Phenotype characterisation of TBX4 mutation and deletion carriers with neonatal and paediatric pulmonary hypertension

Discussion

TBX4 variant carriers are at risk for abnormal distal lung development, PPHN, paediatric-onset PH, multiple congenital anomalies including congenital heart defects and a typical foot malformation, and developmental disabilities. The majority of our patients (63%) presented with a biphasic clinical course consisting of PPHN and neonatal respiratory failure with apparent resolution around 1 month of age, followed by chronic PH later in infancy or early childhood. It is notable that this form of PH fits a precapillary phenotype; however, given the degree of concurrent lung irregularities, these individuals would not technically meet traditional criteria for World Health Organization Classification Group 1 PH (PAH), and might well be classified as Group 3 (PH due to chronic lung disease and/or hypoxia) [6]. Our description of developmental lung disease in patients with TBX4 variants suggests that associated PH may have several causal associations including chronic respiratory disease and hypoxia in addition to idiopathic PH. Given the difficulty of defining these aetiologies in our retrospective series, we are using the term PH (versus PAH) for TBX4-associated vascular disease.

Diffuse developmental lung disorders are rare diseases related to aberrations in primary mechanisms of lung airway and vascular development, and include such diagnoses as AD, congenital alveolar dysplasia and alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV), a lethal neonatal disease associated with FOXF1 variants [28]. Emerging evidence shows that developmental lung disorders are phenotypically heterogeneous. ACDMPV was recently reported in older infants with seemingly precapillary PH, suggesting that FOXF1-related disease has a broader clinical spectrum than initially thought [29, 30]. TBX4 variants were reported in a neonate presenting with lethal AD [15], one with lethal congenital alveolar dysplasia and one with an undefined alveolar growth abnormality and survival beyond 8 months of age [16]. Our pathology findings, with a broader range of age and clinical manifestations, shed light on the pathogenesis of PH in TBX4 mutants, even though we cannot exclude a selection bias because the biopsies were obtained on a clinical basis without unified criteria. This study confirms that various developmental abnormalities affecting alveolar, interstitial and vascular structures underlie TBX4-associated PH. These features imply compromised growth of pulmonary endoderm severely affecting airway/alveolar development, and mesenchymal maldevelopment, reflected by hypertensive remodelling of pulmonary arteries, prominent IBA and other findings of interstitial disease. Our longitudinal observation suggests that these pathological fetal processes continue after birth and progress with age in certain cases, with gradual vascular and lymphatic remodelling and development of the collateral circulation, leading to progressive PH and end-stage lung disease in childhood or young adulthood.

Imaging studies suggest a combination of lung hypoplasia and alveolar dysfunction associated with the neonatal presentation, and progressive bronchial and interstitial changes developing over time in certain cases. The combination of prominent interstitial lung disease and PH in a subset of patients (cases 2, 12, 13, 18 and 19) suggests that mechanisms related to the lung disease, perhaps including chronic hypoxia, may be a contributing factor. However, RHC data suggest severe pulmonary vascular disease in most cases regardless of parenchymal disease, with earlier age at diagnosis and more severe functional values compared to a reference paediatric PAH cohort (the REVEAL cohort [31]). Lack of response to vasoreactivity testing has also been described in children with BMPR2 mutations compared to those without [32], suggesting that genetic forms of PH have distinct vascular pathophysiology.

Kerstjens-Frederikse et al. [10] first described TBX4 mutations in 30% of a cohort of children diagnosed with idiopathic/familial PAH and SPS, as opposed to only 2.5% of a control adult cohort. Zhu et al. [11] calculated a 7.7% prevalence of TBX4-related disease in a larger cohort of paediatric PAH. Levy et al. [12] also estimated a 7.5% TBX4 mutation prevalence in 3 out of 40 infants with PAH. Eyries et al. [8] found a TBX4 variant prevalence of one out of 36 (2.8%) and four out of 168 (2.4%) in French children and adults with PAH, respectively, which was lower than the prevalence of a BMPR2 variant of 19.4% and 14.3%, respectively, in that cohort. A lower frequency of TBX4 variants has been detected in adult-onset PAH than in paediatric-onset PAH, with an overall mutation frequency estimated at 1.5% (25 out of 1633 cases) [8, 10, 11, 33, 34]. Overall, the lack of standardised inclusion and diagnostic criteria among centres in this paediatric series precludes any inference on prevalence or comparison with BMP-related and other PAH genes.

TBX4 variants are associated with multiple anomalies, consistent with disruption of key developmental processes beyond the lung. Not all phenotypic features have the same expressivity. Whereas SPS has a high penetrance, that of PH appears lower [10]. This selective penetrance may putatively depend on the variant itself and its effect on protein dosage and function. However, phenotypical heterogeneity between relatives with a common variant, some having SPS alone and others having a combination of SPS and PH, suggests that TBX4-related PH is not purely monogenic, and that multiple innate and environmental factors may be at play, similarly to what is observed in other genetic forms of PH [6]. In BMPR2-related disease, PAH penetrance is only 20%, and secondary factors modulate expressivity and disease progression [6]. We observed a 2:1 female prevalence, presumably attributable to selective wastage of male fetuses or an abnormal primary sex ratio. Such disparity was observed in adult PH prior to the identification of causative genes [35], and confirmed in large registries [36]. The role of sex-dependent hormonal factors [37] and modifier genes [38] was subsequently demonstrated in BMPR2-related PAH, accounting for female predominance. Putatively, similar mechanisms also exist for TBX4.

The severity of PH does not necessarily correlate with the predicted level of protein expression, as observed in FOXF1-related ACDMPV [39]. Some CNV cases with complete TBX4 haploinsufficiency have less severe PH than others with less gene-disruptive SNVs in our and other series [10], suggesting either a dominant-negative effect or interactions with genetic or environmental factors, which makes genetic-based prognosis challenging.

There was a greater incidence of developmental disability among our patients with CNVs (cases 1–6) than those with SNVs (cases 7–19), suggesting a role for neighbouring genes, although postnatal factors such as PPHN or ECMO cannot be excluded in the CNV group. Comparing our cases with previously published 17q22–q23.2 deletions (supplementary table S3), deletions involving the contiguous TBX2 and TBX4 loci more frequently resulted in PH and congenital heart defects than did those sparing these two genes, suggesting a major role for these two genes in the cardiovascular components of the syndrome, whereas developmental disability had a homogeneous prevalence regardless of TBX2/4 involvement, suggesting again a role for other neighbouring genes.

TBX2 contributes to airway growth and branching [40] and to endocardial cushion formation, critical in the pathogenesis of septal defects [41]. TBX2 missense mutations were identified in individuals with cardiac septal defects, developmental delays and skeletal anomalies, but no PH [42]. We can speculate that, in the complex pathogenesis of congenital heart defect, TBX4 and TBX2 play significant yet distinct roles as causative or modifier genes, with TBX4 contributing to PH onset and severity in this disease group. Conversely, developmental delay, hearing loss and skeletal defects were equally represented independently of TBX2/TBX4 involvement, suggesting multiple gene interactions in the pathogenesis of non-cardiovascular anomalies.

Although TBX4 was initially identified as a critical actor in hind limb development [43], it is highly expressed in developing lung mesenchyme [44], with a highly conserved TBX4 enhancer sequence regulating its spatiotemporal expression [45]. Homozygous TBX4 mutant mouse embryos die at embryonic day 10.5 from defective allantois formation and placental insufficiency [46], and conditional lung mesenchymal TBX4 reduction leads to impaired lung development [44]. TBX4 interacts with fibroblast growth factor 10 (FGF10), an essential regulator of the limb and lung bud growth and airway branching [47], which may account for combined lower limb/pulmonary phenotype in TBX4-associated disease. The FGF10 pathway also regulates epithelial expression of thyroid transcription factor 1 (TTF1, encoded by the NKX2.1 gene), a key factor in alveolar development and surfactant synthesis [48], which may contribute to neonatal respiratory symptoms in TBX4 mutants.

Limitations of this study include a recruitment bias towards paediatric and neonatal forms of TBX4-linked PH; a lack of standardised inclusion criteria that precludes estimating the prevalence of TBX4 variants among infants affected with PPHN, infantile/paediatric PH and congenital heart defects; and variable timing of follow-up precluding outcome comparisons.

In summary, this study confirms that TBX4 variants underlie a complex variety of developmental lung disorders, resulting in a spectrum of clinical manifestations including PPHN, neonatal hypoxic respiratory failure, interstitial lung disease and chronic/progressive paediatric PH, often associated with multisystem anomalies. The variability and complexity of the phenotype and its potential overlap with other PH-associated gene defects warrant thorough molecular genetic testing, involving TBX4-inclusive diagnostic panels combined with CNV microarrays, in order to capture both small and large variants. The biphasic evolution we describe, characterised by hypoxic respiratory failure at birth followed by later-onset PH, suggests that infants with severe PPHN, especially if idiopathic, should undergo an appropriate echocardiography follow-up during infancy and early childhood, and should be tested for TBX4 variants when positive and/or in the presence of suggestive features such as congenital heart defects, foot anomalies and SPS. Larger cohort- and population-based studies are needed to better delineate genotype–phenotype correlations and determine future diagnostic and therapeutic strategies.