An intronic ABCA3 mutation that is responsible for respiratory disease

Abstract

Introduction: Member A3 of the ATP-binding cassette family of transporters (ABCA3) is essential for surfactant metabolism. Nonsense, missense, frameshift, and splice-site mutations in the ABCA3 gene (ABCA3) have been reported as causes of neonatal respiratory failure (NRF) and interstitial lung disease. We tested the hypothesis that mutations in noncoding regions of ABCA3 may cause lung disease.

Methods: ABCA3-specific cDNA was generated and sequenced from frozen lung tissue from a child with fatal lung disease with only one identified ABCA3 mutation. ABCA3 was sequenced from genomic DNA prepared from blood samples obtained from the proband, parents, and other children with NRF.

Results: ABCA3 cDNA from the proband contained sequences derived from intron 25 that would be predicted to alter the structure and function of the ABCA3 protein. Genomic DNA sequencing revealed a heterozygous C>T transition in intron 25 trans to the known mutation, creating a new donor splice site. Seven additional infants with an ABCA3-deficient phenotype and inconclusive genetic findings had this same variant, which was not found in 2,132 control chromosomes.

Discussion: These findings support that this variant is a disease-causing mutation that may account for additional cases of ABCA3 deficiency with negative genetic studies.

Figure 1. ABCA3 Exons 25–26 Aberrant Splicing

Representative diagram of the ABCA3 cDNA focused on the region of exon 25–26 in the proband. Two additional products can be appreciated migrating at higher molecular weights than observed in the control. Excerpt of DNA sequence shown verifying the presence of the beginning of the intronic sequence corresponding to the additional product.

Figure 2. Transcript-specific RT-PCR and Ban II RE Digest

ABCA3 cDNA showing allele with inserted intronic sequence corresponding to the non p.E690K allele. Using a BanII restriction digest, the known p.E690K mutation eliminates a restriction site in the normally spliced amplicon, generating a larger product (645bp vs. 586bp), confirming the intronic insertion is in trans to the previously identified mutation.

Figure 3. Genomic Sequence Analysis

Genomic sequence with emphasis on the intervening sequence between exons 25 and 26. A C>T transition at the −98 position of IVS25 is present, creating a new donor splice site. An existing upstream potential splice acceptor site is present at the −251 position, giving rise to the 150bp product. DNA sequence analysis is shown verifying the heterozygous C>T transition.