Genetic variant characterization in intron 4 of the surfactant protein B gene.

Genetic variants in intron 4 of the surfactant protein B gene SFTPB have been associated with pulmonary morbidity in newborn infants and adults. Genetic variant discovery in intron 4 requires high fidelity polymerase amplification due to a variable number of intermotif dinucleotide repeats and reliable characterization of alleles genetically distinct due to insertion, deletion, or both of 11 conserved sequence motifs. To optimize genetic variant discovery, we selected a high fidelity polymerase enzyme by replicate amplification and compared automated sequencing with agarose gel electrophoresis for all variant alleles (N=68) in a cohort of Missouri infants with (N=187) and without (N=53) respiratory distress. We identified and characterized six new alleles based on sequence motifs and two pairs of variant alleles with similar mobilities by agarose gel electrophoresis but distinct motif sequences. We confirmed uniformity of invariant alleles by sequencing (N=77). Logistic regression using race and intron 4 genotype for infants > or = 35 weeks gestation suggested that the invariant allele was independently associated with increased risk of respiratory distress (OR for the invariant allele 2.7, 95% CI 1.0-7.2). Reliable characterization of genetic variants in intron 4 requires both a high fidelity polymerase and sequencing of variant alleles.

Informed consent for genetic research.

BACKGROUND: Rapid technological advances in genetic research and public concern about genetic discrimination have led to anticipatory safeguards in the informed consent process in the absence of legal examples of proven discrimination. Despite federal and state regulations to restrict access to personal health information, including genetic information, institutional review boards have required the addition of language to informed consent documents that warns about the risks of discrimination with participation in genetic research. OBJECTIVE: To determine the reasons that families refused consent for their infant's participation in a study evaluating a genetic cause of respiratory distress syndrome. DESIGN: Survey conducted between February 1, 2002, and March 31, 2003. SETTING: Academic, tertiary free-standing children's hospital. PARTICIPANTS: A convenience sample of 465 families were approached for consent. The 135 families who refused consent were surveyed. MAIN OUTCOME MEASURES: Reasons for refusal. RESULTS: Of the nonconsenting families, 79% spontaneously and specifically identified institutionally required language in our consent form concerning the risk of denial of access to health insurance and employment as the primary reason for refusal; 97% indicated that their fears resulted directly from language in our consent form. Only 20% of families who refused consent cited inadequate time to consider the study. CONCLUSIONS: The institutionally required description of risk of genetic discrimination due solely to participation in genetic research was the primary reason for refusal to consent in this cohort. Information about federally and institutionally mandated protections for confidentiality of participants in genetic research should be included in the informed consent document to balance the description of hypothetical risks and more accurately inform subjects.

Developmental and genetic regulation of human surfactant protein B in vivo.

BACKGROUND: Genetic and developmental disruption of surfactant protein B (SP-B) expression causes neonatal respiratory distress syndrome (RDS). OBJECTIVES: To assess developmental and genetic regulation of SP-B expression in vivo. METHODS: To evaluate in vivo developmental regulation of SP-B, we used immunoblotting to compare frequency of detection of mature and pro-SP-B peptides in developmentally distinct cohorts: 24 amniotic fluid samples, unfractionated tracheal aspirates from 101 infants >or=34 weeks' gestation with (75) and without (26) neonatal RDS, and 6 nonsmoking adults. To examine genetic regulation, we used univariate and logistic regression analyses to detect associations between common SP-B (SFTPB) genotypes and SP-B peptides in the neonatal RDS cohort. RESULTS: We found pro-SP-B peptides in 24/24 amniotic fluid samples and in 100/101 tracheal aspirates from newborn infants but none in bronchoalveolar lavage from normal adults (0/6) (p < 0.001). We detected an association (p = 0.0011) between pro-SP-B peptides (M(r) 40 and 42 kDa) and genotype of a nonsynonymous single nucleotide polymorphism at genomic position 1580 that regulates amino-terminus glycosylation. CONCLUSIONS: Pro-SP-B peptides are more common in developmentally less mature humans. Association of genotype at genomic position 1580 with pro-SP-B peptides (M(r) 40 and 42 kDa) suggests genetic regulation of amino terminus glycosylation in vivo.

Recombination as a mechanism for sporadic mutation in the surfactant protein-C gene.

OBJECTIVE: To determine haplotype background of common mutations in the genes encoding surfactant proteins B and C (SFTPB and SFTPC) and to assess recombination in SFTPC. STUDY DESIGN: Using comprehensive resequencing of SFTPC and SFTPB, we assessed linkage disequilibrium (LD) (D'), and computationally inferred haplotypes. We computed average recombination rates and Bayes factors (BFs) within SFTPC in a population cohort and near SFTPC (+/-50 kb) in HapMap cohorts. We then biochemically confirmed haplotypes in families with sporadic SFTPC mutations (n = 11) and in individuals with the common SFTPB mutation (121ins2, n = 30). RESULTS: We detected strong evidence (weak LD and BFs > 1,400) for an intragenic recombination hot spot in both genes. The 121ins2 SFTPB mutation occurred predominantly (89%) on 2 common haplotypes. In contrast, no consistent haplotypes were associated with mutated SFTPC alleles. Sporadic SFTPC mutations arose on the paternal allele in four of five families; the remaining child had evidence for somatic recombination on the mutated allele. CONCLUSIONS: In contrast to SFTPB, disease alleles at SFTPC do not share a common haplotype background. Most sporadic mutations in SFTPC occurred on the paternal allele, but somatic recombination may be an important mechanism of mutation in SFTPC.

Comprehensive genetic variant discovery in the surfactant protein B gene.

Completely penetrant mutations in the surfactant protein B gene (SFTPB) and >75% reduction of SFTPB expression disrupt pulmonary surfactant function and cause neonatal respiratory distress syndrome. To inform studies of genetic regulation of SFTPB expression, we created a catalogue of SFTPB variants by comprehensive resequencing from an unselected, population-based cohort (n = 1,116). We found an excess of low-frequency variation [81 SNPs and five small insertion/deletions (in/dels)]. Despite its small genomic size (9.7 kb), SFTPB was characterized by weak linkage disequilibrium (LD) and high haplotype diversity. Using the HapMap Yoruban and European populations, we identified a recombination hot spot that spans SFTPB, was not detectable in our focused resequencing data, and accounts for weak LD. Using homology-based software tools, we discovered no definitively damaging exonic variants. We conclude that excess low-frequency variation, intragenic recombination and lack of common disruptive exonic variants favor complete resequencing as the optimal approach for genetic association studies to identify regulatory SFTPB variants that cause neonatal respiratory distress syndrome in genetically diverse populations.