A new synonymous variant involving an mRNA splicing site in CYP21A2 detected in 12 unrelated patients with deficiency of 21‐hydroxylase

Abstract

To the Editor Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is an inherited autosomal recessive disorder caused by alterations in the CYP21A2 gene. CAH shows a strong genotype–phenotype correlation and, since most pathological alleles are due to mechanisms of asymmetric recombination and gene conversion events between the functional gene and its pseudogene, a small panel of point pathological variants together with a study of deletions and conversions allow to detect a large majority of alleles.Missense, nonsense, frameshift, or intronic rare variants almost complete the characterization afterwhole gene Sanger sequencing, but a small percentage of patients remain uncharacterized. We present 13 patients (12 families) affected with 21-OHD in which CYP21A2 Sanger sequencing together with MLPA did not detect alterations that fulfilled genotypes expected in a recessive disease: neither two severe ones in those with classical forms of CAH nor the second mild or severe alteration in those patients with a non-classical form. This partial characterization led us to expand the genetic studies by adding complementary analyses in order to elucidate other variants detected. Table 1 shows the available biochemical/clinical data and the genotypes identified. Curiously, a synonymous variant was present in these 13 individuals in compound heterozygosity with the previously characterized CYP21A2 alteration (Table 1). This variant, consisting in a thymine instead of a cytosine (c.1116C>T; p.Ser372=, NM_000500.9) involves the 3 coding base of the consensus splicing donor site conserved in about 80% of mammalians and it likely causes abnormal mRNA processing. GenScan reveals that the mutated sequence codes for a different protein in which three new amino acids (Threonine, Proline, and Phenylalanine) are introduced in phase in a region that is coding for the active center of the enzyme. The highest population minor allele frequency observed in any population including 1000 Genomes Phase 3, ESP and genomAD is <0.01. In silico tools such as UMD-Predictor, Human Splicing Finder, Mutation Taster classify the change as potentially pathogenic, while others such as ClinGen and Varsome consider it as of uncertain significance. This alteration has never been reported in ClinVar or a recent revision collecting an exhaustive listing of known CYP21A2 variants. Other data bases such as HGMD, LOVD, and Decipher neither have available data for the variant. Although different haplotypes were detected through analyses of CYP21A2 gene microsatellite markers in 50 and 30 (D6S2792-D6S273CYP21A2-D6S1014-D6S439), the closest marker (D6S273) was identical in the five segregated alleles carrying the change c.1116C>T, suggesting a common origin of the variant. Also supporting this hypothesis, the D6S273-shared-allelewas besides detected in 7 of the8 remaining patients studied (Table 1). The patient not carrying this D6S273-shared-allele (CAH2194) might be explained by a different origin, a phenomenon of slippage (one additional dinucleotide repeat) or the occurrence of recombination phenomena. We seem to be facing a novel pathogenic variant of CAH that might allow us to explain the phenotype observed in our patients. The degree of pathogenicity of this new variant deduced from the clinical and/or biochemical features exhibited by most patients point towards a moderate–severe effect on functionality since classical phenotypes (patients 4223, 9361, 9062 and CAH-2194) had the variant in compound heterozygosity with severe alleles. But a mild effect should also be assumed attending to the clinical signs observed in the non-classical forms with the variant in hemizygosis (patients 7634, CAH-4312 and CAH-4581; Table 1). This behavior could be explained by different degrees of alternative correct splicing acting similarly to how the most frequent pathogenic variant c.293-13C>G does. To our knowledge, this is the first synonymous coding base change involving the splicing site in CYP21A2. The variant would potentially activate a cryptic splice donor site leading to an alternative splicing. Functional studies could help to assess its pathogenicity and understand the degree of severity associated. Also, demonstration of its presence in other populations would contribute to strengthen the evidence of their potential involvement in the disease. Its routinely determination should be recommended in those populations with significant stablished recurrence.