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Splice site mutations are a common cause of X-linked chronic granulomatous disease
M de Boer1, B G Bolscher, M C Dinauer
1Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Insights
Splicing errors in gp91-phox messenger RNA (mRNA) cause X-linked chronic granulomatous disease (CGD). These mutations, affecting correct mRNA splicing, are a common cause of CGD in European patients.
Area of Science:
- Immunology
- Genetics
- Molecular Biology
Background:
- Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by defective phagocyte respiratory burst.
- X-linked CGD, the most common form, results from mutations in the CYBB gene encoding the gp91-phox subunit of NADPH oxidase.
- Understanding the molecular basis of CGD is crucial for diagnosis and potential therapeutic strategies.
Purpose of the Study:
- To investigate the molecular defects in the gp91-phox gene in patients with X-linked CGD.
- To identify the specific mutations leading to the absence of gp91-phox mRNA sequences.
- To determine the frequency of splicing errors as a cause of X-linked CGD.
Main Methods:
- Reverse transcription of gp91-phox messenger RNA (mRNA) into complementary DNA (cDNA).
- Polymerase chain reaction (PCR) amplification of the cDNA coding region.
- DNA sequencing to analyze mutations and identify splice site alterations.
Main Results:
- Sequence analysis revealed the absence of specific exon sequences (exons 7, 5, 3, and 2) in four X-linked CGD patients.
- A carrier patient exhibited cDNA lacking 57 nucleotides from exon 6 due to a novel splice site mutation.
- Single nucleotide substitutions at splice sites or within exon 6 were identified as the cause of aberrant mRNA splicing.
Conclusions:
- The absence of gp91-phox mRNA exons in X-linked CGD patients is caused by splicing errors.
- Mutations affecting mRNA splicing are a common cause of X-linked CGD, identified in five out of 30 studied patients.
- These findings highlight the importance of analyzing mRNA splicing in the genetic diagnosis of CGD.
Abstract:
Chronic granulomatous disease (CGD) is characterized by the absence of a respiratory burst in activated phagocytes. Defects in at least four different genes lead to CGD. Patients with the X-linked form of CGD have mutations in the gene for the beta-subunit of cytochrome b558 (gp91-phox). We studied the molecular defect in four patients with X-linked CGD. In a fifth family, we studied the mother of a patient with X-linked CGD who had died before our investigations. Gp91-phox messenger RNA (mRNA) was reverse transcribed into cDNA and the coding region was amplified by polymerase chain reaction into three fragments. Sequence analysis showed the absence of the exon 7, 5, 3, and 2 sequences in patients 1, 2, 3, and 4, respectively. In carrier 5, we found both normal cDNA and cDNA that lacked 57 3'-nucleotides of exon 6. We analyzed the splice sites of the flanking introns of the missing exons. In patients 1, 2, and 3, we found single nucleotide substitutions within the first five positions of the down-stream 5' donor splice sites. In patient 4, a similar substitution was found at position -1 of the 3' acceptor splice site of intron 1. In carrier 5, no mutation was found in the exon 6-intron 6 boundary sequence. Instead, a single substitution was observed in exon 6 (C----A at nucleotide 633) that created a new donor splice site. Apparently, mRNA splicing occurs preferentially at this newly created splice site. We conclude that the absence of the exon sequences in the gp91-phox mRNA of these patients is due to splicing errors. Of 30 European X-linked CGD patients studied by us so far, five appear to be caused by mutations that affect correct mRNA splicing. Thus, such mutations appear to be a common cause of X-linked CGD.