doi: 10.1161/CIRCRESAHA.108.178079
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© 2008 American Heart Association, Inc.
Editorials |
Holt–Oram Syndrome and Atrial Fibrillation
Opening the (T)-Box
From the Center of Molecular Medicine (C.I.M.M.B.A.), University of Florence, Italy.
Correspondence to Elisabetta Cerbai, PhD, Center of Molecular Medicine, University of Florence, Viale G. Pieraccini 6, 50139 Firenze, Italy. E-mail elisabetta.cerbai{at}unifi.it
See related article, pages 1433–1442
Key Words: T-box genes • cardiac development • atrial fibrillation • Holt-Oram Syndrome
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Introduction |
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 Top Introduction T-Box Genes and Heart...Gene Dosage: Too Less...References |
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Cardiogenesis is a complex phenomenon: its success—and ultimately life births—depends on factors acting in a combinatorial or hierarchical fashion and turning on and off gene transcription. Actually, the incidence of cardiac defects at birth is relatively high (1% to 2%), and our comprehension of these phenomena very limited. Hence, the role of transcription factors in cardiac specification and maturation has claimed increasing attention in recent years, providing a complex and evolving picture of the molecular and cellular processes involved but leaving many questions unanswered. In this issue of Circulation Research, Postma et al1 add to a growing list of regulatory factors/functions a step forward in our understanding of cardiac morphogenesis and disclosing novel features of Tbx5.
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T-Box Genes and Heart Development |
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TopIntroductionT-Box Genes and Heart...Gene Dosage: Too Less...References |
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Tbx5 belongs to the T-box gene family; the first member was identified in 1927 by a genetist who selected a mouse strain with truncated tail carrying a heterozygous mutation in a locus called T (reviewed in2). More than 60 years later the gene was cloned and named Brachyury (short-tail in Greek),3 but its functional role remained obscure because the T-gene product lacked homology to any previously characterized protein. Between 1993 and 1997, it was described as a novel DNA-binding protein and the crystallographic structure of the domain, termed the T-box, revealed a new feature of protein-DNA interaction.4 In recent years, studies in transgenic mice lines and in patients carrying spontaneous mutations have demonstrated that T-box genes act as crucial regulators for the morphogenesis of a wide range of tissues and organs (skeletal or genital apparatus, cardiovascular or endocrine system) and, when defective, as major contributors to several human syndromes.
To set T-box genes in their functional scenario, it is worthwhile to recall that they have been detected from the oocyte to the adulthood and are expressed in each compartment of the heart and limbs at critical stages of development.5 By binding to palindromic DNA sequences termed TBE (T-box Binding Element), Tbx proteins can either activate or repress gene transcription in varied ways: via distinct DNA-binding domains (eg, Tbx20) or depending on the cellular environment (eg, Tbx2), either alone or acting synergistically with other transcription factors of the GATA and LIM protein family (eg, Tbx4 and Tbx5). Among the Tbx proteins expressed in the human and mouse embryonic heart (namely Tbx1-5, Tbx18, and Tbx20), the partnership between Tbx5 and the transcription factor Nkx2-5 is by far the best characterized and more pertinent to the present issue.
Mutations in Tbx5 cause Holt–Oram Syndrome (HOS), a rare dominant inherited disease characterized by upper limb and multiple heart defects including atrial and ventricular septal defects, tetralogy of Fallot, hypoplastic left heart, and conduction anomalies (OMIM number 142900). Whereas some patients display complete loss of function attributable to Tbx5 truncation mutations, others carry only point mutations. Protein analysis has shown that, even when the DNA-binding domain is intact, mutations in the Tbx5 sequence responsible for interaction with cardiac transcription factor Nkx2-5 or GATA4 are sufficient to cause disease.6 A similar pattern of cooperativity has been postulated for Tbx1 and Nkx2-5; Tbx1 mutations are associated with the DiGeorge Syndrome, another human congenital disease displaying heart malformations besides craniofacial and glandular abnormalities.7
The expression of Tbx proteins undergoes a precise temporal and spatial regulation in developing cardiac structure and overlapping expression of "activators" (A) or "repressors" (R) is suggestive of cooperative and competitive regulatory mechanisms on Tbx target gene(s). The Figure shows a schematic representation of the T-box family with emphasis on factors playing a key role in cardiac development and chamber specification. The most extensively characterized target gene in the heart is Nppa, coding for the atrial natriuretic peptide (ANP) precursor protein. Tbx5, Tbx2, Tbx3, and Tbx20 all potentially participate in the global regulation of Nppa expression, although with different actions (Figure). Overall, studies in mouse embryos reveal that each gene has unique developmental functions in cardiac lineage determination, chamber specification, formation of cardiac substructures such as valves, epicardium, septum, and conduction system. Among the other genes of the T-box family, brachyury is important in the initial mesoderm formation,8 as also shown in cardiac differentiation from human embryonic stem cells9 but not directly implicated in heart development.
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Gene Dosage: Too Less or Too Much |
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TopIntroductionT-Box Genes and Heart...Gene Dosage: Too Less...References |
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Most HOS patients carry mutations leading to protein truncation and—from a phenotypic point of view—to haploinsufficiency. Missense mutations, also extensively characterized, give rise to proteins with reduced ability to bind DNA or synergize with GATA4 or Nkx2-5.
All of the known autosomal human T-box gene syndromes but one (recessive isolated ACTH deficiency) occur in heterozygous individuals, presumably attributable to embryonic lethality in homozygotes. This may imply a gene dosage-related severity of the disease, although the genotype–phenotype relationship between Tbx5 mutations and cardiac disease in HOS patients is unclear, and posttranscriptional processes may also be involved.10 Notwithstanding the molecular mechanism, the mutations described so far are caused by protein loss-of-function.
What is the effect of Tbx upregulation on cardiac development and function? To date, what we have learned derives mostly from overexpression in chicken or in transgenic mouse models with cardiac-specific promoters. Over- or underexpression of Tbx1 gives mice with almost identical phenotypes11 alike, Tbx5 overexpression in mice inhibits ventricular chamber maturation.12 A human correlate, the chromosome 12q2 duplications resulting in increased Tbx5 dosage, has been reported to cause HOS, as observed in haploinsufficiency. Altogether, these studies suggest that a balanced level of Tbx5 protein is critical for its function, as under- and overexpression result in similar phenotypes: the concept of the right gene dosage, effectively synthesized by Hatcher and Basson.13
Given these premises, the article by Postma et al1 is somehow surprising. The starting point is an observation reported more than 10 years ago of a family with atypical HOS.14 The affected members had skeletal anomalies but only a few of them displayed cardiac malformation, and it was concluded that the phenotype did not satisfy the HOS features. Evidence of muscular-skeletal anomalies also in individuals previously assigned as not affected, led to renewed and thorough examination of the family, enlarged by newborns. Interestingly, most of them developed paroxysmal atrial fibrillation as early as 9 years old and in the absence of cardiac disease. From this observation, the authors develop an elegant approach to get insights into the mechanisms of such atypical association, by combining functional and molecular studies strengthened by the experience of years of excellence in the research field of cardiac morphogenesis. The mutation consists in a nucleotide change in a very conserved region of the gene leading to a Glycine for Arginine substitution at position 125 (p.G125R). The result, as shown by heterologous expression in HEK293 cells, is a protein maintaining the ability to bind DNA and Nkx2-5 similarly to the wild-type Tbx5. However, as for target gene transcription (Nppa, Cx40; see Figure), the mutant Tbx5 displayed increased activation of target promoters than the wild-type protein, suggesting that the mutation was associated with a gain-of-function. The next step was to verify the effect of mutant Tbx5, transfected in immortalized cardiac cell lines, over a wide range of proteins being somehow associated with atrial fibrillation. Besides Cx40 and Nppa, also Tbx3 and Kcnj2 (the gene coding for the inward rectifier channel, IK1) resulted to be overexpressed, whereas others (Hcn4, Snc5A, and Cx43) were unaffected.
Unfortunately, genetics of atrial fibrillation is too initial to speculate further on these results. Nevertheless, an immediate and obvious consideration is that the majority of mutations linked to atrial fibrillation and described so far consists in potassium channel gain-of-function (Kcnj2 being one of them), which shortens action potential duration and facilitates reentry.15 Therefore, it sounds conceivable that IK1 channel overexpression may predispose to atrial arrhythmias. A similar yet less consistent evidence exists for Cx40, the dominant connexin isoform in the fast conduction system coexpressed in atrial myocytes with Cx43 (typical of the working myocardium): Cx40 polymorphisms have been associated with atrial fibrillation in humans,16 however no animal models of Cx40 overexpression are available yet. As for Nppa, unfortunately authors did not obtain ethical consent to measure ANP levels in patients—the most straightforward proof-of-concept; however, in human atrial cardiomyocytes ANP exerts direct electrophysiological effects,17 which may favor the appearance of arrhythmogenic mechanisms. Present knowledge on cellular electrophysiology of atrial arrhythmias derives largely from patients with chronic atrial fibrillation, which causes electric remodeling and anomalies in channel, pump, and connexin properties. Yet, also in this setting Kcnj2 is upregulated whereas Hcn4 (coding for the major sinoatrial isoform of f-channels) and Snc5A (coding for the sodium channel) expressions are unchanged or diminished.18 Because electrophysiological remodeling promotes arrhythmia chronification,15 it is tempting to speculate that—despite diverse pathogenetic mechanisms (intrinsic channel mutations, enhanced transcription attributable to mutant Tbx5, or remodeling)—potassium channel gain-of-function is a common substrate of atrial fibrillation.
The work by Postma et al1 opens a new perspective on the genetic background of HOS and the genotype–phenotype relationship in patients. At the same time, it challenges generating new animal models, where Tbx5 overexpression is more thinly regulated, as expected in case of the gain-of-function mutation described hitherto. Finally, aside from the developmental cardiology field, it instills new doubts and hypotheses on the pathogenesis of (atrial) arrhythmias in humans, awaiting to be investigated.
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Acknowledgments |
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Sources of Funding
The authors’ work is supported by the European Union, Normacor project (LSH-M/CT/2006/018676).
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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References |
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TopIntroductionT-Box Genes and Heart...Gene Dosage: Too Less...References |
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