Splicing analysis of CYP11B1 mutation in a family affected with 11β-hydroxylase deficiency: case report

Abstract

BackgroundCongenital adrenal hyperplasia (CAH) due to steroid 11β-hydroxylase deficiency (11β-OHD) is a rare form of CAH associated with low renin hypertension, hypokalemia, hyperandrogenemia and ambiguous genitalia in affected females. Herein we describe the clinical, hormonal and molecular characteristics of two Uzbekistan siblings with 11β-OHD and analyze the effects of a splicing mutation.Case presentationA 46,XX girl presented with genital ambiguity and low renin hypertension; her 46,XY brother presented with precocious puberty. Hormonal studies suggested 11β-OHD. Mutation analysis was performed by PCR followed by Sanger sequencing of the entire coding regions and their flanking introns of the CYP11B1 gene. Mutation analysis showed that both patients were compound heterozygous for IVS7 + 1G > A, and c.421C > T. Although the identified mutations have been previously described, this is, to our knowledge, the first report of these mutations in compound heterozygotes. A minigene assay was used to determine the effects of the splicing mutation. The constructs containing either the wild-type or the splice-site mutant CYP11B1 genomic DNA of exons-introns 6–9 were transfected into COS-7 cells; subsequently, RNA splicing was assessed by reversed transcribed-PCR of CYP11B1 complementary DNA. The minigene assay revealed that the IVS7 + 1G > A mutation resulted in two shorter incorrectly spliced products; one skipping the exon 7 and the other skipping the exons 7–8. The c.421C > T mutation leads to the introduction of a premature stop codon at residue 141 (p.R141X). These mutations are expected to code non-functional proteins.ConclusionCompound heterozygous mutations (IVS7 + 1G > A and p.R141X) in the CYP11B1 gene were found to cause 11β-OHD. The IVS7 + 1G > A mutation causes aberrant splicing of CYP11B1 leading to exon skipping. This finding could facilitate the future novel therapies targeted on splicing modulation to treat human disease.

CDHR1 mutations in retinal dystrophies

“…It has been demonstrated before that the design of a minigene construct can influence the splicing outcome. Two recent studies that analyzed the very same canonical splice site mutation in the CYP11B1 gene but utilized different minigene amplicons (one containing only two exons, the other four exons) observed very different missplicing outcomes: whereas the shorter minigene amplicon revealed intron retention for the mutant allele, the longer minigene amplicon led to either skipping of one exon or two exons 35 , 36 . To add even more complexity, we observed a small amount of correctly spliced transcript for the c.2040 + 5 G > T variant which might lead to residual protein function.…”

46,XX DSD due to Androgen Excess in Monogenic Disorders of Steroidogenesis: Genetic, Biochemical, and Clinical Features

“…The CYP11B1 gene is located on the long arm of chromosome 8 (8q21–q22), and it is in very close proximity to the highly homologous CYP11B2 gene, which encodes for aldosterone synthase. About 130 mutations of the CYP11B1 gene have been described and are localized all over the gene, with a minor presence in exons 1 and 9 [128,129,130,131,132,133,134,135,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150]. Figure 5 summarizes the majority of allelic variants that have been described so far in the literature for females (62 patients) together with their influence on genital phenotype (see also Table S2).…”