Respiratory failure in a term infant with cis and trans mutations in ABCA3.

A full-term female neonate presented with persistent respiratory failure and radiologic studies consistent with surfactant deficiency. Sequencing of the ATP-binding cassette transporter A3 gene (ABCA3) revealed three mutations: R280C, V1399M and Q1589X. The infant underwent bilateral lung transplantation at 9 months of age and is alive at 3 years of age. Parental sequencing demonstrated that two of the mutations (R280C and Q1589X) were oriented on the same allele (cis), whereas V1399M was oriented on the opposite allele (trans). As more than one mutation in ABCA3 can be present on the same allele, parental studies are needed to determine allelic orientation to inform clinical decision making and future reproductive counseling.

Increased risk for respiratory distress among white, male, late preterm and term infants.

OBJECTIVE: To determine whether race/ethnicity and sex independently increase risk of respiratory distress syndrome (RDS) in late preterm and term infants. STUDY DESIGN: Using a cohort design, we studied the risk of RDS associated with race/ethnicity and sex in infants with gestational age (GA) 34 to 42 weeks born between 1 January 2000 and 31 December 2009 (n=286 454) within 12 hospitals in the Northern California Kaiser Permanente Medical Care Program. RESULT: Male sex (adjusted odds ratio (aOR) 1.68; 95% confidence interval 1.45 to 1.93) and White race/ethnicity (vs Asians (aOR 0.57; 95% confidence interval 0.47 to 0.70), Blacks (aOR 0.66; 95% confidence interval 0.50 to 0.87), and Hispanics (aOR 0.76; 95% confidence interval 0.64 to 0.90)) independently increase risk for RDS regardless of GA. A GA <39 weeks, operative delivery, maternal diabetes, and chorioamnionitis also increased RDS risk in this cohort. CONCLUSION: Male sex and White race/ethnicity independently increase risk for RDS in late preterm and term infants. Timing of elective delivery should acknowledge these risks.

Pilot trial of late booster doses of surfactant for ventilated premature infants.

OBJECTIVE: Many premature infants at risk for bronchopulmonary dysplasia experience episodes of surfactant dysfunction with reduced surfactant protein B (SP-B). In this study, we investigated the safety and responses to booster doses of surfactant. STUDY DESIGN: A total of 87 infants, 500 to 1250 g birth weight, who were ventilated at 7 to 10 days received 2 or 3 doses of Infasurf (Calfactant, Forest Pharmaceuticals, St Louis, MO, USA) within a 1-week period. RESULT: For 184 doses, occurrence rates of transient bradycardia (13) and plugged endotracheal tube (5) were low, and no other adverse effects were noted. Treatment transiently improved the respiratory severity score (FiO(2) × mean airway pressure), SP-B content (+75%) and surface properties of isolated surfactant. Levels of eight proinflammatory cytokines in tracheal aspirate were interrelated and unchanged from baseline after surfactant treatment. CONCLUSION: Booster doses of surfactant for premature infants with lung disease are safe and transiently improve respiratory status as well as composition and function of endogenous surfactant.

The age of necrotizing enterocolitis onset: an application of Sartwell's incubation period model.

OBJECTIVE: Model age of necrotizing enterocolitis (NEC) onset applying Sartwell's model of incubation periods, and examine its relationship to gestational age (GA). STUDY DESIGN: Retrospective chart review of St Louis Children's Hospital neonates diagnosed with NEC (≥Bell's stage II) from 2004 to 2008, inclusive. RESULT: The relationship between age of NEC (N=84 cases) onset and GA best fits a non-linear model, with infants ≤28 weeks having a disproportionately longer time to onset than older GA groups and explained 50.3% of the variability in age of NEC onset. Additional clinical variables provided no improvement in explaining age of NEC onset. Application of Sartwell's model to age of NEC onset proved a good fit, when birth is used as the common exposure episode, and age is equivalent of the incubation period. CONCLUSION: The relationship between day of NEC diagnosis and GA is non-linear, with lower GA infants having disproportionately longer time to onset. Despite these GA differences, the fit to Sartwell's model for incubation periods model is consistent with NEC being a consequence of an event that occurs at or soon after birth.

Pulmonary surfactant kinetics of the newborn infant: novel insights from studies with stable isotopes.

Deficiency or dysfunction of the pulmonary surfactant plays a critical role in the pathogenesis of respiratory diseases of the newborn. After a short review of the pulmonary surfactant, including its role in selected neonatal respiratory conditions, we describe a series of studies conducted by applying two recently developed methods to measure surfactant kinetics. In the first set of studies, namely 'endogenous studies', which used stable isotope-labeled intravenous surfactant precursors, we have shown the feasibility of measuring surfactant synthesis and kinetics in infants using several metabolic precursors, including plasma glucose, plasma fatty acids and body water. In the second set of studies, namely 'exogenous studies', which used a stable isotope-labeled phosphatidylcholine (PC) tracer given endotracheally, we estimated the surfactant disaturated phosphatidylcholine (DSPC) pool size and half-life. The major findings of our studies are presented here and can be summarized as follows: (a) the de novo synthesis and turnover rates of the surfactant (DSPC) in preterm infants with respiratory distress syndrome (RDS) are very low with either precursor; (b) in preterm infants with RDS, pool size is very small and half-life much longer than what has been reported in animal studies; (c) patients recovering from RDS who required higher continuous positive airway pressure pressure after extubation or reintubation have a lower level of intrapulmonary surfactant than those who did well after extubation; (d) term newborn infants with pneumonia have greatly accelerated surfactant catabolism; and (e) infants with uncomplicated congenital diaphragmatic hernia (CDH) and on conventional mechanical ventilation have normal surfactant synthesis, but those requiring extracorporeal membrane oxygenated (ECMO) do not. Information obtained from these studies in infants will help to better tailor exogenous surfactant treatment in neonatal lung diseases.

Surfactant metabolism in the neonate.

With the use of stable isotope-labeled intravenous precursors for surfactant phosphatidylcholine (PC) synthesis, it has been shown that the de novo synthesis rates in preterm infants with respiratory distress syndrome (RDS) are very low as are turnover rates. This is consistent with animal data. Surfactant therapy does not inhibit endogenous surfactant synthesis, and prenatal corticosteroids stimulate it. With the use of stable isotope-labeled PC given endotracheally, surfactant pool size was estimated. It turned out to be low in RDS, as expected. Similar studies were performed in term neonates with severe lung diseases. In general, patients with lung injury show a lower surfactant synthesis. The controversy around surfactant in congenital diaphragmatic hernia (CDH) persists: studies on CDH with and without extracorporeal membrane oxygenation yielded different results. In severe meconium aspiration syndrome surfactant synthesis was found to be decreased but surfactant pool size was maintained. It is possible and safe to study surfactant metabolism in human neonates with the use of stable isotopes. This can help in answering clinical questions and has the potential to bring new in vitro and animal findings about surfactant metabolism to the patient.

Improving survival of vulnerable infants increases neonatal intensive care unit nosocomial infection rate.

OBJECTIVE: To determine the factors associated with an increasing rate of nosocomial infections in infants with very low birth weights. METHODS: Retrospective review of clinical and nosocomial infection databases for all infants with birth weights of 1500 g or less admitted to an academic neonatal intensive care unit between January 1, 1991, and December 31, 1997 (N = 1184). Two study periods were compared: 1991-1995 and 1996-1997. RESULTS: Among the 1085 infants who survived beyond 48 hours, the proportion who developed nosocomial infections increased from 22% to 31% (P =.001) and the infection rate increased from 0.5 to 0.8 per 100 patient-days (P<.001) during the period from 1996 to 1997. In that same period, the median duration of indwelling vascular access increased from 10 to 16 days (P<.001), and the median duration of mechanical ventilation increased from 7 to 12 days (P<.001). Although the device-specific rate of bloodstream or respiratory infections did not change, the increase in infections was directly attributable to the increasing proportion of infants who required these devices. In both study periods, the peak incidence of initial infection occurred between 10 and 20 days of age. For the entire sample, proportional hazard models identified birth weight, duration of vascular access, and postnatal corticosteroid exposure as significant contributors to the risk of infection. CONCLUSIONS: The increasing number of technology-dependent infants was the primary determinant in the increase of nosocomial infections. Because these infections occur in a small proportion of infants, understanding the host factors that contribute to this vulnerability is necessary to decrease nosocomial infections in neonatal intensive care units.

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.

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.

Retinal and intraventricular cerebral hemorrhages in the preterm infant born at or before 30 weeks' gestation.

PURPOSE: To determine the prevalence of retinal hemorrhages and their association with cerebral intraventricular hemorrhages (IVH) in low-birth-weight preterm neonates born at or before 32 weeks' gestation. METHODS: We prospectively studied a consecutive series of 22 neonates (24-30 weeks' gestation; mean gestational age, 27 weeks; mean weight, 1065 g) admitted to the neonatal intensive care unit. Anterior segment and indirect ophthalmoscopic examination, as well as cranial ultrasonographic examination, were performed on day 1 and day 10 of life. The prevalence of retinal and intraventricular hemorrhage was tested statistically for association with obstetric and neonatal clinical variables. RESULTS: The prevalence of retinal hemorrhage was 9% (2/22; 95% CI, 3%-21%) on day 1 and 2% (1/22) on day 10. The prevalence of IVH was 27% (6/22; 95% CI, 9%-46%): 14% (3/22) on day 1 and 23% (5/22) on day 10. Retinal hemorrhages occurred with greater frequency in neonates born to women who had intrauterine infection (chorioamnionitis, P =.043) and low umbilical cord pH levels (P =.027). No association was found between the presence of retinal hemorrhage and IVH (P = 1.000), mode of delivery (ie, vaginal vs cesarean section, P = 1.000), birth weight (P =.476), or gestational age (P = 1.000). The presence of subconjunctival hemorrhage was associated with IVH (P =.046). CONCLUSIONS: Retinal hemorrhages occur in less than 10% of low-birth-weight neonates, ie, a prevalence one half that observed in term neonates (22%). The hemorrhages tend to resolve without sequelae in the first 10 days of life and occur more commonly in infants born to women with uterine infection. Retinal hemorrhages in very premature neonates are not predictive of IVH-related brain damage.

High-frequency oscillation and paralysis stabilize surfactant protein-B--deficient infants.

OBJECTIVE: To determine if high-frequency oscillatory ventilation and neuromuscular blockade improve oxygenation and chest radiographic appearance more effectively than high-frequency oscillation alone for surfactant protein-B (SP-B)--deficient infants. STUDY DESIGN: We reviewed medical records and chest radiographs of five SP-B--deficient infants awaiting lung transplantation. Changes in FiO2 and radiographic scores were analyzed with respect to neuromuscular blockade status. RESULTS: FiO2 consistently increased 0.20 (SD 0.11) during high-frequency ventilation without neuromuscular blockade (p = 0.02) and decreased 0.14 (SD 0.11) during high-frequency ventilation with neuromuscular blockade (p = 0.05). Chest radiographic appearance, quantified by an expansion/aeration index, consistently deteriorated without neuromuscular blockade (p = 0.01) and consistently improved with neuromuscular blockade (p = 0.03). Changes in FiO2 correlated with changes in radiograph scores (r = 0.7, p < 0.001). CONCLUSIONS: High-frequency ventilation with neuromuscular blockade optimizes oxygenation for SP-B--deficient infants. This ventilatory strategy should be considered while awaiting the diagnosis of SP-B deficiency or lung transplantation.

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.

Use of stable isotope labeling technique and mass isotopomer distribution analysis of [(13)C]palmitate isolated from surfactant disaturated phospholipids to study surfactant in vivo kinetics in a premature infant.

Pulmonary surfactant is a complex mixture of phospholipids and proteins which lowers surface tension and maintains alveolar expansion at end expiration. Developmental and genetic disruption of pulmonary surfactant metabolism leads to respiratory distress in newborns. Stable isotope labeling of metabolic precursors of disaturated phospholipids, the most abundant and specific component of pulmonary surfactant, permits the measurement of the kinetics of surfactant metabolism in vivo. We measured [U-(13)C(6)]glucose incorporation into palmitic acid derived from disaturated surfactant phospholipids. A 24 h infusion of [U-(13)C(6)]glucose (140 mg kg(-1)) was administered to a premature infant who required mechanical ventilation for respiratory distress syndrome; tracheal aspirate samples were obtained at the start of the infusion and at regular intervals for the next 70 h. Each tracheal aspirate sample was incubated with osmium tetroxide to isolate disaturated surfactant phospholipids. Methyl esters of the fatty acids in the disaturated phospholipids were prepared and the enrichment of [(13)C]methyl palmitate was measured by gas chromatography/mass spectrometry (GC/MS) and gas chromatography/combination/isotope ratio mass spectrometry (GC/C/IRMS). Mass isotopomer distribution analysis (MIDA) was used to calculate the fractional synthetic rate (FSR) of palmitate synthesized from acetate. With both GC/MS and GC/C/IRMS, palmitate (13)C enrichment was first detected 12.3 h after the start of the tracer infusion. The enrichment increased in a linear fashion, reached a peak at 47 h and remained constant in the remainder of the samples. The FSR of palmitate from acetate was 5.2% per day. Stable isotope techniques and MIDA will provide insights into the kinetics of surfactant metabolism in newborns with respiratory dysfunction.

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.

Lung transplantation in very young infants.

INTRODUCTION: Established successes with adult lung transplantation have laid the foundation for extension of this therapeutic modality to infants and children dying of end-stage pulmonary disease. The purpose of this report is to convey our experience with 19 infants undergoing lung transplantation before the age of 6 months. METHODS: Six patients with predominantly pulmonary vascular disease and 13 patients with primarily pulmonary parenchymal disease have undergone bilateral sequential lung transplantation at our institution since 1990. Mean age at transplant was 104 +/- 44 days, and mean weight was 4.9 +/- 1.6 kg. RESULTS: Although early mortality (32%, 6/19) was higher than that previously reported for older pediatric age groups, long-term survival was similar (44% at a maximum follow-up of 6 years). Although anastomotic complications and infections occurred at a rate approximating that seen in older pediatric age groups, episodes of acute rejection appear to occur with decreased frequency. Similarly, at a mean follow-up of 3 years, only 2 (15%) of 13 long-term survivors have evidence of bronchiolitis obliterans. The functional residual capacity, as measured on infant pulmonary function tests, has gradually increased as the children have grown, suggesting that lung growth is occurring. CONCLUSIONS: Bilateral lung transplantation is a viable alternative in infants dying of end-stage pulmonary disease. Efforts directed toward avoiding the complications that lead to early posttransplant mortality combined with the seemingly lower incidence of early and late rejection may provide long-term results better than those in other age groups.

Measurement of protein flux with positron emission tomography in neonates.

AIM: To determine whether abnormal transvascular protein flux can be measured with positron emission tomography (PET) in neonates with respiratory distress syndrome (RDS). METHODS: Fourteen infants with normal gas exchange (non-RDS group) underwent one PET measurement and 12 infants with RDS (the RDS group) underwent two measurements of protein flux, as determined by the pulmonary transcapillary escape rate for 68Gallium labelled transferrin (PTCER). RESULTS: The mean PTCER for the RDS infants (132 +/- 39 10(-4)/min) was significantly greater than that for infants without RDS (75 +/- 27 10(-4)/min). PTCER did not change between measurements in the infants with RDS, including five who received and responded to surfactant replacement between the two scans. CONCLUSIONS: Increased transvascular flux of large molecular weight proteins complicates RDS in preterm infants. PET provides a tool with which to evaluate the processes that contribute to pulmonary dysfunction in neonates.

Normal lung function in subjects heterozygous for surfactant protein-B deficiency.

Inherited deficiency of surfactant protein-B (SP-B) is a fatal autosomal recessive disorder of lung cell metabolism caused most frequently by a frameshift mutation in codon 121 of the SP-B gene (121ins2) and is characterized by rapidly progressive respiratory failure immediately after birth. Lungs from genetically engineered heterozygous SP-B-deficient mice exhibit decreased compliance and mild air trapping. To determine whether pulmonary function of heterozygous SP-B-deficient humans is similarly affected, we studied nine heterozygous subjects 16 to 44 yr of age and two unaffected subjects 7 and 23 yr of age from five families of 121ins2 SP-B-deficient infants. An increase in residual volume was noted in one heterozygous family member and one unaffected family member. Compliance, maximal transpulmonary pressure, air flow, and gas exchange were normal in all heterozygous subjects tested. These data suggest that humans heterozygous for the 121ins2 mutation have normal pulmonary function through the first four decades of life. The impact of advancing age and environmental exposures on the lung function of heterozygotes remains to be determined.