Surfactant protein B deficiency in neonatal respiratory disease

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Surfactant protein B deficiency is an inherited disease of full-term newborn infants which leads to lethal respiratory failure within the first year of life and is refractory to mechanical ventilation, surfactant therapy, glucocorticoid induction of SP-B production, and extracorporeal membrane oxygenation. The inheritance is autosomal recessive. Two mutations, l2lins2 and R236C have been identified in exons of SP-B gene to date. The l2lins2 mutation is a substitution of three bases (GAA) for the single nucleotide (C) at position 375. The net gain of two bases causes a frameshift and introduces a premature signal for termination of translation after codon 214. The R236C mutation was observed in an infant with a compound heterozygous deficiency, l2lins2 mutation in one allele and the new point mutation in the other allele, a T for C substitution in codon 236, resulting in the substitution of a cysteine for arginine normally encoded by codon 236.We have developed a genetic diagnosis of th … More e mutations, l2lins2 and R236C, in congenital SP-B deficiency. A rapid procedure for microextraction of genomic DNA from whole blood was developed. Using site-directed mutagenesis by overlap extension, the l2lins2 mutation (C->GAA) was introduced into a PCR product. The R236C mutation (C->T) was also introduced using the same technique. The introduction of the mutations in SP-B genome was confirmed by the DNA sequencing after thermal cycling. The mutants were used as a positive control of the mutations. We also developed a rapid procedure for microextraction of genomic DNA from a paraffin-embedded lung tissue. We examined the indicated genomic SP-B mutations in patients with congenital alveolar proteinosis. Furthermore, hydrophilic surfactant proteins, SP-A and SP-D, in sera were determined in a patient with congenital alveolar proteinosis. Those surfactant proteins were measured using enzyme-linked immunosorbent assay with monoclonal antibodies against human SP-A and SP-D, respectively. We could detect a significant amount of SP-A and SP-D in sera, suggesting that the measurement of the surfactant proteins may provide a useful tool to evaluate the respiratory disorder. Less