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Background: The molecular genetic basis of pulmonary arterial hypertension (PAH) is heterogeneous, with at least 26 genes displaying putative evidence for disease causality. Heterozygous variants in the ATP13A3 gene were recently identified as a new cause of adult-onset PAH. However, the contribution of ATP13A3 risk alleles to child-onset PAH remains largely unexplored., Methods and Results: We report three families with a novel, autosomal recessive form of childhood-onset PAH due to biallelic ATP13A3 variants. Disease onset ranged from birth to 2.5 years and was characterised by high mortality. Using genome sequencing of parent-offspring trios, we identified a homozygous missense variant in one case, which was subsequently confirmed to cosegregate with disease in an affected sibling. Independently, compound heterozygous variants in ATP13A3 were identified in two affected siblings and in an unrelated third family. The variants included three loss of function variants (two frameshift, one nonsense) and two highly conserved missense substitutions located in the catalytic phosphorylation domain. The children were largely refractory to treatment and four died in early childhood. All parents were heterozygous for the variants and asymptomatic., Conclusion: Our findings support biallelic predicted deleterious ATP13A3 variants in autosomal recessive, childhood-onset PAH, indicating likely semidominant dose-dependent inheritance for this gene., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

To determine the population-based frequency of a rare mutation (the 121ins2 mutation in the surfactant protein B gene), we developed high-throughput techniques to extract reliably and rapidly amplifiable DNA from Guthrie cards. Using a 3-mm punch from each of 10,044 Guthrie cards obtained from the Missouri Department of Health, we extracted DNA with deionized water by heating in the presence of 2% Chelex in a 96-well format. Average yield of DNA from each punch was 52.6 +/- 21 microg. Using 36mer primers and a 10-microL reaction volume, we amplified a 354-bp fragment of the surfactant protein B gene that contained the mutation and identified the mutation by its susceptibility to restriction enzyme digestion with SfuI. The procedure required 5 h per 96 samples but only 2 h of technician time. The amplification rate on the first attempt was 99.2%. Based on detection of eight individuals heterozygous for the mutation (confirmed by direct sequencing), we estimate the allele frequency to be 0.8/1000 individuals, an estimate not significantly different from previous estimates based on independent methods. High-throughput DNA extraction and amplification will permit establishment of DNA banks as well as efficient estimation of population-based genotype frequency for both rare and common genetic disorders.

Genetic risk for respiratory distress in infancy has been recognized with increasing frequency in neonatal intensive care units. Reports of family clusters of affected infants and of ethnic- and gender-based respiratory phenotypes point to the contribution of inheritance. Similarly, different outcomes among gestationally matched infants with comparable exposures to oxygen, mechanical ventilation, or nutritional deficiency also suggest a genetic risk for respiratory distress. Examples of inherited deficiency of surfactant protein B in both humans and genetically engineered murine lineages illustrate the importance of identifying markers of genetic risk. In contrast to developmental, inflammatory, or nutritional causes of respiratory distress that may resolve as infants mature, genetic causes result in both acute and chronic (and potentially irreversible) respiratory failure. The availability of clinically useful genetic markers of risk for respiratory distress in infancy will permit development of rational strategies for treatment of genetic lung disorders of infancy and more accurate counseling of families whose infants are at genetic risk for development of respiratory distress at birth or during early childhood. We review examples of genetic variations known to be associated with or cause respiratory distress in infancy.

Objective: Determine whether end-tidal CO(2) (ETCO(2)) monitoring allows for more rapid discrimination of tracheal versus esophageal intubation than standard clinical assessment during neonatal resuscitation in the delivery room., Study Design: Endotracheal tube (ETT) placement was assessed using either a hand-held monitor that displayed graphic and quantitative ETCO(2) by an investigator not involved in the resuscitation, or using clinical parameters by the resuscitation team unaware of the ETCO(2) data. The time differences between ETCO(2) and clinical determinations of ETT placement were compared., Results: Capnography correctly identified all 16 tracheal and 11 esophageal intubations performed on 16 study infants. The median times (and range) in seconds required for capnographic and clinical determination of tracheal intubation were 9 (4 to 26) vs. 35 (18 to 70), p<.001, and for esophageal intubation were 9 (4 to 17) vs. 30 (25 to 111), p=.001., Conclusion: Capnography allowed more rapid determination of both tracheal and unintended esophageal intubation than clinical assessment.

Objective: To determine the contribution of surfactant protein abnormalities to the development of chronic lung injury in a familial form of interstitial lung disease., Study Design: An 11-year-old girl, her sister, and their mother who were diagnosed with chronic interstitial lung disease underwent laboratory investigation of surfactant protein expression in bronchoalveolar lavage fluid and lung biopsy specimens. Nineteen patients with idiopathic pulmonary fibrosis and 9 patients who were investigated for pulmonary malignancy but who did not have interstitial lung disease served as control subjects., Results: The 3 family members were found to have absent surfactant protein C (SP-C) and decreased levels of SP-A and SP-B in bronchoalveolar lavage fluid (BALF). Immunostaining for pulmonary surfactant proteins in lung biopsy specimens obtained from both children demonstrated a marked decrease of pro-SP-C in the alveolar epithelial cells but strong staining for pro-SP-B, SP-B, SP-A, and SP-D. No deviations from published surfactant protein B or C coding sequences were identified by DNA sequence analysis. All control subjects had a detectable level of SP-C in the BALF., Conclusion: The apparent absence of SP-C and a decrease in the levels of SP-A and SP-B are associated with familial interstitial lung disease.

Hereditary surfactant protein B (SP-B) deficiency has been lethal in the first year of life without lung transplantation. We tested the hypothesis that SP-B gene mutations may result in milder phenotypes by investigating the mechanisms for lung disease in two children with less severe symptoms than have been previously observed in SP-B deficiency. Immunostaining patterns for pulmonary surfactant proteins were consistent with SP-B deficiency in both children. DNA sequence analysis indicated that both children were homozygous for a mutation in exon 5 that created an alternative splice site. Reverse transcriptase PCR and sequence analysis confirmed use of this splice site, which resulted in a frameshift and a premature termination codon in exon 7. The predominant reverse transcriptase PCR product, however, lacked exon 7, which restored the reading frame but would not allow translation of the exons that encode mature SP-B. Western blot analysis detected reduced amounts of mature SP-B as well as an aberrant SP-B proprotein that corresponded to the size expected from translation of the abnormal transcript. We conclude that a novel splicing mutation was the cause of lung disease in these children and that hereditary SP-B deficiency can be the cause of lung disease in older children.

Two female sibling full-term newborns developed respiratory distress shortly after birth, which progressed to respiratory failure. Tracheal lavage demonstrated presence of surfactant protein A (SP-A), but little surfactant protein B (SP-B), without aberrant surfactant protein C (SP-C). On a lung biopsy performed in both infants, prominent type II pneumocyte hyperplasia was evident. Through ultrastructural examination an absence of normally formed lamellar bodies was determined, with numerous irregular electron dense bodies within the type II pneumocytes. These electron dense bodies could also be identified in the alveolar spaces and alveolar macrophages. No alveolar tubular myelin was present. Abnormally high immunoreactivity for surfactant proteins SP-A, proSP-B, SP-B, and proSP-C was demonstrated by light microscopy. Presence of incompletely processed immunopositive proSP-B, but not proSP-C was observed in the alveolar lumina. No mutations in either the SP-B or SP-C gene were identified by sequence analysis of amplified cDNA. We conclude that these siblings exhibit an inherited surfactant deficiency characterized by abnormal accumulations of surfactant proteins within the pneumocytes. This abnormal accumulation may be due to a primary secretory defect, a defect in surfactant phospholipids, or an abnormal interaction between the phospholipids and surfactant proteins.

Infants with inherited deficiency of pulmonary surfactant protein (SP) B develop respiratory failure at birth and die without lung transplantation. We examined aspects of surfactant metabolism in lung tissue and lavage fluid acquired at transplantation or postmortem from ten infants born at term with inherited deficiency of SP-B; comparison groups were infants with other forms of chronic lung disease (CLD) and normal infants. In pulse/chase labeling studies with cultured deficient tissue, no immunoprecipitable SP-B was observed and an approximately 6-kD form of SP-C accumulated that was only transiently present in CLD tissue. SP-B messenger RNA (mRNA) was approximately 8% of normal in deficient specimens, and some intact message was observed after, but not before, explant culture. Transcription rates for SP-B, assessed by nuclear run-on assay using probes for sequences both 5' and 3' of the common nonsense mutation (121ins2), were comparable in all lungs examined. The minimal surface tension achieved with lavage surfactant was similarly elevated in both deficient and CLD infants (26-31 mN/m) compared with normal infants (6 mN/m). Both SP-B-deficient and CLD infants had markedly decreased phosphatidylglycerol content of lavage and tissue compared with normal lung, whereas synthetic rates for phospholipids, including phosphatidylglycerol, were normal. We conclude that the mutated SP-B gene is transcribed normally but produces an unstable mRNA and that absence of SP-B protein blocks processing of SP-C. Chronic infant lung disease, of various etiologies, reduces surfactant function and apparently alters phosphatidylglycerol degradation.

Objective: Surfactant protein B deficiency is a lethal cause of respiratory distress in infancy that results most commonly from a homozygous frameshift mutation (121ins2). Using independent clinical ascertainment and molecular methods in different populations, we sought to determine allele frequency., Study Design: Using clinical characteristics of the phenotype of affected infants, we screened the Missouri linked birth-death database (n = 1 052 544) to ascertain potentially affected infants. We used molecular amplification and restriction enzyme digestion of DNA samples from a metropolitan New York birth cohort (n = 6599) to estimate allele frequency., Results: The point estimate and 95% confidence interval of the 121ins2 allele frequency in the Missouri cohort are 1/1000 individuals (.03-5.6/1000) and in the New York cohort are.15/1000 (. 08-.25/1000). These estimates are not statistically different., Conclusions: The close approximation of these independent estimates suggests accurate gene frequency (approximately one 121ins2 mutation per 1000-3000 individuals) despite its rare occurrence and that this mutation does not account for the majority of full-term infants with lethal respiratory distress.

Inability to produce surfactant protein B (SP-B) causes fatal neonatal respiratory disease. A frame-shift mutation (121ins2) is the predominant but not exclusive cause of disease. To determine the range of mechanisms responsible for SP-B deficiency, both alleles from 32 affected infants were characterized. Sixteen infants were homozygous for the 121ins2 mutation, 10 infants were heterozygous for the 121ins2 and another mutation, and six infants were homozygous for other mutations. Thirteen novel SP-B gene mutations were identified, which were not found in a control population. One novel mutation was found in two unrelated families. Surfactant protein expression was evaluated by immunohistochemistry and/or protein blotting. Absence of proSP-B and mature SP-B was associated with nonsense and frame-shift mutations. In contrast, proSP-B expression was associated with missense mutations, or mutations causing in-frame deletions or insertions, and low levels of mature SP-B expression were associated with four mutations. Extracellular staining for proSP-C and/or aberrantly processed SP-C was observed in lungs of all infants with SP-B gene mutations. Hereditary SP-B deficiency is caused by a variety of distinct mutations in the SP-B gene and may be associated with reduced, as well as absent, levels of mature SP-B, likely caused by impaired processing of proSP-B.

We report a case of a full-term female infant who presented with severe respiratory distress shortly after birth and died at 23 d of age with unremitting respiratory failure. Infectious and other known causes of respiratory disease in this clinical setting were excluded. Examination of a lung biopsy showed abnormal lung parenchyma with features reminiscent of desquamative interstitial pneumonitis. Ultrastructural studies revealed that alveolar type II cells lacked cytoplasmic lamellar bodies, while other organelles appeared normal. Histochemical and immunohistochemical investigations indicated normal alveolar type II cell marker expression including surfactant proteins (SP-A, SP-B, pro-SP-B, and pro-SP-C). Mutations in the coding sequences of the SP-B gene were excluded as a cause of disease. This case appears to be a novel congenital defect affecting the pulmonary surfactant system. The cellular abnormality may involve the assembly of cytoplasmic lamellar bodies in alveolar type II cells-the principal storage site of pulmonary surfactant.

Hereditary surfactant protein B (SP-B) deficiency is an uncommon autosomal recessive lung disorder that causes hypoxemic respiratory failure in mature, morphologically normal infants. Recognition and diagnosis of this condition is of paramount importance, as it has significant implications for future pregnancies with a recurrence risk of 25%. In a family with three neonatal deaths over 20 years, SP-B deficiency was diagnosed following the death of the fourth affected infant. Previous deaths were mistakenly attributed to hyaline membrane disease (HMD), congenital Mycoplasma hominis infection, and pulmonary hypertension, however, following the diagnosis in the proposita, SP-B deficiency was also confirmed in her deceased siblings by immunohistochemical staining of autopsy specimens. This case highlights the presentation, postnatal course, diagnosis, and therapeutic options of SP-B deficiency in addition to the mode of inheritance and the possibility of antenatal diagnosis. Genetic consultation is imperative in the investigations of recurrent neonatal deaths, especially in cases of remote events. The recent enormous advances in human genetics have shown that many conditions previously ascribed to environmental agents have a genetic basis.

The hydrophobic surfactant proteins, SP-B and SP-C, serve important roles in surfactant function and metabolism. Both proteins are encoded by single genes, located on human chromosomes 2 and 8 respectively, which have been characterized and extensively studied. Mutations in the SP-B gene have been shown to cause severe lung disease, and polymorphisms in the SP-B gene may be associated with the development of RDS in premature infants. In contrast, mutations in the SP-C gene have not yet been identified or shown to cause lung disease, although given the apparent importance of SP-C in surfactant function, this remains a possibility.

A 38-day-old male infant with persistent pulmonary hypertension and respiratory failure since birth was found to have a complete absence of surfactant protein B (SP-B) along with an aberrant form of SP-C in his tracheal aspirate fluid, findings consistent with the diagnosis of hereditary SP-B deficiency. Surprisingly, SP-B and SP-B messenger ribonucleic acid were present in lung biopsy tissue. However, DNA sequence analysis demonstrated a point mutation in exon 5 of one of the SP-B gene alleles. The infant's mother was found to be a carrier of this mutation. The infant's other SP-B allele did not differ from the published DNA sequence for the SP-B gene. We conclude that this patient had a transient deficiency of SP-B, in contrast to that of previously described infants with irreversible respiratory failure caused by hereditary SP-B deficiency. We recommend that infants with suspected SP-B deficiency have serial analysis of tracheal fluid samples for both SP-B and SP-C before lung biopsy, along with genetic analysis for the known SP-B mutations. We speculate that the new mutation found in one of this patient's SP-B genes was in part responsible for the transient deficiency of SP-B.

Objective: To evaluate lung transplantation for treatment of surfactant protein B (SP-B) deficiency., Study Design: We compared surfactant composition and function from pretransplantation and posttransplantation samples of bronchoalveolar lavage fluid, somatic and lung growth, neurodevelopmental progress, pulmonary function, and pulmonary immunohistology in 3 infants with SP-B deficiency who underwent bilateral lung transplantation at 2 months of age and 3 infants who underwent lung transplantation for other reasons., Results: Two years after transplantation, the 2 surviving infants with SP-B deficiency exhibited comparable somatic growth and cognitive development to the comparison infants. All infants had delays in gross motor development that improved with time. Both groups have exhibited normal gas exchange, lung growth, and pulmonary function. The SP-B-deficient infants have also exhibited normal SP-B expression and pulmonary surfactant function after lung transplantation. In two SP-B-deficient infants antibody to SP-B developed. No pathologic consequences of this antibody were identified., Conclusions: Apart from the development of anti-SP-B antibody, the outcomes for SP-B-deficient infants after lung transplantation are similar to those of infants who undergo lung transplantation for other reasons. Lung transplantation offers a successful interim therapy until gene replacement for this disease is available.

Objective: To evaluate components of pulmonary surfactant and identify mutations in the surfactant protein B gene (SP-B) of a term infant with severe respiratory distress and chronic lung disease. PATIENT AND TESTING: Respiratory distress developed in an infant delivered at term, and he required extracorporeal bypass support for 2 weeks. Until his unexpected death at 9.5 months, he was ventilator and oxygen dependent and required continual dexamethasone therapy. Tracheobronchial lavage samples were analyzed for content of surfactant proteins (SPs), and DNA from blood samples were sequenced and analyzed by polymerase chain reaction restriction analysis for the presence of SP-B gene mutations. Surfactant lipid composition and function, the contents of SPs and their messenger RNAs (mRNAs), and the immunostaining pattern for SPs were determined in postmortem lung tissue., Results: The lavage sample contained SP-A but not SP-B, and DNA restriction analysis indicated that the patient and his mother were heterozygous for the previously described 121ins2 mutation of SP-B. Postmortem lung tissue contained normal levels of SP-A and its mRNA, a low but detectable level of SP-B, and near normal content of SP-B mRNA. SP-C was abundant on staining, and some 6-kd precursor was present in tissue. A surfactant fraction was deficient in phosphatidylglycerol and was not surface active. On DNA sequencing, a point mutation was found in exon 7 of the patient's SP-B gene allele without the 121ins2 mutation, resulting in a cysteine for arginine substitution, and the father was a carrier for the same mutation., Conclusions: We describe a patient who is a compound heterozygote with a new mutation and only a partial deficiency of SP-B. Some forms of inherited SP-B deficiency may have low expression of immunoreactive and possibly functional SP-B with milder lung disease and longer survival. These infants may benefit from glucocorticoid therapy and may not develop antibodies to SP-B after either lung transplant or gene therapy.

Surfactant protein B (SP-B) deficiency is an inherited disease of full-term newborn infants which leads to lethal respiratory failure within the first year of life. Genetic analysis of affected infants has permitted identification of a mutation in the SP-B gene found in several unrelated kindreds which disrupts pulmonary surfactant composition and function. Lung transplantation has resulted in reconstitution of pulmonary surfactant function and long-term survival. SP-B deficiency represents the first opportunity to link physiologic characteristics of respiratory failure in infancy with specific molecular and cellular defects. This linkage will facilitate development of novel strategies for the treatment of neonatal respiratory diseases.

An infant with a family history of congenital alveolar proteinosis associated with surfactant protein B (SP-B) deficiency was identified when SP-B was not detected in amniotic fluid obtained at 37, 38, and 40 weeks of gestation. Surfactant replacement with commercially available preparations that contained SP-B was begun soon after delivery. Progressive respiratory failure developed despite continued surfactant replacement, corticosteroid therapy, and extracorporeal membrane oxygenation. The infant died at 54 days of age while awaiting lung transplantation. Surfactant extracted from amniotic fluid, bronchoalveolar lavage fluid, and lung tissue had no phosphatidylglycerol; surface tension was 24 dynes/cm (normal, < 10 dynes/cm) and did not decrease with in vitro addition of exogenous SP-B. Pulmonary vascular permeability measured with positron emission tomography was twice normal. At autopsy the alveolar proteinosis pattern was less prominent than that seen in affected siblings. Immunoreactivity of SP-B was absent in type II cells, but numerous foreign body granulomas with central immunoreactivity for SP-B and surfactant protein C were present. We conclude that exogenous surfactant replacement did not normalize surfactant composition, activity, or pulmonary vascular permeability. These findings suggest that endogenous SP-B synthesis is necessary for mature surfactant metabolism and function.