Identification and functional characterization of compound heterozygous CYP11B1 gene mutations

Area of Science:

• Genetics
• Endocrinology
• Molecular Biology

Background:

• 11β-Hydroxylase deficiency (11β-OHD) is a primary cause of congenital adrenal hyperplasia (CAH).
• Mutations in the CYP11B1 gene are responsible for 11β-OHD.
• Previous analysis of a Chinese patient revealed a splice-site mutation (c.595+1G>A) in CYP11B1.

Purpose of the Study:

• To investigate the genetic basis of 11β-OHD in a family with a previously identified splice-site mutation.
• To analyze the functional impact of the c.595+1G>A mutation on CYP11B1 gene expression and enzyme activity.

Main Methods:

• DNA extraction and quantitative real-time PCR (qPCR) for gene copy number analysis.
• Whole-exome sequencing (WES) followed by Sanger sequencing for mutation identification.
• In vitro minigene assay to assess pre-mRNA splicing alterations.

Main Results:

• A novel 2840-bp deletion (c.395+661_c.1121+180del) in CYP11B1 was identified in the patient and his father.
• The patient presented compound heterozygous mutations: the novel deletion and the previously identified c.595+1G>A splice-site mutation.
• Minigene assay confirmed c.595+1G>A causes exon 3 deletion, altering the reading frame and leading to premature stop codon.

Conclusions:

• A novel large deletion and a splice-site mutation in CYP11B1 were identified in 11β-OHD patients.
• Both identified mutations disrupt CYP11B1 enzyme activity by altering the reading frame.
• These findings expand the known mutation spectrum of CYP11B1 and aid in accurate molecular diagnosis of 11β-OHD.