A CYP11A1 homozygous exonic variant inducing an alternative splicing, frameshift and truncation in a family with congenital adrenal hyperplasia

Abstract

Background: Congenital adrenal hyperplasia (CAH) is a heterogeneous group of adrenal steroidogenesis disorders with variable degrees of glucocorticoid, mineralocorticoid and sex steroid deficiencies. CYP11A1 gene encodes the mitochondrial cholesterol side-chain cleavage enzyme (P450scc), which initiates the first reaction in steroidogenesis by converting cholesterol to pregnenolone. Variants in this gene are extremely rare but associated with severe forms of CAH due to its early and critical function in various steroid biosynthesis pathways. Here, we report a CYP11A1 exonic homozygous variant that, although exonic in location, affects splicing by creating an additional aberrant splicing site with frameshift and truncation of the gene.

Patients and methods: The proband is a 23-year old 46,XY patient raised as a girl. She was a product of normal pregnancy for first-degree relative parents. Soon after birth, she had vomiting, dehydration, hypotension, hyponatremia and hyperkalemia. She was started on glucocorticoids and mineralocorticoids with prompt recovery. Apart from a chronic need for these medications, her neonatal and childhood history was unremarkable. She sought medical advice at age 19 years for delayed puberty with primary amenorrhea and lack of breast development. On evaluation, she had normal external female genitalia, no breast development, undescended testes and absent uterus and ovaries. Her hormonal evaluation revealed very low estrogen, testosterone, cortisol, aldosterone, 17-hydroxyprogesterone, and androstenedione levels. ACTH, LH, FSH and renin were very high consistent with primary gonadal and adrenal failure. Her parents are healthy first-degree cousins. She has three sisters, all with 46,XX karyotype. One of them is clinically and biochemically normal while the other two sisters have normal female phenotype, normal uterus and ovaries, similar hormonal profile to the proband but different karyotype (46,XX) and absence of undescended testes. gDNA was used for whole exome sequencing (WES). Sanger sequencing was performed to confirm the detected variant and its segregation with the disease.

Results: WES identified a homozygous missense variant in CYP11A1 changing the second nucleotide (GCG > GTG) at position 189 in exon 3 and resulting in a change of Alanine to Valine (p.Ala189Val). This variant was confirmed by PCR and Sanger sequencing. It was found in a homozygous form in the proband and her two affected sisters and in a heterozygous form in the unaffected sister. In-silico analysis predicted this variant to create a new splicing site with frameshift and truncation of the gene transcript. This was confirmed by isolation of RNA, cDNA synthesis, gel electrophoresis and sequencing.

Conclusion: We describe a family with a very rare form of CAH due to a CYP11A1 variant leading to creation of a new splice site, frameshift and premature truncation of the protein.

Keywords: CYP11A1; Congenital adrenal hyperplasia; Missense variant; Whole exome sequencing, Disorders of sex development.

Fig. 1

Figure 1.(A) Pedigree of the consanguineous family who had 3 affected daughters with severe adrenal insufficiency and complex sexual development. Pedigree generated using (https://www.progenygenetics.com); squares (males); circles (females); annotated symbols (affected individuals); open symbols (unaffected individuals); arrowheads (proband). (B) Summary of the clinical, cytogenetic and genital evaluation of the four siblings.

Fig. 2

Figure 2.Molecular characterization of the identified c.566C>T variant in CYP11A1 gene. (A) Schematic diagram and annotation of the identified variant in the CYP11A1 (c.566C>T, p.A189V is located on chromosome chromosome 15q24.1. The variant falls in the third exon. (B) Chromatogram Sanger sequencing segregation analysis of the proband (III-1), affected siblings (III-2 and III-3) and the healthy unaffected sister (III-4). Blue highlight represents the identified variant.

Fig. 3

Figure 3. Characterization of c.566C>T splicing variant in CYP11A1 gene. (A) Schematic representation of the CYP11A1 genomic region. Square represents the location of the identified variant. (B) Agarose gel (2%) demonstrating the results of the RT‐PCR performed on the RNA extracted from the proband and an unrelated normal control, using primers located in exon 3 and exon 4. PCR amplification of the region around the variant showed a decrease in the size of the CYP11A1 gene product in the proband (408 bp) compared the control sample (469 bp). (C) Alignment of the cDNA sequences of the proband and the control. The alignment file was generated using clustal Omega (EMBL-EBI) (https://www.ebi.ac.uk/Tools/msa/clustalo/).

Fig. 4

Figure 4. Sanger sequencing of the wild‐type control and proband’s cDNA. (A) Schematic representation of the variant within exon 3 that creates a donor-splice site that leads to a defective transcript. Dashed lines above the gene structure represent different alternative splicing event. (B) Chromatograms sequence analysis showing a deletion of 61 bp on exon 3 (blue highlight) in the affected individual compared to the control.