Single nucleotide polymorphism in 5'-flanking region reduces transcription of surfactant protein B gene in H441 cells.

Surfactant protein (SP)-B is expressed in a cell-specific manner and is essential for surfactant function and survival. Abnormal surfactant function occurs in humans and genetically engineered mice with SP-B levels well below 50% of normal. SP-B mRNA levels vary in fetal lung explants among individuals, possibly due to genetic variety. Polymorphisms within the SP-B gene have been described extensively; however, some of their functional relevance remains unclear. Mutations within the SP-B gene may affect mRNA content, but altered gene transcription or mRNA-stability has not been clearly demonstrated. We characterized a single nucleotide polymorphism (SNP) found in the upstream enhancer of SP-B, consisting of a single base pair change in the consensus sequence of the most downstream-located thyroid transcription factor 1 binding element in the upstream enhancer of the SP-B 5'-flanking region and located at position 384 upstream of the transcriptional start site of the SP-B gene. In a small patient population (n = 53) we found 70% were homozygous for the wild type (WT), one individual (2%) was homozygous for the polymorphism (Pm), and 28% were heterozygous. To further elucidate possible functions we performed electromobility shift assays with extracts from H441 cells that showed a reduced binding affinity of the mutated sequence compared with WT. In reporter gene assays the Pm caused a reduction of 53% in transcriptional activity compared with WT in transfected H441 cells. Stimulation of these constructs with retinoic acid resulted in enhanced reporter gene activity of both constructs. After stimulation the Pm still exhibited a reduced activity compared with the WT sequence. We conclude that the described SNP causes differences in SP-B transcriptional activity and thus may contribute to individually different SP-B mRNA levels.