0031-3998/00/4803-0275 PEDIATRIC RESEARCH Vol. 48, No. 3, 2000 Copyright © 2000 International Pediatric Research Foundation, Inc. Printed in U.S.A. ARTICLES Prolonged Survival in Hereditary Surfactant Protein B (SP-B) Deficiency Associated with a Novel Splicing Mutation ALSTON E. DUNBAR III, SUSAN E. WERT, MACHIKO IKEGAMI, JEFFREY A. WHITSETT, AARON HAMVAS, FRANCES V. WHITE, BRUNO PIEDBOEUF, CATHERINE JOBIN, SUSAN GUTTENTAG, AND LAWRENCE M. NOGEE Department of Pediatrics [A.E.D., L.M.N.], Division of Neonatology, Johns Hopkins University, Baltimore, Maryland 21287, U.S.A.; Department of Pediatrics [S.E.W., M.I., J.A.W.], Divisions of Pulmonary Biology and Neonatology, Children’s Hospital Medical Center, Cincinnati, Ohio 45229, U.S.A.; Department of Pediatrics [A.H.], Division of Newborn Medicine, Washington University School of Medicine and St. Louis Children’s Hospital, St. Louis, Missouri 63110, U.S.A.; Department of Pathology [F.V.W.], Lauren V. Ackerman Laboratory of Surgical Pathology, Washington University School of Medicine, St. Louis, Missouri 63110, U.S.A.; Pediatric Unit [B.P., C.J.], Research Center, Laval University, St. Foy, Quebec, Canada G1V 4G2; and Department of Pediatrics [S.G.], Division of Neonatology, Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, U.S.A. ABSTRACT Hereditary surfactant protein B (SP-B) deficiency has been SP-B as well as an aberrant SP-B proprotein that corresponded to lethal in the first year of life without lung transplantation. We the size expected from translation of the abnormal transcript. We tested the hypothesis that SP-B gene mutations may result in conclude that a novel splicing mutation was the cause of lung milder phenotypes by investigating the mechanisms for lung disease in these children and that hereditary SP-B deficiency can disease in two children with less severe symptoms than have be the cause of lung disease in older children. (Pediatr Res 48: been previously observed in SP-B deficiency. Immunostaining 275–282, 2000) patterns for pulmonary surfactant proteins were consistent with SP-B deficiency in both children. DNA sequence analysis indi- Abbreviations cated that both children were homozygous for a mutation in exon SP-B, surfactant protein B 5 that created an alternative splice site. Reverse transcriptase proSP-B, SP-B proprotein PCR and sequence analysis confirmed use of this splice site, SP-C, surfactant protein C which resulted in a frameshift and a premature termination codon proSP-C, SP-C proprotein in exon 7. The predominant reverse transcriptase PCR product, RG-PCR, restriction site generating PCR however, lacked exon 7, which restored the reading frame but LB, lamellar body would not allow translation of the exons that encode mature TM, tubular myelin SP-B. Western blot analysis detected reduced amounts of mature Sat PC, saturated phosphatidylcholine Pulmonary surfactant is a complex of lipids and proteins genetically engineered to be homozygous for SP-B null alleles essential for normal lung function. SP-B facilitates spreading and in human infants with an inherited deficiency of SP-B of surfactant lipids, formation of TM, and metabolism of results in lethal respiratory distress (2, 3). Hereditary SP-B surfactant components (1). The absence of SP-B in mice deficiency is an autosomal recessive disorder that typically presents in neonates shortly after birth with unrelenting respi- Received December 29, 1999; accepted February 2, 2000. ratory distress and a chest radiographic appearance similar to Correspondence: Lawrence Nogee, M.D., CMSC 210, 600 North Wolfe St., Baltimore, respiratory distress syndrome. The gene for SP-B has 11 exons MD 21287, U.S.A. spanning 9.5 kb on the short arm of chromosome 2 (4, 5). Supported by NIH HL54703 (L.M.N.), HL54187 (L.M.N.), HL56387 (S.E.W., M.I., J.A.W.), and HL38859 (J.A.W.). A.E.D. and L.M.N. are supported by the Eudowood Translation of SP-B mRNA yields a preproprotein (proSP-B) Foundation. that is processed to a 79-amino acid mature SP-B protein 275 276 DUNBAR ET AL. encoded by exons 6 and 7 (6). The most common mutation A protocol for the evaluation of infants with unexplained causing SP-B deficiency is an insertion in exon 4 (121ins2) that neonatal respiratory disease was approved by the Institutional results in a frameshift and a premature codon for the termina- Review Board of the respective institutions. Informed consent tion of translation and is associated with a marked decrease in was obtained before obtaining blood for genetic studies. SP-B mRNA and the complete absence of proSP-B and mature Antisera, immunohistochemistry, and protein analysis. An- SP-B proteins (3). Although a child who was a compound tisera specific for mature SP-B (28031), full-length proSP-B heterozygote for SP-B gene mutations resulting in a partial (55522), the amino terminal portion of proSP-B (55019), the deficiency of SP-B has been reported, there has been little carboxy terminal portion of proSP-B (96189), full-length variation in the phenotype of SP-B deficiency (7). With ag- proSP-C (76983), and the amino terminal portion of proSP-C gressive respiratory support, SP-B–deficient infants have sur- (68514) were generated in rabbits as previously described (3). vived outside the neonatal period, but, without lung transplan- Immunostaining of formalin-fixed paraffin-embedded lung bi- tation, the disease has been lethal in the first year of life in all opsy specimens for surfactant proteins was performed with and children identified to date (8). We tested the hypothesis that without antigen retrieval using methods that have been previ- mutations in the SP-B gene may result in milder lung disease ously described (9). A Vectastain ABC peroxidase elite rabbit by investigating the mechanisms for lung disease in two unre- IgG kit was used to detect the antigen-antibody complexes lated children of French-Canadian descent. The first was a (Vector Laboratories, Inc., Burlingame, CA, U.S.A.). Lung full-term infant who was oxygen but not ventilator dependent homogenates were prepared from sections of right lower and at 1 mo of age, and the second was an ambulatory 6-y-old with left lower lobe from patient 1 that were obtained at the time of interstitial lung disease and a lifelong oxygen requirement. transplant and snap-frozen in liquid nitrogen. Homogenates were separated by SDS-PAGE, transferred to nitrocellulose paper, and immunoreacted for surfactant proteins (3). Proteins METHODS were detected by enhanced chemiluminescence using a horse- Case Histories radish peroxidase conjugated secondary antibody (Amersham Life Sciences, Arlington Heights, IL, U.S.A.) with exposure to Patient 1. Patient 1 was a 3.5-kg male infant born at 40 wk Kodak x-ray film. The relative amounts of SP-B detected by gestation via cesarean section because of fetal distress who Western blot analysis were quantified by densitometric analy- developed respiratory distress at8hofage. Chest radiographic sis using a commercially available imager and software pack- appearance was consistent with respiratory distress syndrome. age (Gel-Doc 2000 and Quantity One, Bio-Rad, Hercules, CA, He developed a spontaneous pneumothorax at 18 h of age that U.S.A.) under exposure conditions that provided a linear re- required thoracostomy drainage and conventional mechanical sponse to the amount of protein loaded. ventilation. He did not receive surfactant replacement. He was Electron microscopy. Tissue for electron microscopy was extubated but remained tachypneic and required supplemental fixed in 3% glutaraldehyde and postfixed in 1% osmium oxygen. Dexamethasone did not improve his respiratory symp- tetroxide (OsO4) in 0.1 M sodium cacodylate buffer. Tissue toms. There was no family history of neonatal respiratory cubes were stained with 3% uranyl acetate, dehydrated in distress, and the parents were not related. Because of persistent increasing concentrations of alcohol, and embedded in spurr respiratory symptoms, he underwent an open lung biopsy and plastic. Thin sections were mounted on plain copper grids and had blood drawn to evaluate for surfactant protein deficiency at stained with uranyl acetate and lead citrate. 5 wk of life. While he remained extubated, his respiratory DNA analysis. Genomic DNA was isolated from peripheral function progressively declined, and he underwent bilateral blood (Puregene, Gentra Systems, Minneapolis, MN, U.S.A.). lung transplantation at 4 mo of age. Coding regions and flanking intron sequences of the SP-B gene Patient 2. Patient 2 was a full-term female born after an were amplified by PCR as previously described (9). PCR uncomplicated pregnancy who developed respiratory distress products were sequenced using SP-B specific primers end 33 shortly after birth but did not require mechanical ventilation. labeled with P-dATP using T4 polynucleotide kinase (New She had a persistent oxygen requirement prompting an open England Biolabs, Beverly, MA, U.S.A.) and thermocycle se- lung biopsy at 2 mo of age, with histopathologic changes that quencing (Thermosequenase, Amersham Life Sciences, Cleve- included interstitial fibrosis and alveolar proteinosis, although land, OH, U.S.A.). Sequencing reaction products were sepa- no clinical diagnosis was established. Her respiratory symp- rated on 6% polyacrylamide gels and visualized by toms persisted despite diuretic and steroid therapy. She was autoradiography after exposure to Kodak x-ray film. Patient discharged at 6 mo of life with home oxygen therapy. Her SP-B genomic sequences were compared with published SP-B course has been complicated by the progressive development gene sequence (4). of pulmonary hypertension and right ventricular hypertrophy. RG-PCR. An antisense primer (AGGTTTGGGCAGGG- A trial of cyclophosphamide was not effective in treating her GGTCTGACGT; nucleotides 2480–2503) was designed with lung disease. She required a brief period of mechanical venti- a single base pair difference (A3 G) from the wild-type lation at2yofageasaresult of pneumonia.