The work presented in this thesis describes; (1) development of an in vivo model of human fetal lung development; (2) development of a pyrosequencing genotyping method; and (3) an association of candidate genes with neonatal pulmonary lung disease.
The focus here has been on the role of surfactant proteins in lung development and neonatal lung disease. These proteins are good candidate genes for these studies due to their essential role in lung development, lung maturation, and host defense. Development of a functional alveolar epithelium capable of gas exchange and surfactant secretion is essential for successful adaptation of the fetus to extra-uterine life. Premature birth is commonly accompanied by a deficiency and perturbation of the surfactant system. Pulmonary surfactant is a lipoprotein complex produced by type II pneumocytes that acts to reduce at low lung volumes surface tension at the air-liquid interface in the alveolus and, thereby, prevents atelectasis. Surfactant proteins (SP)-A, SP-B, SP-C, and SP-D have multiple important roles within the alveolus and are subject to developmentally and hormonally regulated expression.
Specific aim I of this thesis describes an in vivo xenograft model of human fetal lung development. Tissues were analyzed from 3 to 42 days post-grafting for morphological alterations. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, a significant increase was observed in both the number of lamellar bodies per cell and lamellar body positive cells. Cellular proliferation, as marked by proliferating cell nuclear antigen (PCNA) ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. Tissues that were grafted longer than 28 days, started to undergo distention of alveoli, presumably due to the accumulation of fluid. These findings indicate that the fetal lung xenograft model can serve as a valuable tool in the study of human fetal lung development. This model can provide the means to study the impact of various pharmacological agents on the development of human fetal lungs in general, and on the surfactant proteins in particular.
In order to better study the role of surfactant protein genetic variants in neonatal respiratory disease, rapid and accurate methods of genotyping are necessary. In this respect, Specific aim IIa of this thesis describes the development of a novel pyrosequencing based method for genotyping of single nucleotide polymorphisms of SP-A, SP-B, and SP-D.
This high-throughput method of genotyping was applied to the study of surfactant protein genetic variants in Bronchopulmonary dysplasia (BPD) in Specific aim IIb. BPD is a chronic lung disease of light weight prematurely born infants that are mechanically ventilated from birth. It has been suggested that genetic factors contribute to BPD pathogenesis. We hypothesized that genetic variants of surfactant proteins are differentially responsive to disruption of surfactant homeostasis in premature birth and are either protection or susceptibility factors for BPD.
In order to study genetic associations of surfactant protein (SP) genetic variants and microsatellite markers linked to SP-B, a family based association study was conducted using the Transmission Disequilibrium Test (TDT) and Family Based Association Test (FBAT). Haplotype analysis revealed two SP-A-SP-D susceptibility haplotypes, and ten susceptibility and one protective haplotypes for SP-B and SP-B-linked microsatellite markers.
Taken together, these studies provide a model that may be used to study lung development and response of the developing lung to therapeutic interventions, and associations of surfactant protein genetic variants with BPD. (Abstract shortened by UMI.)