Mutations in the thyroid transcription factor gene NKX2-1 result in decreased expression of SFTPB and SFTPC.

BACKGROUND: Mutations in the NK2 homeobox 1 (NKX2-1) gene are associated with lung disease in infants and children. We hypothesize that disruption of normal surfactant gene expression with these mutations contributes to the respiratory phenotypes observed. METHODS: To assess transactivational activity, cotransfection of luciferase reporter vectors containing surfactant protein B or C (SFTPB or SFTPC) promoters with NKX2-1 plasmids was performed and luciferase activity was measured. To assess the binding of mutated proteins to target DNA, electrophoretic mobility shift assays (EMSA) were performed using nuclear protein labeled with oligonucleotide probes representing NKX2-1 consensus binding sequences followed by gel electrophoresis. The effect of overexpression of wild-type (WT) and mutant NKX2-1 on SFTPB and SFTPC was evaluated with quantitative real-time PCR. RESULTS: Decreased transactivation of the SFTPB promoter by both mutants and decreased transactivation of the SFTPC promoter by the L197P mutation was observed. EMSA demonstrated decreased DNA binding of both mutations to NKX2-1 consensus binding sequences. Transfection of A549 cells with NKX2-1 expression vectors demonstrated decreased stimulation of SFTPB and SFTPC expression by mutant proteins compared with that of WT. CONCLUSION: Disruption of transcriptional activation of surfactant protein genes by these DNA-binding domain mutations is a plausible biological mechanism for disruption of surfactant function and subsequent respiratory distress.

Functional characterization of biallelic RTTN variants identified in an infant with microcephaly, simplified gyral pattern, pontocerebellar hypoplasia, and seizures.

BACKGROUND: Biallelic deleterious variants in RTTN, which encodes rotatin, are associated with primary microcephaly, polymicrogyria, seizures, intellectual disability, and primordial dwarfism in human infants. METHODS AND RESULTS: We performed exome sequencing of an infant with primary microcephaly, pontocerebellar hypoplasia, and intractable seizures and his healthy, unrelated parents. We cultured the infant's fibroblasts to determine primary ciliary phenotype. RESULTS: We identified biallelic variants in RTTN in the affected infant: a novel missense variant and a rare, intronic variant that results in aberrant transcript splicing. Cultured fibroblasts from the infant demonstrated reduced length and number of primary cilia. CONCLUSION: Biallelic variants in RTTN cause primary microcephaly in infants. Functional characterization of primary cilia length and number can be used to determine pathogenicity of RTTN variants.

Functional Characterization of ATP-Binding Cassette Transporter A3 Mutations from Infants with Respiratory Distress Syndrome.

Mutations in the ATP-binding cassette transporter A3 gene (ABCA3) result in severe neonatal respiratory distress syndrome and childhood interstitial lung disease. As most ABCA3 mutations are rare or private, determination of mutation pathogenicity is often based on results from in silico prediction tools, identification in unrelated diseased individuals, statistical association studies, or expert opinion. Functional biologic studies of ABCA3 mutations are needed to confirm mutation pathogenicity and inform clinical decision making. Our objective was to functionally characterize two ABCA3 mutations (p.R288K and p.R1474W) identified among term and late-preterm infants with respiratory distress syndrome with unclear pathogenicity in a genetically versatile model system. We performed transient transfection of HEK293T cells with wild-type or mutant ABCA3 alleles to assess protein processing with immunoblotting. We used transduction of A549 cells with adenoviral vectors, which concurrently silenced endogenous ABCA3 and expressed either wild-type or mutant ABCA3 alleles (p.R288K and p.R1474W) to assess immunofluorescent localization, ATPase activity, and organelle ultrastructure. Both ABCA3 mutations (p.R288K and p.R1474W) encoded proteins with reduced ATPase activity but with normal intracellular localization and protein processing. Ultrastructural phenotypes of lamellar body-like vesicles in A549 cells transduced with mutant alleles were similar to wild type. Mutant proteins encoded by ABCA3 mutations p.R288K and p.R1474W had reduced ATPase activity, a biologically plausible explanation for disruption of surfactant metabolism by impaired phospholipid transport into the lamellar body. These results also demonstrate the usefulness of a genetically versatile, human model system for functional characterization of ABCA3 mutations with unclear pathogenicity.

Surfactant protein-C promoter variants associated with neonatal respiratory distress syndrome reduce transcription.

Dominant mutations in coding regions of the surfactant protein-C gene, SFTPC, cause respiratory distress syndrome (RDS) in infants. However, the contribution of variants in noncoding regions of SFTPC to pulmonary phenotypes is unknown. By using a case-control group of infants > or =34 weeks gestation (n = 538), we used complete resequencing of SFTPC and its promoter, genotyping, and logistic regression to identify 80 single nucleotide polymorphisms (SNPs). Three promoter SNPs were statistically associated with neonatal RDS among European descent infants. To assess the transcriptional effects of these three promoter SNPs, we selectively mutated the SFTPC promoter and performed transient transfection using MLE-15 cells and a firefly luciferase reporter vector. Each promoter SNP decreased SFTPC transcription. The combination of two variants in high linkage dysequilibrium also decreased SFTPC transcription. In silico evaluation of transcription factor binding demonstrated that the rare allele at g.-1167 disrupts a SOX (SRY-related high mobility group box) consensus motif and introduces a GATA-1 site, at g.-2385 removes a MZF-1 (myeloid zinc finger) binding site, and at g.-1647 removes a potential methylation site. This combined statistical, in vitro, and in silico approach suggests that reduced SFTPC transcription contributes to the genetic risk for neonatal RDS in developmentally susceptible infants.

Risk factors and prognostic significance of altered left ventricular geometry in preterm infants.

OBJECTIVE: Left ventricular (LV) hypertrophy (LVH) predicts adverse cardiac events in adults. We sought to determine the risk factors and prognostic significance of altered LV geometry in preterm infants. STUDY DESIGN: In an echocardiographic, single-center, retrospective case-control study we investigated the risk factors and outcomes in patients with altered LV geometry (either increased left ventricular mass index (LVMI) or increased relative wall thickness (RWT)) from a cohort of 503 preterm infants ≤2 kg. RESULT: Altered LV geometry was seen in 180 patients and was predicted by postnatal steroids and small for gestational age. Hospital stay was longer in the elevated RWT cases. Altered LV geometry resolved in 129 of the 131 cases with follow-up echocardiogram. Fifteen of 94 patients with elevated RWT died compared to 3/90 controls (P = 0.004). CONCLUSION: Altered LV geometry in preterm infants is associated with postnatal steroid use and small for gestational age. Elevated RWT is associated with longer hospital stay and increased mortality.

Formation and malformation of the vertebrate left-right axis.

Despite an externally symmetric body plan, the internal viscera of all vertebrates are asymmetric with respect to the left-right body axis. Determination of the handedness of this asymmetry is nonrandom and highly conserved among vertebrates. Errors in patterning along the left-right axis, which occur in about 1 in 10,000 human births, may result in significant morbidity and mortality. During early embryonic development, midline structures, in particular the node, coordinate patterning of the three main embryonic axes: anterior-posterior, dorsal-ventral, and left-right. A current model for specification of the handedness of left-right axis asymmetry invokes the activity of embryonic cilia in the node that create a net leftward flow of extraembryonic fluid. This flow is proposed to provide a signal for subsequent asymmetric gene expression. Signaling from the node defines patterns of asymmetric gene expression on the left and right sides of the embryo. These signals for "left" and "right" are ultimately interpreted by organ primordia during later development. Complex activating and inhibiting interactions involving TGF-beta family members, as well as homeobox transcription factors, mediate these asymmetric patterns of gene expression. The identification of the genes regulating left-right axis patterning in model organisms has resulted in the characterization of human mutations associated with left-right axis malformations.

IL-13 regulates cilia loss and foxj1 expression in human airway epithelium.

Mucociliary clearance is essential to the defense mechanisms of the respiratory system. Loss of normal mucociliary clearance contributes to the pathogenesis of genetic and acquired lung diseases. Treatment of cultured differentiated human airway epithelial tissue with IL-13 resulted in a loss of ciliated epithelial cells and an increase in mucus-secreting cells. The loss of ciliated cells was characterized by mislocation of basal bodies and loss of ezrin from the apical cell compartment. In addition to the loss of ciliated cells and increase in mucous cells after IL-13 treatment, cells with characteristics of both ciliated and mucous cells were observed in the airway epithelium. In association with the decrease in ciliated cells after IL-13 treatment, there was noted a decrease in foxj1 expression in the airway epithelium, characterized by a decrease in the number of foxj1-expressing cells. Within the foxj1 promoter, a STAT-binding element was identified and inhibition of foxj1 expression by STAT-6 and IL-13 was demonstrated. These findings suggest molecular and cellular mechanisms for cilia loss in pulmonary disease. Inhibition of foxj1 expression results in loss of apical localization of ezrin and basal bodies with subsequent loss of axonemal structures. These findings have important implications for the pathogenesis and treatment of airway diseases.

Roles of the Foxj1 and Inv genes in the left-right determination of internal organs in mice.

In Foxj1 knockout mice, half show situs solitus while the other half show situs inversus, which means a random determination of the left-right axis. In contrast, the inv mutant mice show a mirror-image configuration of the internal organs, which means a reversal of the left-right axis. Although these two mutant mice have primary cilia on the nodal cells, their phenotypes are different in laterality determination. We thus made Foxj1/inv double mutant mice and analyzed their phenotype. We found the phenotypes of Foxj1/inv double mutant mice to be more similar to those of the Foxj1 mutant mice than those of the inv mutant mice. We also found right pulmonary isomerism to be a major phenotype of the Foxj1 mutant mice and the Foxj1/inv double mutant mice, which is likely due to the absence of the Pitx2 expression at both lateral plate mesoderms. These results indicate that a random signal of laterality (Foxj1) is dominant over the reversal signal of laterality (Inv).

Foxj1 regulates basal body anchoring to the cytoskeleton of ciliated pulmonary epithelial cells.

The forkhead box transcription factor Foxj1 is required for cilia formation and left-right axis determination. To define the role of Foxj1 in ciliogenesis, microarray analysis was performed to identify differentially expressed genes in the pulmonary epithelium of foxj1(+/+) and foxj1(-/-) mice. In the absence of Foxj1, the expression of calpastatin, an inhibitor of the protease calpain, decreased. RNase protection confirmed the decrease in calpastatin expression and decreased calpastatin was detected in the proximal pulmonary epithelium of foxj1(-/-) mice by immunohistochemistry. No change was detected in the expression of calpain 2 in the pulmonary epithelium by western blot or immunohistochemistry. By western blot and immunofluorescence, ezrin, a substrate for calpain, was also found to decrease in the pulmonary epithelium of foxj1(-/-) mice. No change in ezrin gene expression was found by RT-PCR. A decrease in ezrin binding phosphoprotein-50 (EBP-50) was also detected by immunofluorescence in the foxj1(-/-) mouse pulmonary epithelium. Immunoelectron microscopy demonstrated ezrin associated with the basal bodies of cilia in the pulmonary epithelium. Treatment of tracheal explants from foxj1(-/-) mice with a calpain inhibitor resulted in a partial reappearance of cilia observed in these mice. Additionally, following treatment of foxj1(-/-) tracheal explants with calpain inhibitor, basal bodies were observed in an apical location along with relocalization of ezrin and EBP-50. Regulation of calpain activity by calpastatin thus provides a mechanism for regulating the anchoring of basal bodies to the apical cytoskeleton in ciliated cells. In the absence of Foxj1, decreased calpastatin expression with decreased ezrin and EBP-50 results in an inability of basal bodies to anchor to the apical cytoskeleton and subsequent failure of axonemal formation.

Foxj1 regulates asymmetric gene expression during left-right axis patterning in mice.

Mice with a targeted mutation of the foxj1 gene demonstrate either D- or L-looping of the embryonic cardiac tube. Foxj1 is expressed in ventral cells of the embryonic node prior to asymmetric, left-right expression of other genes. Despite an absence of 9+2 cilia in foxj1(-/-) mice, 9+0 cilia are present in the node of foxj1(-/-) embryos. In foxj1(-/-) embryos, the patterns of expression of the TGF-beta family member nodal and the homeobox family member pitx2 are randomized. No expression of the TGF-beta family member lefty-2 is observed in any foxj1(-/-) early somite stage embryos. Foxj1 thus acts early in left-right axis patterning and regulates asymmetric gene expression. This regulation does not appear to be the result of a direct interaction between Foxj1 and the genes examined.

Progressive lung disease and surfactant dysfunction with a deletion in surfactant protein C gene.

Mutations in the surfactant protein (SP)-C gene are responsible for familial and sporadic interstitial lung disease (ILD). The consequences of such mutations on pulmonary surfactant composition and function are poorly understood. To determine the effects of a mutation in the SP-C gene on surfactant, we obtained lung tissue at the time of transplantation from a 14-mo-old infant with progressive ILD. An in-frame 9-bp deletion spanning codons 91-93 in Exon 3 of the SP-C gene was present on one allele; neither parent carried this deletion. SP-C mRNA was present in normal size and amount. By immunofluorescence, proSP-C was aggregated within alveolar Type II cells in a compartment separate from SP-B. In airway surfactant, there was little or no mature SP-B or SP-C; SP-A content was increased. Minimum surface tension was increased (20 mN/m, normal < 5 mN/m). Type II cells contained normal and disorganized appearing lamellar bodies by electron microscopy. This spontaneous deletion on one allele of the SP-C gene was associated with sporadic ILD and abnormalities in surfactant composition and function. We propose that a dominant negative effect on surfactant protein metabolism and function results from aggregation of misfolded proSP-C and subsequent cell injury and inflammation.