stroke 33(3): 706–711.C. LamyC. GiannesiniM. ZuberC. ArquizanJF Meder2002Clinical and imaging findings in cryptogenic stroke patients with and without patent foramen ovale: the PFO-ASA Study. Atrial Septal Aneurysm.Stroke333706711
  13. 13. Schneider B, Zienkiewicz T, Jansen V, Hofmann T, Noltenius H, et al. (1996) Diagnosis of patent foramen ovale by transesophageal echocardiography and correlation with autopsy findings. Am J Cardiol 77: 1202–1209.B. SchneiderT. ZienkiewiczV. JansenT. HofmannH. Noltenius1996Diagnosis of patent foramen ovale by transesophageal echocardiography and correlation with autopsy findings.Am J Cardiol7712021209
  14. 14. Hagen PT, Scholz DG, Edwards WD (1984) Incidence and size of patent foramen ovale during the first 10 decades: An autopsy study of 965 normal hearts. Mayo Clin Proc 59: 17–20.PT HagenDG ScholzWD Edwards1984Incidence and size of patent foramen ovale during the first 10 decades: An autopsy study of 965 normal hearts.Mayo Clin Proc591720
  15. 15. Bridges ND, Hellenbrand W, Latson L, Filiano J, Newburger JW, et al. (1992) Transcatheter closure of patent foramen ovale after presumed paradoxical embolism. Circulation 86: 1902–1908.ND BridgesW. HellenbrandL. LatsonJ. FilianoJW Newburger1992Transcatheter closure of patent foramen ovale after presumed paradoxical embolism.Circulation8619021908
  16. 16. Schuchlenz HW, Weihs W, Horner S, Quehenberger F (2000) The association between the diameter of a patent foramen ovale and the risk of embolic cerebrovascular events. Am J Med 109: 456–462.HW SchuchlenzW. WeihsS. HornerF. Quehenberger2000The association between the diameter of a patent foramen ovale and the risk of embolic cerebrovascular events.Am J Med109456462
  17. 17. De Castro S, Cartoni D, Fiorelli M, Rasura M, Anzini A, et al. (2000) Morphological and functional characteristics of patent foramen ovale and their embolic implications. Stroke 31(10): 2407–2413.S. De CastroD. CartoniM. FiorelliM. RasuraA. Anzini2000Morphological and functional characteristics of patent foramen ovale and their embolic implications.Stroke311024072413
  18. 18. Aslam F, Shirani J, Haque AA (2006) Patent foramen ovale: assessment, clinical significance and therapeutic options. South Med J 99(12): 1367–1372.F. AslamJ. ShiraniAA Haque2006Patent foramen ovale: assessment, clinical significance and therapeutic options.South Med J991213671372
  19. 19. Ferrarini G, Malferrari G, Zucco R, Gaddi O, Norina M, et al. (2005) High prevalence of patent foramen ovale in migraine with aura. J Headache Pain 6(2): 71–76.G. FerrariniG. MalferrariR. ZuccoO. GaddiM. Norina2005High prevalence of patent foramen ovale in migraine with aura.J Headache Pain627176
  20. 20. Dalla VG, Guindani M, Zavarise P, Griffini S, Pezzini A, et al. (2005) Prevalence of patent foramen ovale in a large series of patients with migraine with aura, migraine without aura and cluster headache, and relationship with clinical phenotype. J Headache Pain 6: 328–330.VG DallaM. GuindaniP. ZavariseS. GriffiniA. Pezzini2005Prevalence of patent foramen ovale in a large series of patients with migraine with aura, migraine without aura and cluster headache, and relationship with clinical phenotype.J Headache Pain6328330
  21. 21. Anzola GP, Magoni M, Guindani M, Rozzini L, Dalla VG (1999) Potential source of cerebral embolism in migraine with aura: A transcranial Doppler study. Neurology 52: 1622–1625.GP AnzolaM. MagoniM. GuindaniL. RozziniVG Dalla1999Potential source of cerebral embolism in migraine with aura: A transcranial Doppler study.Neurology5216221625
  22. 22. Onorato E, Melzi G, Casilli F, Pedon L, Rigatelli G, et al. (2003) Patent foramen ovale with paradoxical embolism: midterm results of transcatheter closure in 256 patients. J Interv Cardiol 16: 43–50.E. OnoratoG. MelziF. CasilliL. PedonG. Rigatelli2003Patent foramen ovale with paradoxical embolism: midterm results of transcatheter closure in 256 patients.J Interv Cardiol164350
  23. 23. Schwerzmann M, Wiher S, Nedeltchev K, Mattle HP, Wahl A, et al. (2004) Percutaneous closure of patent foramen ovale reduces the frequency of migraine attacks. Neurology 62(8): 1399–1401.M. SchwerzmannS. WiherK. NedeltchevHP MattleA. Wahl2004Percutaneous closure of patent foramen ovale reduces the frequency of migraine attacks.Neurology62813991401
  24. 24. Garg P, Servoss SJ, Wu JC, Bajwa ZH, Selim MH, et al. (2010) Lack of association between migraine headache and patent foramen ovale: results of a case-control study. Circulation 121(12): 1406–1412.P. GargSJ ServossJC WuZH BajwaMH Selim2010Lack of association between migraine headache and patent foramen ovale: results of a case-control study.Circulation1211214061412
  25. 25. Schwerzmann M, Seiler C, Lipp E, Guzman R, Lövblad KO, et al. (2001) Relation between directly detected patent foramen ovale and ischemic brain lesions in sport divers. Ann Intern Med 134(1): 21–24.M. SchwerzmannC. SeilerE. LippR. GuzmanKO Lövblad2001Relation between directly detected patent foramen ovale and ischemic brain lesions in sport divers.Ann Intern Med13412124
  26. 26. Wright DD, Gibson KD, Barclay J, Razumovsky A, Rush J, et al. (2010) High prevalence of right-to-left shunt in patients with symptomatic great saphenous incompetence and varicose veins. J Vasc Surg 51(1): 104–107.DD WrightKD GibsonJ. BarclayA. RazumovskyJ. Rush2010High prevalence of right-to-left shunt in patients with symptomatic great saphenous incompetence and varicose veins.J Vasc Surg511104107
  27. 27. Kirk EP, Sunde M, Costa MW, Rankin SA, Wolstein O, et al. (2007) Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy. Am J Hum Genet 81(2): 280–291.EP KirkM. SundeMW CostaSA RankinO. Wolstein2007Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy.Am J Hum Genet812280291
  28. 28. Wilmshurst PT, Pearson MJ, Nightingale S, Walsh KP, Morrison WL (2004) Inheritance of persistent foramen ovale and atrial septal defects and the relation to familial migraine with aura. Heart 90: 1315–1320.PT WilmshurstMJ PearsonS. NightingaleKP WalshWL Morrison2004Inheritance of persistent foramen ovale and atrial septal defects and the relation to familial migraine with aura.Heart9013151320
  29. 29. Arquizan C, Coste J, Touboul PJ, Mas JL (2001) Is patent foramen ovale a family trait? A transcranial Doppler sonographic study. Stroke 32: 1563–1566.C. ArquizanJ. CostePJ TouboulJL Mas2001Is patent foramen ovale a family trait? A transcranial Doppler sonographic study.Stroke3215631566
  30. 30. Stennard FA, Costa MW, Lai D, Biben C, Furtado MB, et al. (2005) Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development 132(10): 2451–2462.FA StennardMW CostaD. LaiC. BibenMB Furtado2005Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.Development1321024512462
  31. 31. Biben C, Weber R, Kesteven S, Stanley E, McDonald L, et al. (2000) Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene Nkx2-5. Circ Res 87(10): 888–895.C. BibenR. WeberS. KestevenE. StanleyL. McDonald2000Cardiac septal and valvular dysmorphogenesis in mice heterozygous for mutations in the homeobox gene Nkx2-5.Circ Res8710888895
  32. 32. Ng MYM, Andrew T, Spector TD, Jeffery S (2005) Linkage to the FOXC2 region of chromosome 16 for varicose veins in the otherwise healthy, unselected sibling pairs. J Med Genet 42: 235–239.MYM NgT. AndrewTD SpectorS. Jeffery2005Linkage to the FOXC2 region of chromosome 16 for varicose veins in the otherwise healthy, unselected sibling pairs.J Med Genet42235239
  33. 33. Brickner ME, Hillis D, Lange RA (2000) Medical progress: Congenital heart disease in adults (first of two parts). N Engl J Med 342: 256–263.ME BricknerD. HillisRA Lange2000Medical progress: Congenital heart disease in adults (first of two parts).N Engl J Med342256263
  34. 34. McMahon CJ, Feltes TF, Fraley JK, Bricker JT, Grifka RG, et al. (2002) Natural history of growth of secundum atrial septal defects and implications for transcatheter closure. Heart 87(3): 256–259.CJ McMahonTF FeltesJK FraleyJT BrickerRG Grifka2002Natural history of growth of secundum atrial septal defects and implications for transcatheter closure.Heart873256259
  35. 35. Balzer DT (2004) Transcatheter Closure of Intracardiac Shunts. Curr Treat Options Cardiovasc Med 6(5): 417–422.DT Balzer2004Transcatheter Closure of Intracardiac Shunts.Curr Treat Options Cardiovasc Med65417422
  36. 36. McElhinney , Geiger E, Blinder J, Benson DW, Goldmutntz E (2003) NKX2.5 mutations in patients with congenital heart diseases. J Am Coll Cardiol 42: 1650–1655.McElhinneyE. GeigerJ. BlinderDW BensonE. Goldmutntz2003NKX2.5 mutations in patients with congenital heart diseases.J Am Coll Cardiol4216501655
  37. 37. Kasahara H, Benson DW (2004) Biochemical analyses of eight NKX2-5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies. Cardiovasc Res 64: 40–51.H. KasaharaDW Benson2004Biochemical analyses of eight NKX2-5 homeodomain missense mutations causing atrioventricular block and cardiac anomalies.Cardiovasc Res644051
  38. 38. Reamon-Buettner SM, Hecker H, Spanel-Borowski K, Craatz S, Kuenzel E, et al. (2004) Novel NKX2-5 mutations in diseased heart tissues of patients with cardiac malformations. Am J Pathol 164: 2117–2125.SM Reamon-BuettnerH. HeckerK. Spanel-BorowskiS. CraatzE. Kuenzel2004Novel NKX2-5 mutations in diseased heart tissues of patients with cardiac malformations.Am J Pathol16421172125
  39. 39. Hirayama-Yamada K, Kamisago M, Akimoto K, Aotsuka H, Nakamura Y, et al. (2005) Phenotypes with GATA4 or NKX2.5 mutations in familial atrial septal defect. Am J Med Genet A 135: 47–52.K. Hirayama-YamadaM. KamisagoK. AkimotoH. AotsukaY. Nakamura2005Phenotypes with GATA4 or NKX2.5 mutations in familial atrial septal defect.Am J Med Genet A1354752
  40. 40. Sarkozy A, Conti E, Neri C, D'Agostino R, Digilio MC, et al. (2005) Spectrum of atrial septal defects associated with mutations of NKX2.5 and GATA4 transcription factors. J Med Genet 42(2): e16.A. SarkozyE. ContiC. NeriR. D'AgostinoMC Digilio2005Spectrum of atrial septal defects associated with mutations of NKX2.5 and GATA4 transcription factors.J Med Genet422e16
  41. 41. Schott JJ, Benson DW, Basson CT, Pease W, Silberbach GM, et al. (1998) Congenital heart disease caused by mutations in the transcription factor NKX2-5. Science 281: 108–111.JJ SchottDW BensonCT BassonW. PeaseGM Silberbach1998Congenital heart disease caused by mutations in the transcription factor NKX2-5.Science281108111
  42. 42. Srivastava D, Olson EN (2000) A genetic blueprint for cardiac development. Nature 407: 221–226.D. SrivastavaEN Olson2000A genetic blueprint for cardiac development.Nature407221226
  43. 43. Kirk EP, Hyun C, Thomson PC, Lai D, Castro ML, et al. (2006) Quantitative trait loci modifying cardiac atrial septal morphology and risk of patent foramen ovale in the mouse. Circ Res 98(5): 651–658.EP KirkC. HyunPC ThomsonD. LaiML Castro2006Quantitative trait loci modifying cardiac atrial septal morphology and risk of patent foramen ovale in the mouse.Circ Res985651658
  44. 44. Crispino JD, Lodish MB, Thurberg BL, Litovsky SH, Collins T, et al. (2001) Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors. Genes Dev 15(7): 839–844.JD CrispinoMB LodishBL ThurbergSH LitovskyT. Collins2001Proper coronary vascular development and heart morphogenesis depend on interaction of GATA-4 with FOG cofactors.Genes Dev157839844
  45. 45. Garg V, Kathiriya IS, Barnes R, Schluterman MK, King IN, et al. (2003) GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424: 443–447.V. GargIS KathiriyaR. BarnesMK SchlutermanIN King2003GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5.Nature424443447
  46. 46. Poirier O, Nicaud V, McDonagh T, Dargie HJ, Desnos M, et al. (2003) Polymorphisms of genes of the cardiac calcineurin pathway and cardiac hypertrophy. Eur J Hum Genet 11(9): 659–664.O. PoirierV. NicaudT. McDonaghHJ DargieM. Desnos2003Polymorphisms of genes of the cardiac calcineurin pathway and cardiac hypertrophy.Eur J Hum Genet119659664
  47. 47. Charron F, Tsimiklis G, Arcand M, Robitaille L, Liang Q, et al. (2001) Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA. Genes and Development 15: 2702–2719.F. CharronG. TsimiklisM. ArcandL. RobitailleQ. Liang2001Tissue-specific GATA factors are transcriptional effectors of the small GTPase RhoA.Genes and Development1527022719
  48. 48. Morrisey EE, Ip HS, Tang Z, Parmacek MS (1997) GATA-4 activates transcription via two novel domains that are conserved within the GATA-4/5/6 subfamily. J Biol Chem 272: 8515–8524.EE MorriseyHS IpZ. TangMS Parmacek1997GATA-4 activates transcription via two novel domains that are conserved within the GATA-4/5/6 subfamily.J Biol Chem27285158524
  49. 49. Schluterman MK, Krysiak AE, Kathiriya IS, Abate N, Chandalia M, et al. (2007) Screening and biochemical analysis of GATA4 sequence variations identified in patients with congenital heart disease. Am J Med Genet A 143A(8): 817–823.MK SchlutermanAE KrysiakIS KathiriyaN. AbateM. Chandalia2007Screening and biochemical analysis of GATA4 sequence variations identified in patients with congenital heart disease.Am J Med Genet A143A8817823
  50. 50. Sherin C, Francesca F, Karl P, Brendan T, Ron H, et al. (2008) Investigation between the S377G3 GATA-4 polymorphism and migraine. Open Neurol J 2: 35–38.C. SherinF. FrancescaP. KarlT. BrendanH. Ron2008Investigation between the S377G3 GATA-4 polymorphism and migraine.Open Neurol J23538
  51. 51. Martinson JJ, Hong L, Karanicolas R, Moore JP, Kostrikis LG (2000) Global distribution of the CCR2-64I/CCR5-59653T HIV-1 disease-protective haplotype. AIDS 14(5): 483–489.JJ MartinsonL. HongR. KaranicolasJP MooreLG Kostrikis2000Global distribution of the CCR2-64I/CCR5-59653T HIV-1 disease-protective haplotype.AIDS145483489
  52. 52. Chen X, Levine L, Kwok PY (1999) Fluorescence polarization in homogeneous nucleic acid analysis. Genome Research 9: 492–498.X. ChenL. LevinePY Kwok1999Fluorescence polarization in homogeneous nucleic acid analysis.Genome Research9492498
  53. 53. Posch MG, Perrot A, Schmitt K, Mittelhaus S, Esenwein EM, et al. (2008) Mutations in GATA4, NKX2.5, CRELD1, and BMP4 are infrequently found in patients with congenital cardiac septal defects. Am J Med Genet A 146A(2): 251–253.MG PoschA. PerrotK. SchmittS. MittelhausEM Esenwein2008Mutations in GATA4, NKX2.5, CRELD1, and BMP4 are infrequently found in patients with congenital cardiac septal defects.Am J Med Genet A146A2251253
  54. 54. Cavalli-Sforza LL, Menozzi P, Piazza A (1994) The history and geography of human genes. Princeton, NJ: Princeton University Press. LL Cavalli-SforzaP. MenozziA. Piazza1994The history and geography of human genesPrinceton, NJPrinceton University Press
  55. 55. Feldt RH, Avasthey P, Yoshimasu F, Kurland LT, Titus JL (1971) Incidence of congenital heart disease in children born to residents of Olmsted County, Minnesota, 1950–1969. Mayo Clin Proc 46(12): 794–799.RH FeldtP. AvastheyF. YoshimasuLT KurlandJL Titus1971Incidence of congenital heart disease in children born to residents of Olmsted County, Minnesota, 1950–1969.Mayo Clin Proc4612794799
  56. 56. Hanley PC, Tajik AJ, Hynes JK, Edwards WD, Reeder GS, et al. (1985) Diagnosis and classification of atrial septal aneurysm by two-dimensional echocardiography: report of 80 consecutive cases. J Am Coll Cardiol 6: 1370–1382.PC HanleyAJ TajikJK HynesWD EdwardsGS Reeder1985Diagnosis and classification of atrial septal aneurysm by two-dimensional echocardiography: report of 80 consecutive cases.J Am Coll Cardiol613701382
  57. 57. Belkin RN, Waugh RA, Kisslo J (1986) Interatrial shunting in atrial septal aneurysm. Am J Cardiol 57: 310–312.RN BelkinRA WaughJ. Kisslo1986Interatrial shunting in atrial septal aneurysm.Am J Cardiol57310312
  58. 58. Zabalgoitia-Reyes M, Herrera C, Ghandi DK, Mehlman DJ, McPherson DD, et al. (1990) A possible mechanism for neurologic ischemic events in patients with atrial septal aneurysm. Am J Cardiol 66: 761–764.M. Zabalgoitia-ReyesC. HerreraDK GhandiDJ MehlmanDD McPherson1990A possible mechanism for neurologic ischemic events in patients with atrial septal aneurysm.Am J Cardiol66761764
  59. 59. Molkentin JD (2000) The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression. J Biol Chem 275(50): 38949–38952.JD Molkentin2000The zinc finger-containing transcription factors GATA-4, -5, and -6. Ubiquitously expressed regulators of tissue-specific gene expression.J Biol Chem275503894938952
  60. 60. Charron F, Nemer M (1999) GATA transcription factors and cardiac development. Semin Cell Dev Biol 10(1): 85–91.F. CharronM. Nemer1999GATA transcription factors and cardiac development.Semin Cell Dev Biol1018591
  61. 61. Laverriere AC, MacNeill C, Mueller C, Poelmann RE, Burch JB, et al. (1994) GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J Biol Chem 269(37): 23177–23184.AC LaverriereC. MacNeillC. MuellerRE PoelmannJB Burch1994GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut.J Biol Chem269372317723184
  62. 62. Peterkin T, Gibson A, Loose M, Patient R (2005) The roles of GATA-4, -5 and -6 in vertebrate heart development. Semin Cell Dev Biol 16(1): 83–94.T. PeterkinA. GibsonM. LooseR. Patient2005The roles of GATA-4, -5 and -6 in vertebrate heart development.Semin Cell Dev Biol1618394
  63. 63. Reamon-Buettner SM, Borlak J (2005) GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart. J Med Genet 42(5): e32.SM Reamon-BuettnerJ. Borlak2005GATA4 zinc finger mutations as a molecular rationale for septation defects of the human heart.J Med Genet425e32
  64. 64. Negi S, Singh SK, Pati N, Handa V, Chauhan R, et al. (2004) A proximal tissue-specific module and a distal negative regulatory module control apolipoprotein(a) gene transcription. Biochem J 379(Pt 1): 151–159.S. NegiSK SinghN. PatiV. HandaR. Chauhan2004A proximal tissue-specific module and a distal negative regulatory module control apolipoprotein(a) gene transcription.Biochem J379Pt 1151159
  65. 65. Marcovina SM, Koschinsky ML (1998) Lipoprotein(a) as a risk factor for coronary artery disease. Am J Cardiol 82(12A): 57U–66U.SM MarcovinaML Koschinsky1998Lipoprotein(a) as a risk factor for coronary artery disease.Am J Cardiol8212A57U66U
  66. 66. Lippi G, Guidi G (2003) Lipoprotein(a): an emerging cardiovascular risk factor. Crit Rev Clin Lab Sci 40(1): 1–42.G. LippiG. Guidi2003Lipoprotein(a): an emerging cardiovascular risk factor.Crit Rev Clin Lab Sci401142
  67. 67. Butler TL, Esposito G, Blue GM, Cole AD, Costa MW, et al. (2010) GATA4 mutations in 357 unrelated patients with congenital heart malformation. Genet Test Mol Biomarkers 14(6): 797–802.TL ButlerG. EspositoGM BlueAD ColeMW Costa2010GATA4 mutations in 357 unrelated patients with congenital heart malformation.Genet Test Mol Biomarkers146797802