Sequence Analysis of CYP21A1P in a German Population to Aid in the Molecular Biological Diagnosis of Congenital Adrenal Hyperplasia

Abstract

BACKGROUND The high homology between the CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene) genes is the major obstacle to risk-free genetic diagnosis of congenital adrenal hyperplasia, especially regarding the quantification of gene dosage. Because of the lack of a comprehensive study providing useful information about the detailed genetic structure of CYP21A1P, we used a large data set to analyze and characterize this pseudogene. METHODS We amplified and directly sequenced the CYP21A1P and CYP21A2 genes of 200 unrelated individuals. The resulting sequence data were aligned against the manually curated transcript ENST0000448314 from Havana/Vega matching to the genebuild ENSG00000198457; all differences were documented. Copy number was measured by multiplex ligation-dependent probe amplification when necessary. RESULTS We found that 40 potentially variable positions in CYP21A2 were conserved in CYP21A1P in all study participants. In addition, we detected 14 CYP21A1P variants that were not previously reported in either CYP21A2 or CYP21A1P. Unlike CYP21A2, CYP21A1P possessed certain mutation haplotypes. CONCLUSIONS The genetic structure of CYP21A1P and the potential risks of false conclusions it may introduce are essential considerations in designing a PCR-based diagnosis procedure for congenital adrenal hyperplasia.

Analysis of CYP21A1P and the duplicated CYP21A2 genes

“…Therefore, identification of CYP21A2-like genes and duplicated CYP21A2 genes using either a two-step CYP21A2 amplification (Kharrat et al 2011;Kleinle et al, 2009;Koppens et al 2000;Parajes et al 2008;Wedell et al 1994) or an allele-specific PCR amplification of the CYP21A2 gene (Cantürk et al 2011) may produce PCR drop-off, because it might not be able to distinguish whether CYP21A2 exists downstream of the XB or XA gene. Predictably, the MLPA assay might simultaneously "catch" both the CYP21A2-like (or duplicated CYP21A2) downstream of the TNXA and the CYP21A2 genes downstream of the TNXB.…”

“…The PCR described by Cantürk et al used 2 primers in the 5′ flanking sequence specific for CYP21A2 and CYP21A1P genes, whereas the other 2 primers that are positioned in the 3′ end were specific for 2 genes. Although this PCR is indeed suitable for detecting CYP21A2, CYP21A1P, and the copy number of these two genes, it did find out variants via transfer from the CYP21A2 to CYP21A1P gene (Cantürk et al 2011), which is the CYP21A2-like gene with the 5′end of the CYP21A2 sequence and 3′ end of the CYP21A1P sequence (Tsai et al 2011). Cantürk et al (2011) successfully characterized 200 German populations by this method and found there were 40 potentially variable positions in CYP21A2 which were conserved in CYP21A1P, and 14 CYP21A1P variants were detected.…”

“…Although this PCR is indeed suitable for detecting CYP21A2, CYP21A1P, and the copy number of these two genes, it did find out variants via transfer from the CYP21A2 to CYP21A1P gene (Cantürk et al 2011), which is the CYP21A2-like gene with the 5′end of the CYP21A2 sequence and 3′ end of the CYP21A1P sequence (Tsai et al 2011). Cantürk et al (2011) successfully characterized 200 German populations by this method and found there were 40 potentially variable positions in CYP21A2 which were conserved in CYP21A1P, and 14 CYP21A1P variants were detected. No variants occurring via transfer from CYP21A2 to CYP21A1P gene indicates that it may be rare or non-existent in the German population.…”

“…Recently we read with interest the article by Cantürk et al (2011) published in Clinical Chemistry, in which the authors describe an analysis of the CYP21A1P pseudogene and CYP21A2 gene in a German population using polymerase chain reaction (PCR) amplification and a multiple ligation-dependent probe amplification (MLPA) analysis (Concolino et al 2009). The PCR described by Cantürk et al used 2 primers in the 5′ flanking sequence specific for CYP21A2 and CYP21A1P genes, whereas the other 2 primers that are positioned in the 3′ end were specific for 2 genes.…”

“…Therefore, we question that the result implies that German population might be "independent" in race or such a study needs to assess by more specific method. In addition, analysis of alleles 1 and 2 of CYP21A1P (Cantürk et al supplemental data) of the 200 German samples in Cantürk et al (2011) indicated that frequencies of the CYP21A1P gene containing the CYP21A2 sequence including P30, nt 707-714 (GAGACTAC), I172, V281, Q318, and R356 loci were 4%, 0.3%, 3.6%, 31%, 8.5%, and 46%, respectively. There are some differences with the population of ethnic Chinese (i.e., Taiwanese) in Tsai et al (2011) who showed frequencies of P30, V281, Q318, and R356 loci of 24%, 21%, 11%, and 34%, respectively.…”