Hillary B. Heins

Population and Disease-Based Prevalence of the Common Mutations Associated With Surfactant Deficiency

The prevalence of the common mutations in the surfactant protein-B (121ins2), surfactant protein-C (I73T), and ATP-binding cassette member A3 (E292V) genes in population-based or case-control cohorts of newborn respiratory distress syndrome (RDS) is unknown. We determined the frequencies of these mutations in ethnically diverse population and disease-based cohorts using restriction enzyme analysis (121ins2 and E292V) and a 5′ nuclease assay (I73T) in DNA samples from population-based cohorts in Missouri, Norway, South Korea, and South Africa, and from a case–control cohort of newborns with and without RDS (n = 420). We resequenced the ATP-binding cassette member A3 gene (ABCA3) in E292V carriers and computationally inferred ABCA3 haplotypes. The population-based frequencies of 121ins2, E292V, and I73T were rare (<0.4%). E292V was present in 3.8% of newborns with RDS, a 10-fold greater prevalence than in the Missouri cohort (p < 0.001). We did not identify other loss of function mutations in ABCA3 among patients with E292V that would account for their RDS. E292V occurred on a unique haplotype that was derived from a recombination of two common ABCA3 haplotypes. E292V was over-represented in newborns with RDS suggesting that E292V or its unique haplotype impart increased genetic risk for RDS.

Single ABCA3 Mutations Increase Risk for Neonatal Respiratory Distress Syndrome

BACKGROUND AND OBJECTIVE: Neonatal respiratory distress syndrome (RDS) due to pulmonary surfactant deficiency is heritable, but common variants do not fully explain disease heritability. METHODS: Using next-generation, pooled sequencing of race-stratified DNA samples from infants ≥34 weeks’ gestation with and without RDS (n = 513) and from a Missouri population-based cohort (n = 1066), we scanned all exons of 5 surfactant-associated genes and used in silico algorithms to identify functional mutations. We validated each mutation with an independent genotyping platform and compared race-stratified, collapsed frequencies of rare mutations by gene to investigate disease associations and estimate attributable risk. RESULTS: Single ABCA3 mutations were overrepresented among European-descent RDS infants (14.3% of RDS vs 3.7% of non-RDS; P = .002) but were not statistically overrepresented among African-descent RDS infants (4.5% of RDS vs 1.5% of non-RDS; P = .23). In the Missouri population-based cohort, 3.6% of European-descent and 1.5% of African-descent infants carried a single ABCA3 mutation. We found no mutations among the RDS infants and no evidence of contribution to population-based disease burden for SFTPC, CHPT1, LPCAT1, or PCYT1B. CONCLUSIONS: In contrast to lethal neonatal RDS resulting from homozygous or compound heterozygous ABCA3 mutations, single ABCA3 mutations are overrepresented among European-descent infants ≥34 weeks’ gestation with RDS and account for ∼10.9% of the attributable risk among term and late preterm infants. Although ABCA3 mutations are individually rare, they are collectively common among European- and African-descent individuals in the general population.

A major deletion in the surfactant protein-B gene causing lethal respiratory distress

Background: Loss of function mutations in the surfactant protein‐B gene (SFTPB) cause lethal neonatal respiratory distress due to reduced or absent expression of mature surfactant protein B (SP‐B, encoded in exons 6 and 7). No large deletions in SFTPB have been previously identified.

Measurement of human surfactant protein-B turnover in vivo from tracheal aspirates using targeted proteomics

We describe a method to measure protein synthesis and catabolism in humans without prior purification and use the method to measure the turnover of surfactant protein-B (SP-B). SP-B, a lung-specific, hydrophobic protein essential for fetal-neonatal respiratory transition, is present in only picomolar quantities in tracheal aspirate samples and difficult to isolate for dynamic turnover studies using traditional in vivo tracer techniques. Using infusion of [5,5,5-(2)H(3)] leucine and a targeted proteomics method, we measured both the quantity and kinetics of SP-B tryptic peptides in tracheal aspirate samples of symptomatic newborn infants. The fractional synthetic rate (FSR) of SP-B measured using the most abundant proteolytic fragment, a 10 amino acid peptide from the carboxy-terminus of proSP-B (SPTGEWLPR), from the circulating leucine pool was 0.035 +/- 0.005 h(-1), and the fractional catabolic rate was 0.044 +/- 0.003 h(-1). This technique permits high-throughput and sensitive measurement of turnover of low abundance proteins with minimal sample preparation.

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.

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.

Population-based frequency of surfactant dysfunction mutations in a native Chinese cohort

AbstractBackgroundRare mutations in surfactant-associated genes contribute to neonatal respiratory distress syndrome. The frequency of mutations in these genes in the Chinese population is unknown.MethodsWe obtained blood spots from the Guangxi Neonatal Screening Center in Nanning, China that included Han (n=443) and Zhuang (n=313) ethnic groups. We resequenced all exons of the surfactant proteins-B (SFTPB), -C (SFTPC), and the ATP-binding cassette member A3 (ABCA3) genes and compared the frequencies of 5 common and all rare variants.ResultsWe found minor differences in the frequencies of the common variants in the Han and Zhuang cohorts. We did not find any rare mutations in SFTPB or SFTPC, but we found three ABCA3 mutations in the Han [minor allele frequency (MAF)=0.003] and 7 in the Zhuang (MAF=0.011) cohorts (P=0.10). The ABCA3 mutations were unique to each cohort; five were novel. The collapsed carrier rate of rare ABCA3 mutations in the Han and Zhuang populations combined was 1.3%, which is significantly lower than that in the United States (P<0.001).ConclusionThe population-based frequency of mutations in ABCA3 in south China newborns is significantly lower than that in United States. The contribution of these rare ABCA3 mutations to disease burden in the south China population is still unknown.

Synonymous ABCA3 variants do not increase risk for neonatal respiratory distress syndrome.

OBJECTIVE To determine whether synonymous variants in the adenosine triphosphate-binding cassette A3 transporter (ABCA3) gene increase the risk for neonatal respiratory distress syndrome (RDS) in term and late preterm infants of European and African descent. STUDY DESIGN Using next-generation pooled sequencing of race-stratified DNA samples from infants of European and African descent at ≥34 weeks gestation with and without RDS (n = 503), we scanned all exons of ABCA3, validated each synonymous variant with an independent genotyping platform, and evaluated race-stratified disease risk associated with common synonymous variants and collapsed frequencies of rare synonymous variants. RESULTS The synonymous ABCA3 variant frequency spectrum differs between infants of European descent and those of African descent. Using in silico prediction programs and statistical strategies, we found no potentially disruptive synonymous ABCA3 variants or evidence of selection pressure. Individual common synonymous variants and collapsed frequencies of rare synonymous variants did not increase disease risk in term and late-preterm infants of European or African descent. CONCLUSION In contrast to rare, nonsynonymous ABCA3 mutations, synonymous ABCA3 variants do not increase the risk for neonatal RDS among term and late-preterm infants of European or African descent.

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.