Michelle Trusgnich

Population-Based Estimates of Surfactant Protein B Deficiency

Objective. Surfactant protein B deficiency is a lethal cause of respiratory distress in infancy that results most commonly from a homozygous frameshift mutation (121ins2). Using independent clinical ascertainment and molecular methods in different populations, we sought to determine allele frequency. Study Design. Using clinical characteristics of the phenotype of affected infants, we screened the Missouri linked birth–death database (n = 1 052 544) to ascertain potentially affected infants. We used molecular amplification and restriction enzyme digestion of DNA samples from a metropolitan New York birth cohort (n = 6599) to estimate allele frequency. Results. The point estimate and 95% confidence interval of the 121ins2 allele frequency in the Missouri cohort are 1/1000 individuals (.03–5.6/1000) and in the New York cohort are .15/1000 (.08–.25/1000). These estimates are not statistically different. Conclusions. The close approximation of these independent estimates suggests accurate gene frequency (approximately one 121ins2 mutation per 1000–3000 individuals) despite its rare occurrence and that this mutation does not account for the majority of full-term infants with lethal respiratory distress.

Population-Based Screening for Rare Mutations: High-Throughput DNA Extraction and Molecular Amplification from Guthrie Cards

To determine the population-based frequency of a rare mutation (the 121ins2 mutation in the surfactant protein B gene), we developed high-throughput techniques to extract reliably and rapidly amplifiable DNA from Guthrie cards. Using a 3-mm punch from each of 10,044 Guthrie cards obtained from the Missouri Department of Health, we extracted DNA with deionized water by heating in the presence of 2% Chelex in a 96-well format. Average yield of DNA from each punch was 52.6 ± 21 μg. Using 36mer primers and a 10-μL reaction volume, we amplified a 354-bp fragment of the surfactant protein B gene that contained the mutation and identified the mutation by its susceptibility to restriction enzyme digestion with Sfu I. The procedure required 5 h per 96 samples but only 2 h of technician time. The amplification rate on the first attempt was 99.2%. Based on detection of eight individuals heterozygous for the mutation (confirmed by direct sequencing), we estimate the allele frequency to be 0.8/1000 individuals, an estimate not significantly different from previous estimates based on independent methods. High-throughput DNA extraction and amplification will permit establishment of DNA banks as well as efficient estimation of population-based genotype frequency for both rare and common genetic disorders.

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

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.

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.

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 ≥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 (Mr 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 (Mr 40 and 42 kDa) suggests genetic regulation of amino terminus glycosylation in vivo.

YAC/STS map of 15Mb of Xp21.3-p11.3, at 100kb resolution, with refined comparisons of genetic distances and DMD structure.

The 15Mb region between DXS997 and DXS8054 in Xp21.3-p11.3 has been mapped at seven-fold average coverage in yeast artificial chromosomes (YACs) and 100 kb inter-sequence tagged site (STS) distance. YACs from six different collections show self-consistent maps. The STSs include 18 (CA) repeat and one tetranucleotide repeat marker that detect polymorphism, as well as eight well-studied genes, a second site for MXS1 sequences, and three expressed sequence tags (ESTs). One of the ESTs maps to intron 7 of Duchenne muscular dystrophy, and seems to be a processed intronic sequence with a poly(A) tail.

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

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.