ABSTRACT:The prevalence of the common mutations in the surfactant protein-B (121ins2), surfactant protein-C (I73T), and ATP-binding cassette member A3 (E292V) genes in populationbased or case-control cohorts of newborn respiratory distress syndrome (RDS) is unknown. We determined the frequencies of these mutations in ethnically diverse population and disease-based cohorts using restriction enzyme analysis (121ins2 and E292V) and a 5Ј nuclease assay (I73T) in DNA samples from population-based cohorts in Missouri, Norway, South Korea, and South Africa, and from a case-control cohort of newborns with and without RDS (n ϭ 420). We resequenced the ATP-binding cassette member A3 gene (ABCA3) in E292V carriers and computationally inferred ABCA3 haplotypes. The population-based frequencies of 121ins2, E292V, and I73T were rare (Ͻ0.4%). E292V was present in 3.8% of newborns with RDS, a 10-fold greater prevalence than in the Missouri cohort (p Ͻ 0.001). We did not identify other loss of function mutations in ABCA3 among patients with E292V that would account for their RDS. E292V occurred on a unique haplotype that was derived from a recombination of two common ABCA3 haplotypes. E292V was over-represented in newborns with RDS suggesting that E292V or its unique haplotype impart increased genetic risk for RDS. (Pediatr Res 63: 645-649, 2008)

Genetic variants in intron 4 of the surfactant protein B gene SFTPB have been associated with pulmonary morbidity in newborn infants and adults. Genetic variant discovery in intron 4 requires high fidelity polymerase amplification due to a variable number of intermotif dinucleotide repeats and reliable characterization of alleles genetically distinct due to insertion, deletion, or both of 11 conserved sequence motifs. To optimize genetic variant discovery, we selected a high fidelity polymerase enzyme by replicate amplification and compared automated sequencing with agarose gel electrophoresis for all variant alleles (N=68) in a cohort of Missouri infants with (N=187) and without (N=53) respiratory distress. We identified and characterized six new alleles based on sequence motifs and two pairs of variant alleles with similar mobilities by agarose gel electrophoresis but distinct motif sequences. We confirmed uniformity of invariant alleles by sequencing (N=77). Logistic regression using race and intron 4 genotype for infants > 35 weeks gestation suggested that the invariant allele was independently associated with increased risk of respiratory distress (OR for the invariant allele 2.7, 95% CI 1.0-7.2). Reliable characterization of genetic variants in intron 4 requires both a high fidelity polymerase and sequencing of variant alleles.

SUMMARYObjective-To determine haplotype background of common mutations in the genes encoding surfactant proteins B and C (SFTPB and SFTPC) and to assess recombination in SFTPC.Study design-Using comprehensive resequencing of SFTPC and SFTPB, we assessed linkage disequilibrium (LD) (D'), and computationally inferred haplotypes. We computed average recombination rates and Bayes factors (BFs) within SFTPC in a population cohort and near SFTPC (±50kb) in HapMap cohorts. We then biochemically confirmed haplotypes in families with sporadic SFTPC mutations (n=11) and in individuals with the common SFTPB mutation (121ins2, n=34).Results-We detected strong evidence (weak LD and BFs > 1400) for an intragenic recombination hot spot in both genes. The 121ins2 SFTPB mutation occurred predominantly (89%) on 2 common haplotypes. In contrast, no consistent haplotypes were associated with mutated SFTPC alleles. Sporadic SFTPC mutations arose on the paternal allele in 4 of 5 families; the remaining child had evidence for somatic recombination on the mutated allele.Conclusions-In contrast to SFTPB, disease alleles at SFTPC do not share a common haplotype background. Most sporadic mutations in SFTPC occurred on the paternal allele, but somatic recombination may be an important mechanism of mutation in SFTPC.

Bronchopulmonary dysplasia, a common complication of prematurity, is characterized by inadequate alveolarization. The process of alveolarization, by which the lung forms mature gas-exchanging units, is not well understood. In mice, alveolarizaion occurs during postnatal days four through twelve, when the formation of secondary septa creates thin walled alveoli. The purpose of this study was to investigate the genes involved in this process. RNA was isolated from dissected tips of secondary septa and whole lung tissue of day six postnatal mouse lung. The tips of secondary septa were obtained via laser capture microscopy of frozen sections. Total RNA was isolated from the tip and whole lung samples and amplified in the same manner. Affymetrix gene profiling was then performed, using mouse U74Av2 GeneChips. The signal for galectin-1 mRNA was six fold higher in the secondary septal tips than in whole lung tissue (pϽ0.05). Galectins, or S-type lectins, are beta-galactosidebinding proteins involved in the regulation of cell proliferation, differentiation and apoptosis. Galectin-1 is a homodimer of two 14 kDa subunits. It is the most abundant galectin in the lung. To confirm the relative abundance of galectin-1 in secondary septal tips versus whole lung, immunostaining of sections of day six postnatal and adult mouse lung tissue was performed. Staining for galectin-1 was concentrated in the tips of the secondary septa in the day six postnatal tissue. Furthermore, staining was dramatically increased in the day six mouse lung tissue when compared to staining levels in the adult lung sections. Immunoblot analysis of lung homogenates obtained at different stages of lung development demonstrated that a peak of galectin-1 expression occurs at postnatal days six and twelve, corresponding to the time of alveolarization. The increased expression of galectin-1 at the site and time of ongoing alveolarization suggests that it may play a role in this important aspect of lung development. Supported by NIH grants HL-62861 and HL-07638. SU1498 INHIBITS ALVEOLARIZATION IN NEWBORN MICE.Cho SJ, George CS, Snyder JM, Acarregui MJ. Carver College of Medicine, Univ. of Iowa, Iowa City, IA BACKGROUND: Bronchopulmonary dysplasia (BPD) in premature infants is characterized by inhibited alveolarization and vasculogenesis. Inhibitors of angiogenesis induce emphysema in newborn rats resulting in a phenotype similar to BPD in premature infants. Our goal was to generate a mouse model of inhibited alveolarization that could be employed to explore the mechanisms resulting in, and interventions for BPD. SU1498 is a commercially available compound that inhibits vascular endothelial growth factor receptors. METHODS: Three day old C3H/HeNHsd mice were injected with a single dose of SU1498 (30mg/kg, SC). Lungs of control (sham-injected) and treated mice were inflation fixed on postnatal day 21. Tissue sections were mounted and morphometric analysis was performed to determine the volume density (VD) of air space, tissue, large blood vessels, conducting air...