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Activation of cryptic splice sites in three patients with chronic granulomatous disease.
Mol Genet Genomic Med 2019; 7(9):e854MG

Abstract

BACKGROUND

Chronic granulomatous disease (CGD) is a primary immune deficiency caused by mutations in the genes encoding the structural components of the phagocyte NADPH oxidase. As a result, the patients cannot generate sufficient amounts of reactive oxygen species required for killing pathogenic microorganisms.

METHODS

We analyzed NADPH oxidase activity and component expression in neutrophils, performed genomic DNA and cDNA analysis, and used mRNA splicing prediction tools to evaluate the impact of mutations.

RESULTS

In two patients with CGD, we had previously found mutations that cause aberrant pre-mRNA splicing. In one patient an exonic mutation in a cryptic donor splice site caused the deletion of the 3' part of exon 6 from the mRNA of CYBB. This patient suffers from X-linked CGD. The second patient, with autosomal CGD, has a mutation in the donor splice site of intron 1 of CYBA that activates a cryptic donor splice site downstream in intron 1, causing the insertion of intronic sequences in the mRNA. The third patient, recently analyzed, also with autosomal CGD, has a mutation in intron 4 of CYBA, 15 bp from the acceptor splice site. This mutation weakens a branch site and activates a cryptic acceptor splice site, causing the insertion of 14 intronic nucleotides into the mRNA.

CONCLUSION

We found three different mutations, one exonic, one in a donor splice site and one intronic, that all caused missplicing of pre-mRNA. We analyzed these mutations with four different splice prediction programs and found that predictions of splice site strength, splice enhancer and splice silencer protein binding and branch site strength are all essential for correct prediction of pre-mRNA splicing.

Authors+Show Affiliations

Sanquin Research and Landsteiner Laboratory, Amsterdam Medical Center, University of Amsterdam, Amsterdam, The Netherlands.Sanquin Research and Landsteiner Laboratory, Amsterdam Medical Center, University of Amsterdam, Amsterdam, The Netherlands.Department of Stem Cell Transplantation Research, University Children's Hospital Zürich, Zürich, Switzerland.Sanquin Research and Landsteiner Laboratory, Amsterdam Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31364312

Citation

de Boer, Martin, et al. "Activation of Cryptic Splice Sites in Three Patients With Chronic Granulomatous Disease." Molecular Genetics & Genomic Medicine, vol. 7, no. 9, 2019, pp. e854.
de Boer M, van Leeuwen K, Hauri-Hohl M, et al. Activation of cryptic splice sites in three patients with chronic granulomatous disease. Mol Genet Genomic Med. 2019;7(9):e854.
de Boer, M., van Leeuwen, K., Hauri-Hohl, M., & Roos, D. (2019). Activation of cryptic splice sites in three patients with chronic granulomatous disease. Molecular Genetics & Genomic Medicine, 7(9), pp. e854. doi:10.1002/mgg3.854.
de Boer M, et al. Activation of Cryptic Splice Sites in Three Patients With Chronic Granulomatous Disease. Mol Genet Genomic Med. 2019;7(9):e854. PubMed PMID: 31364312.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Activation of cryptic splice sites in three patients with chronic granulomatous disease. AU - de Boer,Martin, AU - van Leeuwen,Karin, AU - Hauri-Hohl,Mathias, AU - Roos,Dirk, Y1 - 2019/07/30/ PY - 2019/04/02/received PY - 2019/05/24/revised PY - 2019/06/05/accepted PY - 2019/8/1/pubmed PY - 2019/8/1/medline PY - 2019/8/1/entrez KW - CYBA KW - CYBB KW - chronic granulomatous disease (CGD) KW - cryptic splice site KW - gp91phox deficiency KW - p22phox deficiency SP - e854 EP - e854 JF - Molecular genetics & genomic medicine JO - Mol Genet Genomic Med VL - 7 IS - 9 N2 - BACKGROUND: Chronic granulomatous disease (CGD) is a primary immune deficiency caused by mutations in the genes encoding the structural components of the phagocyte NADPH oxidase. As a result, the patients cannot generate sufficient amounts of reactive oxygen species required for killing pathogenic microorganisms. METHODS: We analyzed NADPH oxidase activity and component expression in neutrophils, performed genomic DNA and cDNA analysis, and used mRNA splicing prediction tools to evaluate the impact of mutations. RESULTS: In two patients with CGD, we had previously found mutations that cause aberrant pre-mRNA splicing. In one patient an exonic mutation in a cryptic donor splice site caused the deletion of the 3' part of exon 6 from the mRNA of CYBB. This patient suffers from X-linked CGD. The second patient, with autosomal CGD, has a mutation in the donor splice site of intron 1 of CYBA that activates a cryptic donor splice site downstream in intron 1, causing the insertion of intronic sequences in the mRNA. The third patient, recently analyzed, also with autosomal CGD, has a mutation in intron 4 of CYBA, 15 bp from the acceptor splice site. This mutation weakens a branch site and activates a cryptic acceptor splice site, causing the insertion of 14 intronic nucleotides into the mRNA. CONCLUSION: We found three different mutations, one exonic, one in a donor splice site and one intronic, that all caused missplicing of pre-mRNA. We analyzed these mutations with four different splice prediction programs and found that predictions of splice site strength, splice enhancer and splice silencer protein binding and branch site strength are all essential for correct prediction of pre-mRNA splicing. SN - 2324-9269 UR - https://www.unboundmedicine.com/medline/citation/31364312/Activation_of_cryptic_splice_sites_in_three_patients_with_chronic_granulomatous_disease L2 - https://doi.org/10.1002/mgg3.854 DB - PRIME DP - Unbound Medicine ER -