Phenotype and outcome of pulmonary arterial hypertension patients carrying a TBX4 mutation

Discussion

We describe a cohort of 20 PAH patients from a nationwide PH registry carrying a TBX4 mutation across a broad age range from birth to late adulthood. Our report describes the phenotype and outcome of a French mixed cohort of childhood-onset and adult-onset PAH associated with TBX4 mutation. PAH due to TBX4 gene mutations seems to occur with a bimodal distribution mode in our cohort, comprising two main peaks of occurrence: an early onset in childhood as described previously [5, 12, 14] and a later presentation in adulthood (figure 2). This observation is supported by the fact that TBX4 mutations are identified in 6% of paediatric index cases and in 3% of adult index cases. This consideration cannot be stated formally given the limited size of our population. However, a recent study of exome sequencing in a large cohort of 2572 adult and pediatric PAH cases finds the same type of presentational distribution in PAH patients carrying a TBX4 mutation [21]. A female predominance was observed, as described in other forms of heritable PAH [22, 23]. Clinical features of SPS were found in most patients but were frequently non-obvious. At diagnosis, patients presented with severe pre-capillary PH, usually associated with bronchial, parenchymal and vascular abnormalities, as demonstrated by HRCT of the chest and lung histology. After initiation of PAH therapy, a statistically significant clinical improvement was observed with no significant haemodynamic changes. Even if prognosis seemed to be better than in PAH associated with BMPR2 mutation it remained severe, with two deaths and two lung transplantations at follow-up.

A high penetrance of TBX4 mutation for skeletal abnormalities has been described [5]. In our cohort, 80% of patients presented with signs of SPS. However, signs were absent or non-obvious in some patients. Indeed, three out of 20 patients had no skeletal abnormalities, illustrating that PAH may be the only clinical manifestation of TBX4 mutation mimicking IPAH. Therefore, personal and familial signs of SPS must be systematically searched in every patient newly diagnosed with idiopathic or familial PAH and TBX4 mutations should be screened even in the absence of skeletal abnormalities. In the literature, this ascertainment was previously made by Zhu et al. [13], who identified 13 cases of PAH associated with a TBX4 mutation, including a single case of SPS and by Navas et al. [24], who identified four PAH patients carrying a TBX4 mutation with no skeletal abnormalities identified. By contrast, the penetrance of TBX4 mutation for PAH is incomplete, as previously reported with other PAH predisposing genes. In the present series, we have identified a small proportion of familial cases (four patients from one family and 16 sporadic cases) in comparison with what is usually observed in PAH associated with BMPR2 mutation [16]. In the reported family, all TBX4 mutation carriers presented with SPS but only four female members developed PAH, at an age of 1 year, 3 years, 17 years and 76 years, respectively, demonstrating that the manifestation of the disease is independent of the mutation type (figure 1).

The haemodynamic characteristics in our population are broadly similar to previous reports, showing severe pre-capillary PH at diagnosis without acute vasodilator response according to current guidelines. However, some patients presented with partial vasoreactivity in accordance with what has been reported in paediatric series [14]. Among the four patients who received CCB therapy only one presented stable disease, while the other three became worse requiring the use of PAH treatments. This haemodynamic profile was similar to the results observed in other heritable PAH populations, such as BMPR2 or ACVRL1 mutation carriers, for whom acute vasoreactivity was classically absent [22, 23].

We observed congenital heart diseases in 15% of our cases (two atrial septal defects and one patent ductus arteriosus): two in the nine childhood-onset PAH cases and one in the 11 adult-onset PAH cases. The paediatric report by Galambos et al. [14] describes 36.8% atrial septal defects, 21% patent ductus arteriosus, 5.3% ventricular septal defects and 5.3% patent foramen ovale. These differences may be explained at least in part by a higher incidence of CNV in the paediatric population. Indeed, the CNVs generally encompassed the whole TBX4 gene plus some other genes, including the TBX2 gene, which plays an important role in the embryologic development of the heart and is associated with cardiac septal defects [25, 26].

One may hypothesise that pre-capillary PH in patients carrying TBX4 mutation could be a consequence of developmental disorders of the lungs and should be classified as PH due to chronic respiratory disease (group 3 of the PH classification) [27]. Analysis of HRCT of the chest demonstrated that 60% of patients presented with bronchial abnormalities, with central and peripheral thickening, irregularity of the bronchial walls and presence of tracheal or bronchial diverticula in one-third of patients. PFTs revealed an obstructive respiratory disease in 28.6% of patients and that 28.6% of patients showed decreased FEV₁ at <80%. Furthermore, four patients had a history of mild asthma. Histological assessment of lung samples in three patients confirmed an abnormal distal lung development with emphysematous-like dystrophia appearing as disruption and rarefaction of the bronchiolar septation, abnormalities of bronchi with the presence of tracheal and bronchial diverticula and interstitial fibrosis. Furthermore, histologic examinations showed the presence of cholesterol cleft inclusions located in the perivascular connective tissue. This aspect is not a common feature of PAH but has been described previously in heterogeneous PH populations in which it is impossible to formally eliminate the presence of underlying TBX4 mutation or any developmental disorder [28–30]. These findings are reminiscent of those reported in the paediatric cohort of Galambos et al. [14]. Moreover, lung explants from our patients had a small size macroscopically, compatible to a certain degree with pulmonary hypoplasia, a lung development disorder classically defined by a ratio of lung weight to body weight of less than 0.012 after 28 weeks of gestation [20]. Finally, this hypothesis may still be supported by the low D_LCO, which is not usually as pronounced in IPAH and heritable PAH [31]. These abnormalities may be related to pulmonary development disorders due to TBX4 mutations. However, some patients did not have CT abnormalities but developed PAH, arguing for the presence of isolated pulmonary vascular disease. Indeed, histologic assessment revealed pulmonary arterial remodelling as classically observed in PAH, including plexiform lesions (figures 3b and 3c) similar to those observed in IPAH or heritable PAH [2]. Moreover, we did not show significant correlations between PVR (reflecting the vascular anomalies) and FEV₁, FVC or P_aO2 (reflecting lung parenchymal involvement) or D_LCO (reflecting the involvement of the alveolo-capillar barrier). This finding argues for a specific involvement of pulmonary arteries in this heritable form of PAH and against a mechanism merely due to parenchymal involvement.

Even if it might have started early, during lung development, pulmonary vascular involvement could progress slowly in TBX4 mutation carriers. This could explain the relative long-term stability with PAH treatments. Despite initially severe PAH, requiring a combination of PAH therapies and, in turn, the use of triple combination therapy in 60% of patients, clinical status improved despite the absence of significant haemodynamic improvement. The overall survival rates of our cohort seem to be better than those observed in other forms of IPAH and heritable PAH, but they must be interpreted with caution because of the retrospective nature of the study, the small size of the studied population and the variable duration in follow-up [32].

Most of the radiologic or histologic abnormalities observed in our patients could be related to the embryologic roles of the TBX4 gene. In utero, TBX4 is abundantly expressed in the pulmonary mesenchyme at critical stages of development, where the transcription factor activates genes that are essential for branching morphogenesis, including FGF10 and Nkx2.1. The association of TBX4 mutations with PAH further raises the possibility that TBX4 regulates genes that influence pulmonary vascular development and function [33]. Using a transgenic mouse line (Tbx4 lung enhancer), it was recently shown that angiogenic vessels originating from the extra-pulmonary site initially serve as a stem, and trigger lung mesenchymal stem cell differentiation and commitment to endothelial progenitor cells, which then coalesce into pre-existing vessels and become the major source for lung vasculature [34]. In alveolar capillary dysplasia, a disease mainly caused by FOXF1 mutations and characterised by pulmonary vein misalignment, capillary paucity and alveolar misdevelopment, decreased FOXF-1 and/or its downstream transcription factor TBX4 disrupt lung microvessel formation and homing to alveolar epithelium [35]. The involvement of FGF signalling in early lung development has been thoroughly investigated. FGF9 positively regulates the expression of Fgf10 via the upregulation of TBX4 in the lung mesenchyme. Epithelium-derived FGF9 and mesothelium-derived FGF9 induce proliferation in both the epithelial and mesenchymal compartments of the developing lung and are also involved in pulmonary vascular development [36]. It has been shown that TBX4 is highly specific in lung fibroblasts and that TBX4 is required for cell proliferation and collagen gel contraction capacity [37]. Transcriptome analysis has revealed that TBX4 could broadly regulate fibroblast-related pathways and partly contribute to super-enhancer-mediated transcriptional programmes. TBX4 knockdown and subsequent RNA-seq were performed in adult fibroblasts. Among the significantly regulated genes, KCNK6, CYP1B1, HMOX1 and ESM1 have been shown previously to be important in PAH pathogenesis [38]. Thus, TBX4 deficiency may compromise lung development and pulmonary vasculature in particular, through insufficient angiogenesis related in part to low FGF expression. In adults, TBX4 is mainly expressed in pulmonary fibroblasts, where it regulates genes and pathways involved in PAH.

The main limitations of this study are first of all the retrospective data collection, with a potential underestimation of paediatric cases due to a lack of exhaustivity in genetic screening in early death. Moreover, the population is of limited size, even if it remains the largest adult PAH population of TBX4 mutation carriers to date. Notably, it is unlikely to gather a much larger cohort of patients, since the prevalence of TBX4 mutation currently ranges from 2.8% to 7.7% in childhood-onset PAH and from 1.5% to 2.4% in adult-onset PAH [12, 13, 17, 18]. Mutations in the TBX4 gene have probably also been underdiagnosed because, until recently, physicians were not systematically looking for skeletal abnormalities in PAH patients. Moreover, mutations in the TBX4 gene have not been tested for in all PAH cases due to its recent association with pulmonary vascular disease. It has been difficult to interpret the haemodynamic response to PAH therapy in this study because of incomplete haemodynamic follow-up in childhood PAH. Last, the pathologic assessments brought major information on pulmonary vascular and parenchymal involvements but this assessment was limited to a small number of patients, which makes it difficult to generalise the results to the whole population.

In conclusion, PAH due to TBX4 mutation is an autosomal dominant disease with incomplete penetrance for PAH and/or SPS, and generally occurs in sporadic cases. PAH in this population is severe and occurs across a broad age range from birth to late adulthood. Heritable PAH associated with TBX4 mutations often presents with bronchial and parenchymal radiological abnormalities and low D_LCO.