Complex Compound Inheritance of Lethal Lung Developmental Disorders Due to Disruption of the TBX-FGF Pathway

Abstract

Primary defects in lung branching morphogenesis, resulting in neonatal lethal pulmonary hypoplasias, are incompletely understood. To elucidate the pathogenetics of human lung development, we studied a unique collection of samples obtained from deceased individuals with clinically and histopathologically diagnosed interstitial neonatal lung disorders: acinar dysplasia (n = 14), congenital alveolar dysplasia (n = 2), and other lethal lung hypoplasias (n = 10). We identified rare heterozygous copy-number variant deletions or single-nucleotide variants (SNVs) involving TBX4 (n = 8 and n = 2, respectively) or FGF10 (n = 2 and n = 2, respectively) in 16/26 (61%) individuals. In addition to TBX4, the overlapping ∼2 Mb recurrent and nonrecurrent deletions at 17q23.1q23.2 identified in seven individuals with lung hypoplasia also remove a lung-specific enhancer region. Individuals with coding variants involving either TBX4 or FGF10 also harbored at least one non-coding SNV in the predicted lung-specific enhancer region, which was absent in 13 control individuals with the overlapping deletions but without any structural lung anomalies. The occurrence of rare coding variants involving TBX4 or FGF10 with the putative hypomorphic non-coding SNVs implies a complex compound inheritance of these pulmonary hypoplasias. Moreover, they support the importance of TBX4-FGF10-FGFR2 epithelial-mesenchymal signaling in human lung organogenesis and help to explain the histopathological continuum observed in these rare lethal developmental disorders of the lung.

Keywords: 17q23.1q23.2 recurrent deletion; 5p12 deletion; T-box transcription factor 4; aplasia of lacrimal and salivary glands; fibroblast growth factor 10; lacrimoauriculodentodigital (LAAD) syndrome; lung hypoplasia; neonatal lung disease.

Figure 1

Phases of Human Lung Development and Histopathological Characterization of the Lung Sections (A) Schematic representation of phases of human lung development and stages of lung growth arrest in particular disorders (adapted from Kimura and Deutsch⁸³). (B–G) Histologic sections of autopsy lung. TBX4 mutations largely resemble the earlier stages of lung development when the majority of lung is composed of conducting airways (pseudoglandular stage). (B) (P026) The distal acinar tubules are dilated and more complex with abundant intervening mesenchyme (canalicular stage). (C) (P006) Despite the immature appearance, well-formed lamellar bodies were seen in a single case by electron microscopy: arrows denote lamellar bodies, original magnification 4,800×) and there was robust expression of surfactant related proteins (thyroid transcription factor 1, surfactant protein B, and pro surfactant protein C by immunostaining [n = 3], data not shown). (D and E) Two case subjects (P025 depicted) showed a marked variation in histologic appearance with areas of acinar dysplasia (D) juxtaposed to more normal saccular spaces (E). (F and G) Lungs from subjects with FGF10 mutations resemble later phases of development when distal airspaces are subdivided by secondary crests containing a double-walled capillary network (saccular stage), suggestive of congenital alveolar dysplasia in a term infant P042 (F), and mature alveoli are polygonal with thin interalveolar septa and a single capillary bed (alveolar stage). (G) (P076) More mature appearing lung architecture, but a reduced number of alveolar spaces, characteristic of pulmonary hypoplasia.

Figure 2

Schematic Representations of SNVs and CNVs Involving TBX4 (A) Topologically associating domains (TADs) detected in fetal lung fibroblasts at 17q23.1q23.2 (B) The 17q23.1q23.2 region depicting deletions identified in nine subjects with pulmonary hypoplasia (red bars) overlapping the enhancers identified in IMR-90 cell line or the super-enhancer in lung fibroblasts (black bars). Complex LCRs flanking the recurrent 17q23.1q23.2 deletions are shown. H3KMe1 and H3KMe3 marks in the fetal lung, conservation scores, and lncRNAs are shown below deletion track. Regions enriched in non-coding variants are highlighted in blue. (C) Distribution of variants in the 17q23.1q23.2 deletion region showing SNV enrichment (variants with MAF < 10% shared by at least two affected subjects with 17q23.1q23.2 deletion and two affected subjects with de novo TBX4 missense variant and absent in 13 control individuals with the same deletion but without lung abnormalities). (D) The TBX4 gene and variants identified in two subjects mapping in CpG island. (E) The TBX4 protein showing T-box domain (blue). Missense mutations and 4 bp deletion identified in three unrelated subjects with lung hypoplasia (red). Previously reported variants identified in individuals with pulmonary hypertension (PAH), ischiocoxopodopatellar syndrome, or PAH with coexisting ischiocoxopodopatellar syndrome (black, blue, and green, respectively)., , , , , ,

Figure 3

Schematic Representations of SNVs and CNVs Involving FGF10 (A–C) Pedigrees of families with 5p12 CNV deletions (A) (P040/P041), (B) (P076), and SNV (C) (P033) involving FGF10 are shown. (D) Topologically associating domains (TADs) detected in fetal lung fibroblasts in the region of 5p12 deletion. (E) The 5p12 genomic region depicting CNV deletions identified in three individuals from two unrelated families with pulmonary hypoplasia (red bars) overlapping the enhancers identified in IMR-90 cell line. H3KMe1 and H3KMe3 marks in the human lung, chromatin state annotation based on ChIP-seq mapping (Roadmap) in the IMR-90 cell line, conservation scores (PhyloP) and lncRNAs are shown below deletion track. (F) The FGF10 gene and variants identified in two subjects with lung hypoplasia. (G) The FGF10 protein showing FGF domain (blue). Variants identified in two AcDys subjects are indicated in red. Previously reported variants identified in individuals with LADD syndrome or aplasia of lacrimal and salivary glands (ALSG) are shown in black., , , ,

Figure 4

Lung-Specific Enhancer Region Located Upstream to TBX2 and TBX4 (A) Chromatin state annotation based on ChIP-seq mapping (Roadmap) in the IMR-90 cell line within the chr17:59,279,024–59,462,062 genomic region. SNVs identified in subjects are presented in the top of chromatin state annotation scheme, while SNVs identified in controls are shown below this track. SNVs with gnomAD (r2.0.2) MAF ≥ 0.2 are shown in red; SNVs with MAF > 0.2 are shown in black, and SNVs with unknown MAF are shown in blue. The variants identified in more than one individual with lung disease are indicated by black dashed rectangles. (B) H3KMe1 and H3KMe3 marks in the IMR-90 cell line and fetal lung, conservation scores (PhyloP), and the enhancers identified in IMR-90 cell line within the chr17:59,279,024–59,462,062 genomic region.