Investigation of CYP21A2 Gene Variants in Patients Pre-diagnosed with Congenital Adrenal Hyperplasia

Objectives:

Investigation of CYP21A2 gene variants in patients pre-diagnosed with congenital adrenal hyperplasia (CAH) (premature pubarche, virilization).

Methods:

Two milliliters of peripheral blood samples were collected from the patients. Genomic DNA isolation from nucleated cells of the peripheral blood was performed according to the manufacturer’s protocol (EZ1 Advanced Instruments, Qiagen, Hilden, Germany). Exons and exon-intron boundaries of CYP21A2 gene were amplified according to the protocol of the kit (GML AG, Wollerau/Switzerland). Band patterns of amplicons were checked by agarose gel electrophoresis. Nucleotide sequences of the amplicons were defined by Sanger sequencing method (ABI 3130 Avant system, Applied Biosystems, Grand Island, NY 14072, USA). Analysis of the results was performed using SeqScape v2.7 programme (Transcript: CYP21A2-002 ENST00000418967).

Results:

Four female patients were included in the study. Mean age of the patients was ±9.75. c.-4C>T [5’UTR, rs6470], c.308G>A [p.Arg103Lys, rs6474], c.806G>C [p.Ser269Thr, rs6472, HGMD: CM994664], c.1360C>T [p.Pro454Ser, rs6445], c.1473G>A [p.Pro491=, rs6446], c.1481G>A [p.Ser494Asn, rs6473], c.293-13C>A [IVS2AS, rs6467, HGMD: CS880069], c.*52C>T [3’UTR, rs1058152] variations were defined during the analysis of CYP21A2 sequences.

Conclusion:

The results were assessed considering the International genetic databases “dbSNP” and “The Human Gene Mutation Database (HGMD)”. rs6470, rs6474, rs6446, and rs1058152 variations of the patients were reported as “polymorphism” in the dbSNP and HGMD. rs6467 (HGMD: CS880069), rs6472 (HGMD: CM994664), and rs6473 variations have been associated with CAH (21-hydroxylase deficiency) and rs6445 variation has been associated with CAH (21-hydroxylase deficiency) non-classical type. We found the following variations of the CYP21A2 gene: rs6473 - in our first patient followed up for premature pubarche, rs6445 and 6473 - in our second patient followed up for virilization, rs6467 and rs6472 - in the third patient, and rs6473 and rs6472 - in the fourth patient. The CYP21A2 gene variations determined in the patients supported the clinical pre-diagnosis.