Functional Studies of p.R132C, p.R149C, p.M283V, p.E431K, and a Novel c.652-2A>G Mutations of the CYP21A2 Gene

Abstract

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism and accounts for 90–95% of CAH cases. In the present work, we analyzed the functional consequence of four novel previously reported point CYP21A2 mutations -p.R132C, p.R149C, p.M283V, p.E431K- found in Argentinean 21-hydroxylase deficient patients. In addition, we report an acceptor splice site novel point mutation, c.652-2A>G, found in a classical patient in compound heterozygosity with the rare p.R483Q mutation. We performed bioinformatic and functional assays to evaluate the biological implication of the novel mutation. Our analyses revealed that the residual enzymatic activity of the isolated mutants coding for CYP21A2 aminoacidic substitutions was reduced to a lesser than 50% of the wild type with both progesterone and 17-OH progesterone as substrates. Accordingly, all the variants would predict mild non-classical alleles. In one non-classical patient, the p.E431K mutation was found in cis with the p.D322G one. The highest decrease in enzyme activity was obtained when both mutations were assayed in the same construction, with a residual activity most likely related to the simple virilizing form of the disease. For the c.652-2A>G mutation, bioinformatic tools predicted the putative use of two different cryptic splicing sites. Nevertheless, functional analyses revealed the use of only one cryptic splice acceptor site located within exon 6, leading to the appearance of an mRNA with a 16 nt deletion. A severe allele is strongly suggested due to the presence of a premature stop codon in the protein only 12 nt downstream.

A rare <i>CYP 21</i> mutation (p.E431K) induced deactivation of CYP 21A2 and resulted in congenital adrenal hyperplasia

“…On the other hand, Taboas et al recently described a patient with NC type with p.V281L/p.D322G-p. E431K, and reported that the p.E431K mutation produced about 20% residual enzyme activity and a reduced protein level of CYP21A2 [14,15]. Although their patient did not have p.E431K on single allele and had a different clinical type and combination of mutations, their presented residual enzyme activity and protein data are not consist with our patient's clinical course, or our functional analysis.…”

“…While the IVS2-13 A/C>G mutation is known as a common mutation in patients with the SW type [12,13], p.E431K is a rare point mutation that arises independently of the pseudogene. Although Minutolo et al reported a patient with NC CAH who had p.V281L/p.D322G-p.E431K [14,15], the mutation (NM_000500.7, c. 1401 G>A) has not been reported in the literature or in the single nucleotide polymorphism (SNP) database of the dbSNP-polymorphism repository (http://www.ncbi.nlm.nih.gov/SNP/) apart from their report. This is the first report of a CYP21A2 p.E431K mutation with no accompanying mutation on the same allele.…”

Structure-based activity prediction of CYP21A2 stability variants: A survey of available gene variations

“…To validate our method, we estimated the in silico enzymatic activity of mutants with experimental activities reported from 2014 up to the present21222324, excluding residues known to impair protein function independently of protein stability. To accomplish this, the predicted residual activity after calculation of ∆∆G was compared with the activity reported in functional assays and with the patients’ phenotypes.…”

“…Indeed, a leucine residue is located at this position in the mouse and the rat proteins. In addition, in vitr o studies have suggested that the presence of two mild mutations in cis is generally associated with a severe impairment of enzymatic activity23495051. However, the patient with the p.P335L variant presented a NC phenotype, carrying a large gene conversion/deletion of the CYP21A2 gene on the homologous allele (null allele) and the mild p.V281L mutation in cis.…”

Genetic characterization of a large cohort of Argentine 21‐hydroxylase Deficiency

“…For patients recruited until 2011, the most frequent pseudogene‐derived pathogenic variants in the CYP21A2 gene were screened by allele‐specific PCR or PCR‐RFLP, following the methodology previously established in our laboratory 19 . Samples with at least one nondetermined allele or a discordant phenotype‐genotype correlation and samples from patients recruited from 2011 to 2018 (N = 343), direct sequencing of the entire coding region and the proximal promoter of the CYP21A2 gene was performed as previously described 20,21 . In addition, the distal regulatory region, named promotor Z, 22 was also assessed in samples with the p.P483S variant as described before 23 .…”