Characterization of Genetic Variation in Intron 4 of The Surfactant Protein B Gene.

Abstract

Expression of the surfactant protein B gene is required for function of the pulmonary surfactant. The 9.5 kb surfactant protein B gene includes 10 translated and exons and 1 untranslated exon. Genetic variants in intron 4 characterized by insertions or deletions of 11 distinct motifs have been associated with respiratory distress in some populations of infants, adult respiratory distress syndrome, and risk of squamous cell carcinoma of the lung. Due to polymerase enzyme stutter, characterization of allelic variation by direct sequencing has been difficult. To examine genetic variation in intron 4 in a cohort of Missouri infants (n=240), we identified a polymerase enzyme with high fidelity for intron 4 amplification and analyzed product length by agarose gel electrophoretic mobility. In 480 alleles, we found 14.4% (69/480) variant alleles, 9.4% with insertions and 5.0% with deletions. Allelic diversity was significantly greater among African-Americans (n=204, 19.1% insertion alleles, 2.9% deletion alleles) than Caucasians (n=244, 2.0% insertion alleles, 7.4% deletion alleles) (p<.001). Insertion variants were strongly associated with African Americans (28/31), and deletions were associated with Caucasians (15/20) (p<.001). We then cloned fragments from a subset of 42 infants (22 African-American, 19 Caucasian, 1 Hispanic). Automated sequencing of the 32 variant alleles revealed 6 previously unreported variants (all insertions) that accounted for an unexpectedly high proportion (18/32) of variant alleles sequenced. Two different insertion variants shared agarose gel electrophoretic mobility (Mr~570 bps), as did 2 other insertion variants (Mr~600 bps). When analyzed by automated sequencing, these variants differed in motif insertion or deletion or in length of dinucleotide (CA) repeat. The C genotype at genomic position 1580, a C/T nonsynonymous, single nucleotide polymorphism in exon 4, was strongly associated with insertion (28/31 = T) (p<.001). We conclude that genotype at genomic position 1580 and race are associated with intron 4 variation. Characterization of intron 4 by agarose gel electrophoretic mobility alone may fail to detect important differences in genetic variation in intron 4 that contribute to risk for respiratory disease.