Familial neuroendocrine cell hyperplasia of infancy.

BACKGROUND: Neuroendocrine cell hyperplasia of infancy (NEHI) is a recently described children's interstitial lung disease (chILD) disorder of unknown etiology. It manifests clinically with tachypnea, retractions, hypoxemia, and crackles. The characteristic radiographic appearance consists of pulmonary hyperexpansion and ground-glass densities on high-resolution computed tomography (HRCT). Lung histology shows hyperplasia of bombesin-immunopositive neuroendocrine cells within distal bronchioles and alveolar ducts without other identifiable lung pathology or developmental anomaly. METHODS: We describe four families with multiple siblings diagnosed with NEHI. Cases were identified at three pediatric centers. Inclusion criteria included clinical findings consistent with NEHI, lung biopsy confirmation in the index case, and a diagnostic HRCT or biopsy in other siblings. RESULTS: Each family had a proband diagnosed with NEHI based upon pathologic review, and at least one additional sibling diagnosed either by pathologic review or HRCT. All patients presented between 2 and 15 months of age. Both male and female children were affected. The majority of the patients underwent both HRCT and lung biopsy. There were no deaths among affected children. No environmental exposures or other potential etiologies were identified as a cause of presenting symptoms. CONCLUSIONS: The familial occurrence of NEHI suggests the possibility of a genetic etiology for this disorder and highlights the importance of taking a complete family medical history for infants presenting with a suggestive clinical picture. Identification of familial NEHI patients allows for the opportunity to further our understanding of this disorder, its natural history, the phenotypic spectrum, and potential genetic causes.

Neonatal respiratory failure due to a novel mutation in the surfactant protein C gene.

A full-term infant developed respiratory distress immediately after birth, requiring a prolonged course of extra-corporeal membrane oxygenation, followed by high-frequency ventilation. She was unable to wean off mechanical ventilation, required tracheostomy, and ultimately lung transplantation. A novel mutation in the surfactant C protein gene was identified as the cause of her lung disease.

Clinical, radiological and pathological features of ABCA3 mutations in children.

BACKGROUND: Mutations in the ABCA3 gene can result in fatal surfactant deficiency in term newborn infants and chronic interstitial lung disease in older children. Previous studies on ABCA3 mutations have focused primarily on the genetic abnormalities and reported limited clinical information about the resultant disease. A study was undertaken to analyse systematically the clinical presentation, pulmonary function, diagnostic imaging, pathological features and outcomes of children with ABCA3 mutations. METHODS: The records of nine children with ABCA3 mutations evaluated at Texas Children's Hospital between 1992 and 2005 were reviewed and their current clinical status updated. Previous diagnostic imaging studies and lung biopsy specimens were re-examined. The results of DNA analyses were confirmed. RESULTS: Age at symptom onset ranged from birth to 4 years. Cough, crackles, failure to thrive and clubbing were frequent findings. Mean lung function was low but tended to remain static. CT scans commonly revealed ground-glass opacification, septal thickening, parenchymal cysts and pectus excavatum. Histopathological patterns included pulmonary alveolar proteinosis, desquamative interstitial pneumonitis and non-specific interstitial pneumonitis, and varied with age. Dense abnormalities of lamellar bodies, characteristic of ABCA3 mutations, were seen by electron microscopy in all adequate specimens. Outcomes varied with the age at which the severity of lung disease warranted open lung biopsy, and some patients have had prolonged survival without lung transplantation. CONCLUSIONS: The presentation and course of interstitial lung disease due to ABCA3 mutations are variable, and open lung biopsy and genetic testing are warranted early in the evaluation of children with a consistent clinical picture.

Ultrastructure of lamellar bodies in congenital surfactant deficiency.

Congenital surfactant deficiency (CSD) is a newly identified neonatal lung disorder associated with a variety of molecular defects affecting surfactant synthesis and secretion in alveolar type II cells. The authors present ultrastructural findings of abnormal lamellar bodies in lung biopsies from 4 infants with CSD. All were term infants presenting shortly after birth with severe respiratory failure that was unresponsive to conventional therapy and all died within the first month of life. Lung biopsies were performed between 8 and 25 days of age. Biochemical and molecular studies in 2 unrelated male infants identified SP-B deficiency, one case with 121 ins 2 mutation and the second with a 209 + 4 A > G mutation. Light microscopy in both cases showed features of alveolar proteinosis. Ultrastructurally, alveolar type II cells lacked mature lamellar bodies, and their cytoplasm contained numerous pleomorphic inclusions with membranous and vesicular structures not seen in normal type II cells. The other 2 infants were a pair of siblings in whom molecular studies identified mutations in ABCA3 transporter gene. Light microscopy showed features of acinar dysplasia and desquamative interstitial pneumonitis. TEM studies revealed absence of mature lamellar bodies in type II cells and instead showed a mixture of cytoplasmic electron-dense inclusions with concentric membranes and distinctive electron dense aggregates. The ultrastructural changes in alveolar type II cells correlated well with specific gene defect. In SP-B deficiency, the absence of mature lamellar bodies is consistent with the postulated role for this protein in the formation of lamellar bodies. The lack of mature lamellar bodies in the ABCA3 gene mutations is due to the dysfunction of this endogenous lipid transporter that targets surfactant lipid moieties to the lamellar bodies. The findings demonstrate the importance of TEM studies of lung biopsies from infants with CSD as it is a critical adjunct in the diagnosis of neonatal lung disease and in defining the underlying cellular defects.

Population-based screening for rare mutations: high-throughput DNA extraction and molecular amplification from Guthrie cards.

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.

Use of capnography in the delivery room for assessment of endotracheal tube placement.

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.

Genetic disorders of neonatal respiratory function.

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.

Surfactant protein deficiency in familial interstitial lung disease.

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.

Recurrent familial neonatal deaths: hereditary surfactant protein B deficiency.

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.

Prolonged survival in hereditary surfactant protein B (SP-B) deficiency associated with a novel splicing mutation.

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.

Absence of lamellar bodies with accumulation of dense bodies characterizes a novel form of congenital surfactant defect.

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.

Pulmonary surfactant metabolism in infants lacking surfactant protein B.

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.

Population-based estimates of surfactant protein B deficiency.

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.

Allelic heterogeneity in hereditary surfactant protein B (SP-B) deficiency.

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.

Deficiency of lamellar bodies in alveolar type II cells associated with fatal respiratory disease in a full-term infant.

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 deficiency resulting from a novel mutation.

Hereditary surfactant protein B (SP-B) deficiency is an autosomal recessive disease in which affected infants are unable to produce normally functional surfactant, resulting in neonatal respiratory failure and death within the first year of life. The most common cause of SP-B deficiency is a frameshift mutation in exon 4 (121ins2) of the SP-B gene. We report a newborn infant who had onset of respiratory distress during the first days, was unresponsive to exogenous surfactant, corticosteroids, prostacyclin, high frequency oscillatory ventilation and inhaled nitric oxide, and died after 27 days. Immunostaining of lung tissue obtained at biopsy demonstrated absent staining for SP-B, and robust extracellular staining for proSP-C, findings characteristic for SP-B deficiency. DNA analysis revealed the 121ins2 mutation on one of her SP-B alleles and a novel mutation, 122delC, on her other SP-B allele. The proximity of the novel mutation in exon 4 allele found in this infant to the 121ins2 supports the notion that this region may represent a "hot spot" for SP-B gene mutations and confirms the heterogeneity of mechanisms which lead to SP-B deficiency. Hereditary SP-B deficiency is a rare, newly diagnosable and probably under-recognized disease, which should be suspected in term newborn infants with unexplained respiratory failure.

Surfactant protein B deficiency: clinical, histological and molecular evaluation.

Congenital alveolar proteinosis due to surfactant protein B deficiency is an inherited disease which results in severe respiratory failure in term infants soon after birth. The pathophysiologic basis of this disease is now known to be an inability to synthesise adequate quantities of normally functioning surfactant protein B. We report a male infant with fatal respiratory failure of neonatal onset, and histopathological features typical of those seen in congenital alveolar proteinosis. Molecular analysis of genomic DNA revealed two mutations, the 'common' 121ins2 mutation in exon 4, and a novel 2bp frameshift mutation in exon 5. We believe this is the first Australian case of surfactant protein B deficiency confirmed by molecular analysis.