Lethal neonatal respiratory failure due to biallelic variants in BBS1 and monoallelic variant in TTC21B.

BACKGROUND: Bardet-Biedl syndrome (BBS) is a rare, autosomal recessive ciliopathy characterized by early onset retinal dystrophy, renal anomalies, postaxial polydactyly, and cognitive impairment with considerable phenotypic heterogeneity. BBS results from biallelic pathogenic variants in over 20 genes that encode key proteins required for the assembly or primary ciliary functions of the BBSome, a heterooctameric protein complex critical for homeostasis of primary cilia. While variants in BBS1 are most frequently identified in affected individuals, the renal and pulmonary phenotypes associated with BBS1 variants are reportedly less severe than those seen in affected individuals with pathogenic variants in the other BBS-associated genes. CASE-DIAGNOSIS: We report an infant with severe renal dysplasia and lethal pulmonary hypoplasia who was homozygous for the most common BBS1 pathogenic variant (c.1169 T > G; p.M390R) and also carried a predicted pathogenic variant in TTC21B (c.1846C > T; p.R616C), a genetic modifier of disease severity of ciliopathies associated with renal dysplasia and pulmonary hypoplasia. CONCLUSIONS: This report expands the phenotypic spectrum of BBS with the first infant with lethal neonatal respiratory failure associated with biallelic, pathogenic variants in BBS1 and a monoallelic, predicted pathogenic variant in TTC21B. BBS should be considered among the ciliopathies in the differential diagnosis of neonates with renal dysplasia and severe respiratory failure.

The common ABCA3E292V variant disrupts AT2 cell quality control and increases susceptibility to lung injury and aberrant remodeling.

ATP-binding cassette class A3 (ABCA3) is a lipid transporter that plays a critical role in pulmonary surfactant function. The substitution of valine for glutamic acid at codon 292 (E292V) produces a hypomorphic variant that accounts for a significant portion of ABCA3 mutations associated with lung disorders spanning from neonatal respiratory distress syndrome and childhood interstitial lung disease to diffuse parenchymal lung disease (DPLD) in adults including pulmonary fibrosis. The mechanisms by which this and similar ABCA3 mutations disrupt alveolar type 2 (AT2) cell homeostasis and cause DPLD are largely unclear. The present study, informed by a patient homozygous for the E292V variant, used an in vitro and a preclinical murine model to evaluate the mechanisms by which E292V expression promotes aberrant lung injury and parenchymal remodeling. Cell lines stably expressing enhanced green fluorescent protein (EGFP)-tagged ABCA3 isoforms show a functional deficiency of the ABCA3E292V variant as a lipid transporter. AT2 cells isolated from mice constitutively homozygous for ABCA3E292V demonstrate the presence of small electron-dense lamellar bodies, time-dependent alterations in macroautophagy, and induction of apoptosis. These changes in AT2 cell homeostasis are accompanied by a spontaneous lung phenotype consisting of both age-dependent inflammation and fibrillary collagen deposition in alveolar septa. Older ABCA3E292V mice exhibit increased vulnerability to exogenous lung injury by bleomycin. Collectively, these findings support the hypothesis that the ABCA3E292V variant is a susceptibility factor for lung injury through effects on surfactant deficiency and impaired AT2 cell autophagy.

Biallelic ASCC1 variants including a novel intronic variant result in expanded phenotypic spectrum of spinal muscular atrophy with congenital bone fractures 2 (SMABF2).

Spinal muscular atrophy with congenital bone fractures 2 (SMABF2), a type of arthrogryposis multiplex congenita (AMC), is characterized by congenital joint contractures, prenatal fractures of long bones, and respiratory distress and results from biallelic variants in ASCC1. Here, we describe an infant with severe, diffuse hypotonia, congenital contractures, and pulmonary hypoplasia characteristic of SMABF2, with the unique features of cleft palate, small spleen, transverse liver, and pulmonary thromboemboli with chondroid appearance. This infant also had impaired coagulation with diffuse petechiae and ecchymoses which has only been reported in one other infant with AMC. Using trio whole genome sequencing, our proband was identified to have biallelic variants in ASCC1. Using deep next generation sequencing of parental cDNA, we characterized alteration of splicing encoded by the novel, maternally inherited ASCC1 variant (c.297-8 T > G) which provides a mechanism for functional pathogenicity. The paternally inherited ASCC1 variant is a rare nonsense variant (c.466C > T; p.Arg156*) that has been previously identified in one other infant with AMC. This report extends the phenotypic characteristics of ASCC1-associated AMC (SMABF2) and describes a novel intronic variant that partially disrupts RNA splicing.

Functional characterization of four ATP-binding cassette transporter A3 gene (ABCA3) variants.

ABCA3 transports phospholipids across lamellar body membranes in pulmonary alveolar type II cells and is required for surfactant assembly. Rare, biallelic, pathogenic ABCA3 variants result in lethal neonatal respiratory distress syndrome and childhood interstitial lung disease. Qualitative functional characterization of ABCA3 missense variants suggests two pathogenic classes: disrupted intracellular trafficking (type I mutant) or impaired ATPase-mediated phospholipid transport into the lamellar bodies (type II mutant). We qualitatively compared wild-type (WT-ABCA3) with four uncharacterized ABCA3 variants (c.418A>C;p.Asn140His, c.3609_3611delCTT;p.Phe1203del, c.3784A>G;p.Ser1262Gly, and c.4195G>A;p.Val1399Met) in A549 cells using protein processing, colocalization with intracellular organelles, lamellar body ultrastructure, and ATPase activity. We quantitatively measured lamellar body-like vesicle diameter and intracellular ABCA3 trafficking using fluorescence-based colocalization. Three ABCA3 variants (p.Asn140His, p.Ser1262Gly, and p.Val1399Met) were processed and trafficked normally and demonstrated well-organized lamellar body-like vesicles, but had reduced ATPase activity consistent with type II mutants. P.Phe1203del was processed normally, had reduced ATPase activity, and well-organized lamellar body-like vesicles, but quantitatively colocalized with both endoplasmic reticulum and lysosomal markers, an intermediate phenotype suggesting disruption of both intracellular trafficking and phospholipid transport. All ABCA3 mutants demonstrated mean vesicle diameters smaller than WT-ABCA3. Qualitative and quantitative functional characterization of ABCA3 variants informs mechanisms of pathogenicity.

Functional Genomics of ABCA3 Variants.

Rare or private, biallelic variants in the ABCA3 (ATP-binding cassette transporter A3) gene are the most common monogenic cause of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 disease-associated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPase-mediated phospholipid transport (type II). Therapies remain limited and nonspecific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus in A549 cells with CRISPR/Cas9 genome editing. Next, to generate a parent cell line (A549/ABCA3-/-) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS cassette, FACS, and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural vesicle phenotype) of two individual ABCA3 mutants (type I mutant, p.L101P; type II mutant, p.E292V) in A549/ABCA3-/- cells and in both A549 cells and primary, human alveolar type II cells that transiently express each cDNA after adenoviral-mediated transduction. We also confirmed pharmacologic rescue of ABCA3 variant-encoded mistrafficking and vesicle diameter in A549/ABCA3-/- cells that express p.G1421R (type I mutant). A549/ABCA3-/- cells provide a scalable, genetically versatile, physiologically relevant functional genomics platform for discovery of variant-specific mechanisms that disrupt ABCA3 function and for screening of potential ABCA3 pharmacologic correctors.

Phenotypic expansion of KMT2D-related disorder: Beyond Kabuki syndrome.

Pathogenic variants in KMT2D, which encodes lysine specific methyltransferase 2D, cause autosomal dominant Kabuki syndrome, associated with distinctive dysmorphic features including arched eyebrows, long palpebral fissures with eversion of the lower lid, large protuberant ears, and fetal finger pads. Most disease-causing variants identified to date are putative loss-of-function alleles, although 15-20% of cases are attributed to missense variants. We describe here four patients (including one previously published patient) with de novo KMT2D missense variants and with shared but unusual clinical findings not typically seen in Kabuki syndrome, including athelia (absent nipples), choanal atresia, hypoparathyroidism, delayed or absent pubertal development, and extreme short stature. These individuals also lack the typical dysmorphic facial features found in Kabuki syndrome. Two of the four patients had severe interstitial lung disease. All of these variants cluster within a 40-amino-acid region of the protein that is located just N-terminal of an annotated coiled coil domain. These findings significantly expand the phenotypic spectrum of features associated with variants in KMT2D beyond those seen in Kabuki syndrome and suggest a possible new underlying disease mechanism for these patients.

Infants with Atypical Presentations of Alveolar Capillary Dysplasia with Misalignment of the Pulmonary Veins Who Underwent Bilateral Lung Transplantation.

OBJECTIVE: To describe disease course, histopathology, and outcomes for infants with atypical presentations of alveolar capillary dysplasia with misalignment of the pulmonary veins (ACDMPV) who underwent bilateral lung transplantation. STUDY DESIGN: We reviewed clinical history, diagnostic studies, explant histology, genetic sequence results, and post-transplant course for 6 infants with atypical ACDMPV who underwent bilateral lung transplantation at St. Louis Children's Hospital. We compared their histology with infants with classic ACDMPV and compared their outcomes with infants transplanted for other indications. RESULTS: In contrast with neonates with classic ACDPMV who present with severe hypoxemia and refractory pulmonary hypertension within hours of birth, none of the infants with atypical ACDMPV presented with progressive neonatal respiratory failure. Three infants had mild neonatal respiratory distress and received nasal cannula oxygen. Three other infants had no respiratory symptoms at birth and presented with hypoxemia and pulmonary hypertension at 2-3 months of age. Bilateral lung transplantation was performed at 4-20 months of age. Unlike in classic ACDMPV, histopathologic findings were not distributed uniformly and were not diffuse. Three subjects had apparent nonmosaic genetic defects involving FOXF1. Two infants had extrapulmonary anomalies (posterior urethral valves, inguinal hernia). Three transplanted children are alive at 5-16 years of age, similar to outcomes for infants transplanted for other indications. Lung explants from infants with atypical ACDMPV demonstrated diagnostic but nonuniform histopathologic findings. CONCLUSIONS: The 1- and 5-year survival rates for infants with atypical ACDMPV are similar to infants transplanted for other indications. Given the clinical and histopathologic spectra, ACDMPV should be considered in infants with hypoxemia and pulmonary hypertension, even beyond the newborn period.

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.

Hepatic steatosis is prevalent in stillborns delivered to women with diabetes mellitus.

OBJECTIVE: Maternal diabetes is a risk factor for pregnancy complications, including stillbirth and macrosomia. Evolving data suggest that diabetes during pregnancy also has long-term consequences for offspring, putting them at risk for obesity and the metabolic syndrome in childhood. Because nonalcoholic fatty liver disease is known to occur in adults and children with insulin resistance, we hypothesized that altered lipid metabolism in fetuses of diabetic mothers may manifest with hepatic steatosis. METHODS: We undertook a retrospective autopsy study to compare the presence and degree of hepatic steatosis between stillborns delivered to women with pregestational or gestational diabetes mellitus (gestational age 20-40 weeks; n = 33) and age-matched nondiabetic control stillbirth cases (n = 48), the latter enriched for maternal obesity, macrosomia, and similar cause of demise. RESULTS: Histopathologic hepatic steatosis was significantly more prevalent and severe in the diabetic subjects (26/33, 78.8%) than in the controls (8/48, 16.6%) (P < 0.001). Within the diabetic cohort, the severity of steatosis was related directly to gestational age, birth weight, and liver weight, with no correlation of presence or severity of steatosis in the control group to maternal or fetal factors, including maternal body mass index or fetal macrosomia. Although macrosomic stillborns were more common in diabetic women with %hemoglobin A1c >6 and body mass index >30 kg/m, fetal steatosis was independent of glycemic control, maternal obesity, type of diabetes, ethnicity, or fetal sex in our cohort. CONCLUSIONS: This study is the first to our knowledge to demonstrate a specific association between fetal hepatic steatosis and maternal diabetes.

Two deletions overlapping a distant FOXF1 enhancer unravel the role of lncRNA LINC01081 in etiology of alveolar capillary dysplasia with misalignment of pulmonary veins.

Position effects due to disruption of distant cis-regulatory regions have been reported for over 40 human gene loci; however, the underlying mechanisms of long-range gene regulation remain largely unknown. We report on two patients with alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) caused by overlapping genomic deletions that included a distant FOXF1 transcriptional enhancer mapping 0.3 Mb upstream to FOXF1 on 16q24.1. In one patient with atypical late-onset ACDMPV, a ∼1.5 Mb deletion removed the proximal 43% of this enhancer, leaving the lung-specific long non-coding RNA (lncRNA) gene LINC01081 intact. In the second patient with severe neonatal-onset ACDMPV, an overlapping ∼194 kb deletion disrupted LINC01081. Both deletions arose de novo on maternal copy of the chromosome 16, supporting the notion that FOXF1 is paternally imprinted in the human lungs. RNAi-mediated knock-down of LINC01081 in normal fetal lung fibroblasts showed that this lncRNA positively regulates FOXF1 transcript level, further indicating that decrease in LINC01081 expression can contribute to development of ACDMPV.

Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype.

BACKGROUND: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. METHODS: We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. RESULTS: We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. CONCLUSION: This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes.

Lysoptosis is an evolutionarily conserved cell death pathway moderated by intracellular serpins.

Lysosomal membrane permeabilization (LMP) and cathepsin release typifies lysosome-dependent cell death (LDCD). However, LMP occurs in most regulated cell death programs suggesting LDCD is not an independent cell death pathway, but is conscripted to facilitate the final cellular demise by other cell death routines. Previously, we demonstrated that Caenorhabditis elegans (C. elegans) null for a cysteine protease inhibitor, srp-6, undergo a specific LDCD pathway characterized by LMP and cathepsin-dependent cytoplasmic proteolysis. We designated this cell death routine, lysoptosis, to distinguish it from other pathways employing LMP. In this study, mouse and human epithelial cells lacking srp-6 homologues, mSerpinb3a and SERPINB3, respectively, demonstrated a lysoptosis phenotype distinct from other cell death pathways. Like in C. elegans, this pathway depended on LMP and released cathepsins, predominantly cathepsin L. These studies suggested that lysoptosis is an evolutionarily-conserved eukaryotic LDCD that predominates in the absence of neutralizing endogenous inhibitors.

Lung transplantation for pulmonary fibrosis in dyskeratosis congenita: Case Report and systematic literature review.

BACKGROUND: Dyskeratosis congenita (DC) is a progressive, multi-system, inherited disorder of telomere biology with high risks of morbidity and mortality from bone marrow failure, hematologic malignancy, solid tumors and pulmonary fibrosis. Hematopoietic stem cell transplantation (HSCT) can cure the bone marrow failure, but it does not eliminate the risks of other complications, for which life-long surveillance is required. Pulmonary fibrosis is a progressive and lethal complication of DC. CASE PRESENTATION: In this report, we describe a patient with DC who developed pulmonary fibrosis seven years after HSCT for severe aplastic anemia, and was successfully treated with bilateral lung transplantation. We also performed a systematic literature review to understand the burden of pulmonary disease in patients with DC who did or did not receive an HSCT. Including our patient, we identified 49 DC patients with pulmonary disease (12 after HSCT and 37 without HSCT), and 509 with no reported pulmonary complications. CONCLUSION: Our current case and literature review indicate that pulmonary morbidity is one of the major contributors to poor quality of life and reduced long-term survival in DC. We suggest that lung transplantation be considered for patients with DC who develop pulmonary fibrosis with no concurrent evidence of multi-organ failure.

Death due to asthma in two adolescents with sickle cell disease.

In one prospective cohort study, a diagnosis of asthma was associated with an increased risk of mortality in individuals with sickle cell anemia or HbSS. However, a direct relationship between death and asthma exacerbation could not be documented. This report examines two adolescents with sickle cell disease (SCD) who died during worsening symptoms consistent with asthma. Autopsies in both individuals demonstrated pulmonary airway smooth muscle hyperplasia, basement membrane thickening, and eosinophilia, consistent with bronchial asthma. Individuals with SCD and asthma warrant ongoing education, treatment, and surveillance for life-threatening complications of asthma.

Granulocyte macrophage colony-stimulating factor ameliorates DSS-induced experimental colitis.

BACKGROUND: Sargramostim, granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor, stimulates cells of the intestinal innate immune system. Clinical trials show that sargramostim induces clinical response and remission in patients with active Crohn's disease. To study the mechanism, we examined the effects of GM-CSF in the dextran sulfate sodium (DSS)-induced acute colitis model. We hypothesized that GM-CSF may work through effects on dendritic cells (DCs). METHODS: Acute colitis was induced in Balb/c mice by administration of DSS in drinking water. Mice were treated with daily GM-CSF or phosphate-buffered saline (PBS). To probe the role of plasmacytoid DCs (pDCs) in the response to GM-CSF, we further examined the effects of monoclonal antibody 440c, which is specific for a sialic acid-binding immunoglobulin (Ig)-like lectin expressed on pDCs. RESULTS: GM-CSF ameliorates acute DSS-induced colitis, resulting in significantly improved clinical parameters and histology. Microarray analysis showed reduced expression of proinflammatory genes including TNF-alpha and IL1-beta; the results were further confirmed by real-time reverse-transcriptase polymerase chain reaction and serum Bio-plex analysis. GM-CSF treatment significantly expands pDCs and type 1 IFN production. Administration of mAb 440c completely blocked the therapeutic effect of GM-CSF. GM-CSF is also effective in RAG1(-/-) mice, demonstrating activity-independent effects on T and B cells. IFN-beta administration mimics the therapeutic effect of GM-CSF in DSS-treated mice. GM-CSF increases systemic and mucosal type 1 IFN expression and exhibits synergy with pDC activators, such as microbial cytosine-phosphate-guanosine (CpG) DNA. CONCLUSIONS: GM-CSF is effective in the treatment of DSS colitis in a mechanism involving the 440c(+) pDC population.

Diffuse lung disease in young children: application of a novel classification scheme.

RATIONALE: Considerable confusion exists regarding nomenclature, classification, and management of pediatric diffuse lung diseases due to the relative rarity and differences in the spectrum of disease between adults and young children. OBJECTIVES: A multidisciplinary working group was formed to: (1) apply consensus terminology and diagnostic criteria for disorders presenting with diffuse lung disease in infancy; and (2) describe the distribution of disease entities, clinical features, and outcome in young children who currently undergo lung biopsy in North America. METHODS: Eleven centers provided pathologic material, clinical data, and imaging from all children less than 2 years of age who underwent lung biopsy for diffuse lung disease from 1999 to 2004. MEASUREMENTS AND MAIN RESULTS: Multidisciplinary review categorized 88% of 187 cases. Disorders more prevalent in infancy, including primary developmental and lung growth abnormalities, neuroendocrine cell hyperplasia of infancy, and surfactant-dysfunction disorders, constituted the majority of cases (60%). Lung growth disorders were often unsuspected clinically and under-recognized histologically. Cases with known surfactant mutations had characteristic pathologic features. Age at biopsy and clinical presentation varied among categories. Pulmonary hypertension, presence of a primary developmental abnormality, or ABCA3 mutation was associated with high mortality, while no deaths occurred in cases of pulmonary interstitial glycogenosis, or neuroendocrine cell hyperplasia of infancy. CONCLUSIONS: This retrospective cohort study identifies a diverse spectrum of lung disorders, largely unique to young children. Application of a classification scheme grouped clinically distinct patients with variable age of biopsy and mortality. Standardized terminology and classification will enhance accurate description and diagnosis of these disorders.

Differentiation of Human Pluripotent Stem Cells into Functional Lung Alveolar Epithelial Cells.

Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.