summarize this : Holt-Oram syndrome (HOS) is a developmental syndrome that affects heart and arm development and occurs in approxi-
mately 1 in 100,000 births. Individuals with HOS show a range of cardiovascular defects, including both atrial and ventricu-
lar septal defects and problems with cardiac conduction, as well as forelimb defects ranging from minor thumb elongation
to severe arm truncation. Physical mapping in affected families linked this disorder to chromosome 12q2, after which a
candidate gene approach identified TBX5 as the causative gene (Basson et al., 1997; Li et al., 1997). Many reports of HOS
cases comprising several dozen TBX5 mutations have been published (Heinritz et al., 2005). The profound effect of TBX5
function on heart development is also illustrated by a study of patients with non-syndromic septal defects in which muta-
tions in TBX5 were found in 9 out of 68 hearts, but not in peripheral blood (Reamon-Buettner et al., 2004), suggesting that
somatic mutation of TBX5 is a relatively common cause of heart malformations.
Expression studies in the mouse reveal that Tbx5 is expressed in both the heart and the forelimb from their inception
through late development. In the heart, Tbx5 was shown to interact with a number of heart-specific transcription factors
including Gata4, Gata6, Mef2c, and Nkx2-5 to activate transcription of target genes (Habets et al., 2002; Ghosh et al.,
2009; Maitra et al., 2009). The necessity of these interactions is supported by the fact that point mutations in Gata4, Tbx5
or Nkx2-5 that affect only their mutual interaction but not their DNA binding capacity are sufficient to recapitulate heart
malformations caused by full deletion (Hiroi et al., 2001; Ding et al., 2006; Fan et al., 2003; Garg et al., 2003; Linhares
et al., 2004). These factors have all been found to co-occupy the enhancers of genes specific to the cardiac gene program
(He et al., 2011). Moreover, transfection of Tbx5, Gata4, and Mef2c was shown to be able to induce cardiomyocyte-like
(A)
(E) (F)
(B) (C)
(D)
FIGURE 34.4 Limb Malformations Caused by Mutations in T-Box Gene Pathways. (A) Holt-Oram patient with a mutation in TBX5 exhibits both
the loss and partial duplication of the anterior digits (He et al., 2004). (B) Mutation of SALL4, a putative TBX5 target, causes similar limb malformations
in Okihiro syndrome (Kohlhase et al., 2003). (C) Patients with small patella syndrome, caused by TBX4 mutations, show a characteristic gap between
the first two toes and short fourth and fifth toes (Bongersv et al., 2004). (D) SALL4 may also be a TBX4 target, as this patient with Okihiro syndrome has
similar foot abnormalities (Kohlhase et al., 2003). (E) Patients with small patella syndrome show abnormal ossifications of the pelvis (arrows) as well
as foot defects (C) and the knee defects for which the syndrome is named (Bongersv et al., 2004). (F) Mice in which Tbx4 has been ablated immediately
after limb development starts also show defects at all axial levels of the hindlimb (Naiche and Papaioannou, 2007). (A: reprinted with permission from
Wiley-Liss, Inc, a subsidiary of John Wiley & Sons, Inc., from He et al., American Journal of Medical Genetics 126:93, © 2004 Wiley-Liss, Inc. B and D
reprinted with permission from the BMJ Publishing Group from Kohlhase et al., J Med Genet 40:473 © 2003. C and E reprinted with permission from the
University of Chicago Press from Bongers et al., Am J Hum Genet 174:1239 © 2004 by the American Society of Human Genetics.)
T-Box Genes and Developmental Anomalies Chapter | 34 643
cells in infarct hearts, indicating the importance of Tbx5 in differentiation of cardiac tissue (Inagawa et al., 2012). Together,
these transcription factors are thought to form a core module for the cardiac differentiation transcriptional program to
become active (Zhou et al., 2012).
Given this role, it is not surprising that mice lacking Tbx5 were found to have severe malformations of the heart (Bru-
neau et al., 2001). Heterozygous mouse mutants closely recapitulate the HOS heart phenotype, with frequent atrial septal
defects, abnormalities of the conduction system, and occasional ventricular septal defects. Several genes involved in heart
development, such as Gja5 (Cx40) and Nppa (Anf), are downregulated in the Tbx5 heterozygotes, thus demonstrating that
these genes are highly sensitive to Tbx5 dosage. Tbx5 homozygous mutant embryos have an even more dramatic phenotype,
in which the heart is severely hypoplastic, does not loop, fails to express Nppa, and has dramatically lower levels of an array
of cardiac differentiation markers. The Tbx5 homozygous mutant heart does nonetheless express cardiac-specific transcrip-
tion factors and contractile proteins, which indicates that Tbx5 is required for cardiac differentiation, but not specification
(Bruneau et al., 2001).
The mouse Tbx5 mutation also revealed the importance of Tbx5 in forelimb development. Mice that are heterozygous
for Tbx5 have hypoplastic wrist bones and elongated phalanges of the first forelimb digit, which is similar to what has been
reported in some HOS patients. In Tbx5 homozygous mutant embryos, the forelimb bud fails to form due to the absence of
Fgf10, a key gene in limb initiation and outgrowth, in the forelimb field (Ahn et al., 2002). This is probably a direct effect,
as Tbx5 can directly regulate the Fgf10 promoter (e.g., Ahn et al., 2002). The forelimb abnormalities seen in HOS patients
may be the result of a partial loss of Fgf10 function, although this remains to be shown.
In general, the mouse model of a human disorder provides opportunities to investigate the molecular function of a
gene with a variety of techniques that are unavailable for use in human patients (e.g., in situ hybridization, tissue or
organ explants). In an unusual twist, the large number of TBX5 mutations found in HOS patients has provided a wealth
of data regarding the molecular function of TBX5 that is comparable to that provided by the mouse model. Comparison
of the severity of limb defects with the severity of heart defects in human pedigrees with mutations in different parts of
TBX5 has provided suggestions regarding which protein domains are key to the phenotypes in each organ (Basson et al.,
1999), although these relationships have not held up in all analyses (Brassington et al., 2003). Several point mutations
in TBX5 have been found to cause HOS, which has allowed investigators to compare normal and disease-causing vari-
ants in biochemical assays to ascertain the mechanistic nature of the causative defect. Not surprisingly, many disease-
causing point mutations that ablate TBX5 DNA binding or activation domains have been linked to HOS. However, other
mutations causing elongated versions of TBX5 or with mutations in the TBX5 regulatory region have also been linked
to HOS or other heart defects (Muru et al., 2011; Shan et al., 2012; Smemo et al., 2012). More remarkably, mutations
in which TBX5 protein binds DNA successfully but is not capable of interaction with the homeobox transcription
factor NKX2–5 or the co-activator protein WWTR1 (TAZ) are also disease-causing, which shows that transcriptional
co-factors are key components of T-box transcriptional function (Fan et al., 2003; Murakami et al., 2005), as has been
shown in the mouse.
Mutations in TBX5 have not been found in all individuals diagnosed with HOS. In some cases, this may be due to caus-
ative mutations in regulatory regions of TBX5, which were not sequenced. In others cases, individuals have mutations in
other genes that produce the same phenotype, which suggests that they may be in the same molecular pathway as TBX5.
Mutations in SALL4, which is normally associated with Okihiro syndrome, are sometimes misdiagnosed as HOS (Kohlhase
et al., 2003). Mouse Sall4 has been shown to be a downstream target of Tbx5 in both the forelimb and the heart (Harvey and
Logan, 2006; Koshiba-Takeuchi et al., 2006).
An interesting family has been identified in which a hemizygous deletion encompasses both TBX3 and TBX5 (Borozdin
et al., 2006). The affected members of this family show symptoms of both HOS and UMS, characterized by the posterior
limb defects and hypoplastic nipples typical of UMS and the anterior limb defects and heart abnormalities typical of HOS.
Despite the fact that TBX3 and TBX5 are co-expressed in regions of the heart and limb, the loss of both genes did not appear
to exacerbate the phenotype of either syndrome. Although it is impossible to draw firm conclusions from a single family,
the combined UMS/HOS phenotype does indicate that there is little or no redundancy between TBX3 and TBX5 in functions
that are sensitive to haplo-insufficiency