Gene 560 (2015) 129–136

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Review

Molecular basis of the clinical features of Holt–Oram syndrome resulting from missense and extended protein mutations of the TBX5 gene as well as TBX5 intragenic duplications☆ Mohammad M. Al-Qattan ⁎, Hussam Abou Al-Shaar Division of Plastic Surgery and Hand Surgery, King Saud University, Riyadh, Saudi Arabia College of Medicine, Alfaisal University, Riyadh, Saudi Arabia

a r t i c l e

i n f o

Article history: Received 6 October 2014 Received in revised form 24 November 2014 Accepted 2 February 2015 Available online 11 February 2015 Keywords: Holt–Oram syndrome Missense mutation TBX5 Pathogenesis

a b s t r a c t This paper reviews the molecular basis of the clinical features of Holt–Oram syndrome resulting from missense and extended protein mutations of the TBX5 gene as well as TBX5 intragenic duplications. First, we review all previously reported cases with these mutations, and then describe the pathogenesis of the clinical features in the heart and upper limb. Special emphasis is given to ‘non-classic’ upper limb features which are known to occur with these mutations. Finally, the molecular basis of other concurrent anomalies (chest wall, craniofacial, vertebral, and lung anomalies) is reviewed. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Holt–Oram syndrome (MIM: 142900) is a rare syndrome (estimated incidence of 1 in 100,000 live births) caused by TBX5 gene mutations. Classically, affected patients have a combination of radial ray longitudinal deficiency and cardiac defects (Basson et al., 1999). The majority of TBX5 mutations (nonsense, frameshift, and splice site mutations) introduce premature stop codons resulting in truncated proteins which are unable to bind to DNA. Hence, these mutations are considered to cause haplo-insufficiency of T-box activity. As expected, these mutations are known to result in severe upper limb and cardiac malformations (Basson et al., 1999). In contrast, three other types of mutations encode a “non-truncated” protein: missense mutations, extended protein mutations and TBX5 intragenic duplications. TBX5 missense mutations are uncommon and encode the full protein with only a single amino acid substitution; TBX5 run-on mutations result in TBX5 extended mutant proteins; and TBX5 intragenic duplications will increase the dose of the

Abbreviations: TBX5, T BOX 5; NKX2-5, Homeobox protein Nkx-2.5; GATA 4, GATA binding protein; SALL1 and SALL4, (Spalt-like proteins 1 and 4); AER, (Apical ectodermal ridge); FGF, (Fibroblast growth factor); WNT, (Wingless Integrated Protein); CX40, (Connexin 40); RRD, Radial ray deficiency; ASD, Atrial septal defect; VSD, Ventricular septal defect; PFO, Patent Foramen Ovale; PAF, Paroxysmal atrial fibrillation; iRBBB, Incomplete right bundle branch block; cRBBB, Complete right bundle branch block; A-V block, Atrioventricular block; PDA, Patent ductus arteriosus; A-V septal defect, Atrioventricular septal defect. ☆ There is no conflict of interest. ⁎ Corresponding author at: P.O. Box 18097, Riyadh 11415, Saudi Arabia. E-mail address: [email protected] (M.M. Al-Qattan).

http://dx.doi.org/10.1016/j.gene.2015.02.017 0378-1119/© 2015 Elsevier B.V. All rights reserved.

TBX5 protein within embryonic cells. Hence, the clinical features of patients with these mutations tend to be more variable and “non-classic”. Furthermore, the T-box binds DNA as a homo-dimer and it is possible that critical TBX5 protein interactions with DNA are different in the development of the upper limb versus the development of the heart. Hence, one hypothesis states that different missense mutations will result in different degrees of severity of limb versus cardiac anomalies (Basson et al., 1999). The aim of this communication is to describe the molecular basis of the clinical features of Holt–Oram syndrome resulting from missense and extended protein mutations of the TBX5 gene as well as TBX5 intragenic duplications.

2. Literature review The English literature was reviewed for previously reported cases with these mutations of the TBX5 gene. Both the radial ray deficiency and the cardiac anomaly were classified into mild or severe according to the criteria shown in Table 1. Table 2 summarizes the clinical features of previously reported cases of missense mutations of the TBX5 gene (Basson et al., 1999; Boogerd et al., 2010; Brassington et al., 2003; Cross et al., 2000; Debeer et al., 2007; Dias et al., 2007; Faria et al., 2008; Furniss et al., 2009; McDermott et al., 2005; Porto et al., 2010; Postma et al., 2008; Yang et al., 2000). Our literature review also revealed three TBX5 run-on mutations (Bohm et al., 2008; Debeer et al., 2007; Muru et al., 2011), and two

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Table 1 Classification of the severity of radial ray deficiency and cardiac anomalies in Holt–Oram syndrome. The anomaly

Mild

Severe

Radial ray deficiency

Type I hypoplastic thumb (intrinsic muscle deficiency) and/or type I radius hypoplasia (short radius) Isolated atrial septal defect ventricular septal defect, conduction block/arrhythmia or valve defect

More severe types of thumb and radius hypoplasia

Cardiac anomaly

TBX5 intragenic duplications (Kimura et al., 2014; Patel et al., 2012); and these are summarized in Table 3. 2.1. The basic structure and function of the TBX5 protein and its interactions A schematic representation of the TBX5 protein is shown in Fig. 1. A normal structure and dose of the TBX5 protein is essential for normal upper limb and cardiac development; and a clear evidence for that is the Holt–Oram phenotypes associated with “non-truncated” proteins reviewed in the current paper (Tables 2 & 3). Most of the missense mutations result in amino acid substitutions within the T-box DNA binding domain (Fig. 1). A description of all TBX5 protein–protein interactions is beyond the scope of the current review and is described elsewhere (Barnett and Postma, 2014). It is important to note that the TBX5 protein has two nuclear localization segments. The first segment (NLS1) is located within the DNA binding domain and the second segment (NLS2) is located at the C-terminal region (Collavoli et al., 2003). The nuclear availability of TBX5 is a measure of its transcriptional activity. The transactivation domain of the TBX5 protein is at the C-terminal part of the protein (amino acids 339–379) (Zaragoza et al., 2004). LMP4 (a PDZ–LIM protein) interacts with the transactivation domain and the TBX5–LMP4 complex mediates the dynamic shuttling of TBX5 between the nucleus and the cytoplasm (Kulisz and Simon, 2008). Transactivation of TBX5 is promoted by a protein known as TAZ. TBX5–TAZ interactions also occur at the carboxyl terminal (Murakami et al., 2005). Except for LMP4 and TAZ (which interact at the C-terminal region beyond the T-box domain), all other TBX5 protein–protein interactions occur within the T-box DNA binding domain (Barnett and Postma, 2014). Interactions with NKX2-5, GATA4, Hdac 3 and CRM1 proteins are relevant to the current review. NKX2-5 and GATA4 are important cardiac proteins and these will be discussed under the pathogenesis of the cardiac phenotype in Holt–Oram patients. Interactions with Hdac 3 will also be discussed later to explain the pathogenesis of “gain-offunction” of the G125R mutation. Kulisz and Simon (2008) identified a “nuclear export segment” (NES) within the DNA binding domain of the TBX5 protein (Fig. 1). The export protein CRM1 binds to this NES in order to relocate the TBX5 protein from the nucleus to the cytoplasm. Bohm et al. (2008) tested the functional consequences of their extended protein mutation (Table 3). The mutation does not impair nuclear localization nor co-localization with SALL4 (SALL4 is partly induced by TBX5 as will be discussed later with the pathogenesis of limb anomalies). Instead, the extended TBX5 protein was found to modulate the configuration of the protein and mask its transactivator domain resulting in abrogation of the activation of the ANF (Atrial Natriuretic Factor) promoter. The crystallographic structure of the T-box domain (Fig. 2) is also important in understanding the pathogenesis of various missense mutations (Basson et al., 1999; Muller and Hermann, 1997). For example, the glycine residue 80 is at an area known as the ab-loop. The adjacent arginine residue 81 interacts with the major groove of target DNA through two hydrogen bonds. The Gly80Arg mutation replaces glycine with arginine at position 80 and this alters the ab-loop and impairs interactions with target DNA (Muller and Hermann, 1997). As seen in Table 2, there are multiple missense mutations at amino acid position 237. Arginine 237 is located at the carboxyl end of the T-

Combined anomalies and more complex cardiac anomalies

box sequences and it is encoded by CGG, a CpG dinucleotide, which are known as mutational hot spots (Barnett and Postma, 2014). Folding of the normal TBX5 protein positions arginine residue 237 near the glutamate residue 228 and this favors the formation of a salt bridge (Basson et al., 1999). This salt bridge is broken in Arg237Trp and Arg237Gln mutations. This results in re-structuring of the terminal T-box helix, altering TBX5 interactions with the minor groove of target DNA. Stirnimann et al. (2010) tested the binding affinity of several TBX5 mutant proteins containing single point mutations. All mutant proteins showed reduced binding affinities to specific DNA target sites. More interesting, mutant proteins were also different in their ability to bind to unspecific DNA. This indicated that both sequence-specific and sequence-unspecific binding might contribute to the dysregulation of target gene expression. 2.2. The cardiac anomalies and their pathogenesis Development of the heart (Moorman et al., 1997) starts with the cardiac tube which is made up of primary myocardium. The tube has an “inflow” and an “outflow”. The normal TBX5 expression displays a gradient along the cardiac tube. This tube will later give rise to three different structures in the heart: the working myocardium, the conduction system and the cardiac septa. The most common cardiac anomalies associated with Holt–Oram syndrome include septal defects (such as atrial and ventricular septal defects) and conduction disease including atrial fibrillation. More complex anomalies such as atrioventricular canal defects and tetralogy of Fallot are also known to occur (Bohm et al., 2008). 2.2.1. Septal development and TBX5 The normal development of the cardiac septa involves the interaction of TBX5 with two important proteins for cardiac septal development NKX2-5 and GATA4 (Misra et al., 2012; Wang et al., 2011). Both NKX2-5 and GATA4 interact with TBX5 within the DNA binding domain (Fig. 1). It is interesting to note that missense mutations outside the DNA binding domain generally produce a mild cardiac phenotype (Table 2). Experimentally, there is also evidence that the Foxf genes integrate Tbx5 and hedgehog pathways for cardiac septation (Hoffmann et al., 2014). 2.2.2. Conduction system development and TBX5 There are three main concepts of cardiac conduction system development (Christoffels and Moorman, 2009). In the “ring” concept, the tubular heart displays 4 rings along the cardiac tube which are considered to be the early regains committed to form the conduction system. Late in development, the conduction system expresses several neural crest markers. These early rings express specific neural crest markers such as human natural killer 1 (HNK1). The “recruitment” concept states that the future conduction system cells are recruited from the primary myocardial cells by the persistent expression of a specific neural protein known as “embryonic avian polypeptide of 300 kDa” (EAP 300). Early myocardial cells are EAP 300 positive. Later, EAP-300 is downregulated in working myocardial cells and hence only the conduction system cells remain positive for EAP-300. Finally, the “specification” concept is based on the fact that TBX2 and 3 are specifically expressed in the precursors of the conduction system

Table 2 Missense mutations of the TBX5 Gene. Number of families (number of members)

The upper limb anomaly (n = number of patients with the anomaly)

The severity of cardiac anomaly (n = number of patients with the anomaly)

Other concurrent anomalies (n = number of affected patients)

References

GLn49Lys

One family (2 members)

Mild: isolated ASD (n = 2)

No

Yang et al. (2000)

Ile54Thr Asp61tyr

One family (1 member) One family (1 member)

Mild RRD (n = 1) Severe RRD with syndactyly (n = 1) Severe RRD (n = 1) Mild RRD (n = 1)

No No

Yang et al. (2000) Dias et al. (2007)

Met74Ile

Two families (2 members)

Mild: isolated ASD (n = 1) Severe: aortic and mitral valve prolapse with agenesis of the left pericardium (n = 1) Severe: ASD, VSD, ventricular tachycardia (n = 2)

No

Gly80Arg

One family (19 members)

No

Val89Glu Leu94Arg Ile106Val

One family (1 member) One family (1 member) Two families (2 members)

The cardiac anomaly was not specified but 4 members had combined defects (severe) No cardiac anomalies (n = 1) Severe: ASD, VSD (n = 1) No cardiac anomalies (n = 2)

Boogerd et al. (2010) Debeer et al. (2007) Basson et al. (1999)

Trp121Gly

One family (5 members)

Gly125Arg

One family (15 members)

Chest wall Hypoplasia (n = 3) Narrow Shoulders (n = 2) Scoliosis (n = 5)

Gly169Arg

One family (8 members)

His170Leu Gly195Ala Thr223Met

One family (1 member) One family (1 member) Three family (8 cases)

Lys226Asn

One family (2 members)

Thr233Met Arg237Gln

One family (1 member) Six families (30 members)

Arg237Trp

Two families (3 members)

Arg237Pro

One family (2 members)

Mild: isolated ASD (n = 4) Severe: ASD, multiple VSD (n = 1) No cardiac anomalies (n = 2) PAF (n = 13) ASD (n = 1) VSD (n = 1) iRBBB (n = 2) Not specified but generally severe and included conotruncal malformation (n = 8) Severe: ASD, VSD (n = 1) Mild: PFO (n = 1) No cardiac anomaly (n = 2) Mild: VSD (n = 2) Severe: ASD, VSD (n = 4) Severe: ASD, pulmonary stenosis, mitral valve prolapse (n = 2) Severe: ASD, VSD (n = 1) No cardiac anomaly (n = 13) Mild: isolated ASD or VSD or PFO (n = 16) Severe: not specified (n = 1) No cardiac anomaly (n = 1) Mild: ASD (n = 1) Severe: AV canal, VSD (n = 1) Severe: ASD, VSD, A-V block, superior vena cava anomaly, atrial flutter (n = 2)

Ser252Ile Ser261Cys

One family (1 member) One family (3 members)

Val263Met

One family (4 members)

Mild RRD (n = 1) Severe RRD (n = 1) Mild RRD (n = 19) Severe RDD (n = 1) Triphalangeal thumb (n = 1) Phocomelia (n = 1) Mild RRD (n = 1) Mild RRD (n = 4) Clinodactyly of little finger (n = 1) Radial head dislocation (n = 15) Carpal synostosis (n = 6) Scapular dysplasia (n = 3)

Mild RRD (n = 1) No limb anomalies (n = 7) Mild RRD (n = 1) Mild RRD (n = 1) No upper limb anomaly (n = 2) Mild RRD with syndactyly (n = 1) Triphalangeal thumb (n = 4) Triphalangeal thumb (n = 2) Mild RRD (n = 1) RRD of variable severity ± triphalangeal thumb (n = 21) Phocomelia (n = 9) Mild RRD (n = 2) Severe RRD (n = 1) No anomaly (n = 1) Long thumbs without muscle or skeletal abnormalities (n = 1) Mild RRD with syndactyly (n = 1) Severe RRD (n = 1) Hypoplastic distal phalanges of all digits with hypoplastic nails (n = 2) No anomaly (n = 3) Postaxial polydactyly (n = 1)

Mild: VSD (n = 1) No cardiac anomaly (n = 2) Severe: A-V canal, double outlet right ventricle (n = 1) Mild: ASD (n = 3) Severe: cRBBB, ASD, tricuspid valve prolapse, hypoplastic left ventricle (n = 1)

No No No

Furniss et al. (2009) Boogerd et al. (2010) Boogerd et al. (2010) McDermott et al. (2005) Brassington et al. (2003) Postma et al. (2008)

No

Cross et al. (2000)

Aortic coarctation (n = 1) Vertebral fusion (n = 1) No

McDermott et al. (2005) Brassington et al. (2003) Brassington et al. (2003)

11 pairs of ribs, short scapula (n = 2)

Porto et al. (2010)

No Hypoplastic chest wall (n = 2)

No

McDermott et al. (2005) Basson et al. (1999) Brassington et al. (2003) Debeer et al. (2007) Brassington et al. (2003)

Persistent left superior vena cava (n = 1)

Boogerd et al. (2010)

Scoliosis (n = 1) Micrognathia with cleft palate (n = 1)

Cross et al. (2000) Brassington et al. (2003)

Postaxial polydactyly of feet (n = 1) Scoliosis (n = 1) Pectus excavatum (n = 1)

Faria et al. (2008)

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TBX5 mutation

RRD = radial ray deficiency, ASD = atrial septal defect, VSD = ventricular septal defect, PFO = Patent Foramen Ovale, PAF = paroxysmal atrial fibrillation, iRBBB = incomplete right bundle branch block, cRBBB = complete right bundle branch block.

131

Kimura et al. (2014) – Severe in both mother and son (ASD and A-V block in the mother; and ASD, VSD, and sick sinus syndrome in the son)

Patel et al. (2012) – Variable but generally severe: A-V septal defect, hypoplastic left heart syndrome, mitral valve disease, pulmonary stenosis, paroxysmal atrial fibrillation, and sick sinus syndrome.

Mild (isolated ASD)

components (Christoffels and Moorman, 2009). In the absence of TBX2 and 3, there precursor cells differentiate into working myocardium instead of conduction system cells. Once specified, the conduction system cells will exhibit an intense expression of TBX5 and NKX2-5. Interactions between TBX5 and NKX2-5 are essential for the development of the conduction system. Furthermore, TBX5 and NKX2-5 both act synergistically to upregulate CX40 expression which is involved in the normal development of atrioventricular conduction (Bevilacqua et al., 2000). Finally, TBX5 is also a crucial activator of Tbx3 (Mori et al., 2006). Hence, it is of no surprise that TBX5 mutations are commonly associated with conduction defects.

ASD = atrial septal defect; VSD = ventricular septal defect; A-V block = atrioventricular block; PDA = patent ductus arteriosus; A-V septal defect = atrioventricular septal defect. a This mutation results in an elongated TBX5 protein of 580 amino acids. The mutant protein contains 74 miscoding amino acids and 62 supernumerary C-terminal amino acids. b This mutation results in an elongated TBX5 protein with 84 miscoding amino acids and 62 supernumerary C-terminal amino acids. c This small duplication results in only two extra amino acids within the T-box domain (K159_L160 dup). d This 48 kb duplication is encompassing exons 2–9 of the TBX5 gene. e This 11 kb duplication is encompassing exons 1–6 of the TBX5 gene.

One family, one member Leu435FsX146b

Bilateral triphalangeal thumb A small (in-frame) duplication of six One family, one member bases (475_480 dup 6)c d One family, nine members Mild radial ray defects (short thumbs, bowing 48-kb Duplication at 12q24.21 of the radius, dislocation of the radial head) with concurrent ulnar ray defects (ulnar hypoplasia) and clinodactyly of little fingers 11 kb duplication at 12q24.1e One family, two members Mild (bilateral thenar atrophy) in the mother; severe (bilateral radial club-hand) in the son

Severe (sinus bradycardia, ASD, VSD, PDA)

Bilateral hypoplastic clavicles Bohm et al. (2008) micrognathia hypoplastic right lung Skull deformity, narrow shoulders cubitus Muru et al. (2011) valgus, pectus excavatum – Debeer et al. (2007) Severe (ASD, VSD, and A-V block) One family, one member His445FsX136a

Bilateral hypoplastic radii and triphalangeal thumbs Bilateral mild thumb hypoplasia

Number of families (number of members) TBX5 mutation

Table 3 Extended protein TBX5 mutations and intragenic TBX5 duplications.

The upper limb anomaly

References Other concurrent anomalies

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The cardiac anomaly

132

2.2.3. Working myocardium development and TBX5 Barnett and Postma (2014) noted that the two most common complex heart defects in Holt–Oram syndrome are atrioventricular canal defects and tetralogy of Fallot. This is not surprising because TBX5 is directly involved in the normal maturation of the atrioventricular canal (Moskowitz et al., 2004). Furthermore, TBX5 interacts with both NKX2-5 and GATA4; and mutations of both NKX2-5 and GATA4 are known to be associated with tetralogy of Fallot (Misra et al., 2012; Wang et al., 2011). Ghosh et al. (2009) showed that there is physical interaction between TBX5 and members of myocyte enhancer factor 2 C (MEF2C). This interaction leads to a synergistic activation of the α-cardiac myosin heavy chain MYH6. MYH6 is one of the structural proteins of cardiomyocytes. 2.2.4. Genotype–cardiac phenotype correlations The GLy80Arg mutation, which is known to produce a severe cardiac phenotype, is known to impair the synergistic activation of MYH6 by TBX5 (Ghosh et al., 2009). As mentioned before, MYH6 is a structural protein of the working myocardial cell; and hence the inactivation of MYH6 is expected to result in severe cardiac anomalies. The Asp61Tyr mutation (Dias et al., 2007) was seen in a patient with severe aortic and mitral valve prolapse requiring valve replacement. During the procedure, agenesis of the left pericardium was noticed. The presence of pericardial defects in Holt–Oram patients is probably underestimated because such defects are not generally diagnosed with echocardiography. The presence of such defects is not surprising because TBX5 is expressed throughout the epicardium of the embryonic heart and is required for both pro-epicardial and epicardial progenitor cell development (Diman et al., 2014; Hatcher et al., 2004). It remains to be seen, however, if agenesis of the pericardium is specific for the Asp61Tyr mutation. The interaction between different mutant proteins and either NKX2-5 or GATA 4 can be tested in mammalian cells using coimmunoprecipitation. For example, the Ile106Val mutant protein retains the capacity to bind to NKX2-5 and has a slightly enhanced binding to GATA 4 (Boogerd et al., 2010). This is translated clinically in the absence of cardiac anomalies in patients with the Ile106Val mutation (Boogerd et al., 2010; McDermott et al., 2005). In contrast, the Met74Ile mutant protein shows a drastic reduction of binding capacity to both NKX2-5 and GATA 4; and affected patients have severe cardiac anomalies (Boogerd et al., 2010). The Gly125Arg mutation reported by Postma et al. (2008) is a unique mutation because it is a “gain-of-function” mutation. In vitro, the Gly125Arg mutant protein displays enhanced DNA binding in the heart. Clinically, most affected patients have isolated paroxysmal atrial fibrillation (Postma et al., 2008). The pathogenesis of gain-of-function in the Gly125Arg mutation was recently detailed by Barnett and Postma (2014). The transcriptional activity of the TBX5 protein is controlled by several TBX5-protein interactions. One of these proteins is Hdac 3 (Lewandowski et al., 2014). Hdac 3 is a histone modifying protein. It normally interacts with TBX5 to reduce its transcriptional activity by de-acetylation of lysines 157 and 159 within the T-box domain. The gain-of-function mutation Gly125Arg will decrease the normal TBX5–

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Fig. 1. Schematic representation of the TBX5 protein NLS1 = nuclear localization segment 1, NLS2 = nuclear localization segment 2, NES = nuclear export segment. Below the protein are the sites of amino acid substitutions in previously reported missense mutations. Note that the majority are within the DNA binding domain. Above the protein are the sites important TBX5 protein–protein interactions.

Hdac 3 interactions resulting in an increased transcriptional activity of TBX5. These in-vitro studies and clinical observations establish the presence of a genotype–phenotype correlation regarding the cardiac anomalies in Holt–Oram syndrome.

TBX5 is expressed in the upper limb bud and not in the lower limb bud. Hence, TBX5 mutations are expected to result in upper limb anomalies while sparing of the lower limbs. The relationship between TBX5 mutations and the upper limb phenotype is much more complex than the relationship to the cardiac phenotype. This is due to the complex interactions of different mesodermal and ectodermal proteins in the limb development (Fig. 3).

2.3.1. TBX5 and development of the radial ray The normal development of the radial ray (radius and thumb) requires the normal expression and function of SALL1 and SALL4 (Spalt-like proteins 1 and 4) in the anterior mesoderm as well as the normal interaction with the anterior part of the apical ectodermal ridge (AER) which expresses fibroblast growth factor 8 (FGF8). TBX5 is expressed in the mesoderm and is essential for the development of the radial ray through two different pathways (Fig. 3). In the early limb bud, TBX5 induces the expression of FGF10 in the mesoderm. FGF10 will then induce the expression of the Wingless Integrated Protein WNT3A in the AER. WNT3A will then induce the expression of FGF8 in the AER and the latter helps to maintain FGF10 in the mesoderm (this is known as the FGF10–FGF8 loop). Hence, a deficiency in TBX5 is expected to result in a deficiency of ectodermal FGF8 and this will lead to phenotypes that are remarkably similar to the classification spectrum of radial ray deficiency as reviewed by Oberg et al. (2010).

Fig. 2. A diagram of the three dimensional structure of the T-box domain binding to DNA. The ab-loop contains Gly80 and Arg81 residues; and the loop interacts (via hydrogen bonds) with the major groove of target DNA. Normally, folding brings Arg237 near glutamate 227 forming a salt bridge; and this mediates the interaction with the minor groove of target DNA.

Fig. 3. Development of the ulnar ray is under the influence of mesodermal sonic hedgehog (SHH) which interacts with the overlying fibroblast growth factor 4 (FGF4) in the apical ectodermal ridge (AER). Development of the radial ray requires the normal expression and function of spalt-like 1 and 2 (SALL 1 and 4) in the mesoderm as well as FGF8 in the AER. TBOX 5 (TBX5) contributes to the development of the radial ray through two pathways: the first pathway is colored in blue and the second in red (see text for details).

2.3. The upper limb phenotypes and their pathogenesis

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In the second pathway (Fig. 3), TBX5 induces the expression of SALL4 which will form a heterodimer with SALL1. This heterodimer will also enhance the WNT canonical signaling by localizing to heterochromatin. An enhanced WNT signaling will result in enhancing the SALL4 promoter leading to an enhanced SALL4 expression. This pathway was recently discussed by Al-Qattan (2013a); and it explains the overlapping clinical features (radial ray deficiency and cardiac anomalies) in patients with Holt–Oram syndrome (TBX5 mutations), Townes–Brocks syndrome (SALL1 mutations), and Okihiro syndrome (SALL4 mutations). There is a debate in the literature regarding the genotype–phenotype relationship in TBX5 missense mutations. Basson et al. (1999) stated that missense mutations that cause significant cardiac anomalies will cause mild upper limb anomalies and vice versa. Brassington et al. (2003) disputed such a relationship. Our review supports the dispute raised by Brassington et al. (2003). Table 2 shows that the same missense mutation may cause either a severe or a mild radial ray deficiency among different family members, although certain mutations (such as Asp61Tyr, Gly80Arg, Trp121Gly, Gly169Arg, and His170Leu) tend to result in a mild degree of radial ray deficiency. It is interesting to note that most patients with extended protein mutations and TBX5 intragenic mutations (Table 3) also have a mild radial ray defect. 2.3.2. TBX5 mutations and non-classic upper limb phenotypes Non-classic upper limb phenotypes in Holt–Oram patients include phocomelia, radial head dislocation, hypoplasia of the distal phalanges/nails, postaxial polydactyly, ulnar ray defects, isolated triphalangeal thumb deformity, and concurrent syndactyly. Phocomelia was seen in two mutations: Ile106Val and Arg237Gln. Phocomelia is usually described as inter-segmental failure of formation which is different from the radial longitudinal deficiency. Hence, the question that arises is: what is the pathogenesis of phocomelia in TBX5 mutations? Tytherleigh-Strong and Hooper (2003) believe that phocomelia is not a pure inter-segmental defect since almost all patients with phocomelia have concurrent longitudinal deficiencies manifested by digital absence or shortening. The authors propose that phocomelia may be a severe form of longitudinal deficiency which is expected to occur in some patients of Holt–Oram syndrome in whom there is severe suppression of FGF8 in the AER. In support of this hypothesis is the phenotype of the patient with Ile106Val (Boogerd et al., 2010). On the right, the patient had “distal” phocomelia (hand attached directly to the humerus) with monodactyly. On the left, there was severe shortening of the radius and ulna with three digits in the hand. The latter longitudinal deficiency phenotype may represent an intermediate step to the manifestation of “distal” phocomelia. Another non-classic upper limb phenotype is radial head dislocation. This was seen in the family reported by Postma et al. (2008). The Gly125Arg mutation noted in this family is a gain-of-function mutation of TBX5 as shown experimentally (Postma et al., 2008). All 15 affected family members had radial head dislocation and only some had mild thenar muscle atrophy. This indicates that gain-of-function mutations of TBX5 will result in radial head dislocation rather than radial ray deficiency. As discussed earlier under cardiac anomalies, this unique mutation will specifically result in paroxysmal atrial fibrillation. Theoretically, TBX5 intragenic duplication may also result in a “functional” gain because of the increased dose of TBX5. It is interesting to note that the only other mutation that resulted in radial head dislocation was the intragenic duplication reported by Patel et al. (2012); and several of these patients also had paroxysmal atrial fibrillation. A third non-classic phenotype is the hypoplastic distal phalanges and hypoplastic nails noted in the Ser261Cys mutation (Brassington et al., 2003). The short distal phalanges may be explained by the mild suppression of FGF8 during late limb bud development. The hypoplastic nails may be secondary to mild deficiency of the canonical pathway of the dorsalizing protein WNT7A (see the second pathway of TBX5 in the limb bud in Fig. 3).

The Val263Met mutation was also associated with a unique upper limb phenotype (Faria et al., 2008). The mutation was confirmed in the four affected family members. All had cardiac defects and three had a normal upper limb phenotype. The fourth member had bilateral rudimentary postaxial polydactyly. A normal upper limb phenotype was also noted in another two TBX5 mutations: Thr 223Met (Brassington et al., 2003) and Arg237Pro (Boogerd et al., 2010). This should increase the awareness that the presence of upper limb anomalies is not mandatory for the clinical diagnosis of Holt–Oram syndrome. Concurrent radial and ulnar ray defects were seen in the TBX5 intragenic duplication reported by Patel et al. (2012). Ulnar ray defects are not expected to occur in patients with TBX5 mutations because such defects occur secondary to deficiency of Sonic Hedgehog activity on the posterior (ulnar) side of the developing limb bud as shown in Fig. 3 (Al-Qattan et al., 2010). The pathogenesis of these ulnar ray defects remains unclear and requires functional studies. The isolated triphalangeal non-opposable thumb anomaly is developmentally considered as an error of the preaxial ray (Al-Qattan, 2013c). Hence, it is of no surprise that this anomaly was seen in several TBX5 mutations (Leu94Arg, Thr223Met, Lys226Asn and Arg237Gln missense mutations; His445FsX136 extended protein mutation; and the intragenic duplication described by Debeer et al. (2007)). Finally, radial ray deficiency was seen with concurrent syndactyly in several TBX5 mutations (Gln49Lys, Thr223Met, and Ser473 Ile) (Table 2). The pathogenesis of syndactyly is beyond the scope of this communication and is well-described in the literature (Al-Qattan, 2013b). One of the molecular pathways causing syndactyly is through a disturbance of ectodermal FGF8 (Al-Qattan, 2013b). TBX5 mutations may affect ectodermal FGF8 (Fig. 3); explaining the syndactyly. 2.4. Sidedness of limb and heart defects Patients with Holt–Oram syndrome have a striking asymmetry of skeletal involvement, with the left side more severely affected than the right side (Smith et al., 1979). In many of the upper limb phenotypes described in our review, the severity of right versus left upper limb involvement was not stated. However the side was documented in cases in which there was unilateral upper limb involvement. We have summarized these in Table 4. Involvement of the left upper limb was seen in all 11 cases with unilateral limb defects. The pathogenesis of this remains unclear because TBX5 expression is not known to be different in the right versus left limb bud. TBX5 expression in the developing heart is not uniform. In the linear heart tube, TBX5 is expressed in a graded fashion; stronger near the posterior and weaker near the anterior end. As the heart tube loops, asymmetric TBX5 expression is also observed: TBX5 is highly expressed in the presumptive left ventricle (Bruneau et al., 1999). This explains why left sided cardiac malformations are more commonly seen than right sided malformations in Holt–Oram syndrome. Our review confirms the predilection for left sided cardiac malformations. Examples include: the agenesis of the left pericardium noted in the Asp61Tyr mutation, the hypoplastic left ventricle noted in the Val263Met mutation, and the hypoplastic left heart syndrome noted in the duplication reported by Patel et al. (2012). 2.5. Chest wall abnormalities, craniofacial defects, vertebral deformities, and lung anomalies in the Holt–Oram patients and their pathogenesis Our literature review (Tables 2 & 3) revealed that chest wall deformities (such as chest hypoplasia and pectus excavatum) and craniofacial defects (such as micrognathia and cleft palate); and vertebral abnormalities (such as scoliosis and vertebral fusion) may be seen in patients with TBX5 mutations. The pathogenesis of these deformities has not been specifically investigated. When we reviewed the literature, we found that TBX5 is also expressed in the sternum (Pizard et al., 2005) explaining the chest wall deformities. Furthermore, Connexin

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Table 4 Unilateral upper limb defects in TBX5 missense mutations. TBX5 mutation

Number of patients with unilateral upper limb involvement

The upper limb defect

The side affected

References

Gln49Lys Ile54Thr Trp121Gly Thr223Met Arg237Trp Arg237Gln Ser261Cys

1 1 4 1 2 1 1

Thumb hypoplasia Absent thumb Thumb/radius hypoplasia Isolated triphalangeal thumb Thumb hypoplasia Thumb hypoplasia Absent index finger with hypoplastic radius

Left Left Left in all 4 cases Left Left in both cases Left Left

Yang et al. (2000) Yang et al. (2000) Brassington et al. (2003) Brassington et al. (2003) Brassington et al. (2003) Brassington et al. (2003) Brassington et al. (2003)

40 (CX40) is a target of TBX5 (Pizard et al., 2005). CX40 is a gap junction protein which has a role in the bone development. Pizard et al. (2005) showed that TBX5 plays a key regulatory role in bone growth and maturation and this regulation occurs through CX40; and this may explain all other bony abnormalities. The tongue of the micrognathic fetus will obstruct the normal movements of the palatal shelves resulting in cleft palate; explaining the phenotype in patients with the Ser261Cys mutation (Table 2). TBX5 is widely expressed in the developing lung and is important for normal lung development (Arora et al., 2012). Furthermore, TBX5 is an important inducer of FGF10; and the latter is an important paracrine factor in the regulation of the branching morphogenesis of bronchial epithelium (Cebra-Thomas et al., 2003). Lung hypoplasia or agenesis was reported in several stop-codon TBX5 mutations (reviewed by Tseng et al., 2007), but this was seen in only one mutation in the current review (the extended protein mutation reported by Bohm et al. (2008)). 3. Conclusions The pathogenesis of classic cardiac defects (atrial and ventricular septal defects; and conduction defects) in Holt–Oram syndrome is through the abnormal interactions of the T-box domain with NKX2-5 and GATA4. Major cardiac anomalies are uncommon and usually involve the left side of the heart; and this is attributed to the preferential expression of TBX5 in the left side of the developing heart. The pathogenesis of the classic radial ray deficiency is through two pathways of TBX5 within the limb bud: one pathway is related to the FGF10-FGF8 loop and the other pathway is related to SALL4. The complex interactions of TBX5 and other mesodermal and ectodermal factors explain the pathogenesis of the non-classic upper limb phenotypes. However, the striking involvement of the left upper limb in cases with unilateral involvement remains a mystery from the pathogenesis point of view. The most unique mutations were the Gly125Arg missense mutation reported by Postma et al. (2008), and the intragenic duplication reported by Patel et al. (2012). The former is a gain-of-function mutation and is associated with unique limb and cardiac phenotypes (radial head dislocation and isolated paroxysmal fibrillation). The latter is also unique because it is associated with concurrent radial and ulnar ray defects. Finally, bone abnormalities in the chest, face and vertebrae are related to TBX5–CX40 interactions in addition to the high expression of TBX5 in the developing sternum. Acknowledgment The work was supported by the College of Medicine Research Center, Deanship of Scientific Research, King Saud University, Riyadh, Saudi Arabia (2015-q10). References Al-Qattan, M.M., 2013a. Fanconi anemia with concurrent thumb polydactyly and dorsal dimelia. A case report with discussion of embryology. Ann. Plast. Surg. 70, 116–118.

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