Sep 01, 2019
Unfortunately, the RCCX module is very complex and current testing only gets at a low percentage of the mutations which can be very expensive. We have not been able to move forward with our research because there are very few labs in the world sophisticated enough to deal with this. I need to find one who will work with us. This region has many kinds of mutations (lots of variety), including point mutations very large insertions (up to 1 million base pairs) and deletions. Additionally, there are copy number issues and expressing genes must be distinguished from pseudo-genes. 23andme sometimes mentions the possibility of a common CYP21A2 mutation, but then it says they can't be sure (because of the pseudogene problem). There need to be several steps, even after WES to figure out if mutations are significant. There are individual probes that you can use: the contiguous overlap mutations (close to $3000 last time I looked) and a couple of others for TNXB meeting EDS criteria and CYP21A2 at Prevention Genetics. There are also common CYP21A2 mutation probes available at most commercial labs. Even after all of this testing, there is still a good chance that an existing CYP21A2 mutation would be missed. The only TNXB mutations anyone is currently interested in are the ones associated with full criteria EDS. There are many more, most of which are likely to have a much wider phenotype then EDS and carry the risk of autoimmune disease (if inherited with C4 mutations) and risk of chronic illness/psych (if co-inherited was CYP21A2). I am diligently trying to get around this problem by writing to geneticists who can handle this region, looking into bioinformatics companies and looking at HPLC to measure 21 hydroxylase.

"The steroid 21-hydroxylase gene, CYP21A2, is located in the human leukocyte antigen class III region on chromosome 6p21.3, closely adjacent in tandem with three other gene(serine/threonine kinase RP, complement C4, and tenascin TNX), forming a genetic module termed RCCX (RP-C4-CYP21-TNX).

The RCCX module shows a high homology between the functional genes (RP1, CYP21A2, and TNXB)and
the corresponding pseudogenes (RP2, CYP21A1P, and TNXA), leading to gene conversions and gene deletions due to homologous recombination, which inactivate the functional genes. Molecular analysis of CYP21A2 gene is of great importance to understanding the etiology of 21-OHD, both in basic science and in clinical diagnosis. Approximately 95% of defective CYP21A2 genes seen in CAH fall into three categories i)approximately 65% to 70% are deleterious mutations derived from pseudogene CYP21A1P due to small gene conversions, including In2G [IVS-13 A/C->G (28%)], p.I172N (9%), p.V281L (9%), p.Q318X (4%), p.R356W(4%), E6 cluster [p.I235N, p.V236E, p.M238K (4%)],p.G110fx21 (3%), p.P30L (2%), and p.L 307fx15 (1%)2,7; ii)approximately 5% are spontaneous point mutations; and iii)approximately 25% to 30% are large gene rearrangements generated by unequal meiotic crossing over.

Large CYP21A2 gene rearrangements have been traditionally detected by Southern blot (SB) analysis. Other strategies including PCR-based restriction fragment length polymorphism (RFLP) and the real-time quantitative PCR based method for CYP21A2 copy number detection have also been described; however, only 82% of studied subjects showed agreement between SB analysis and the quantitative PCR-based method. Recently, multiplex ligation-dependent probe amplification (MLPA) has been increasingly used for identification of CYP21A2 gene deletion and duplication, suggesting that MLPA may replace SB analysis as an efficient strategy to identify gene copy number. Coeli et al combined MLPA and SB in the genetic analysis of 20 Brazilian patients with 21-OHD. However, to our knowledge, a study comparing SB and MLPA in the evaluation of CYP21A2 gene rearrangements in a large cohort of patients representing diverse genotypes with 21-OHD has not been performed. Moreover, distinction among CYP21A2 deletions is clinically significant. Certain junction sites of CYP21A1P/CYP21A2 chimera have been associated with a milder phenotype, the so-called attenuated chimera. In addition, contiguous deletion ofCYP21A2 and TNXB has been associated with Ehlers Danlos syndrome in a significant subset of CAH patients. Identification of these clinically significant chimeras has been incorporated into our mutation analysis strategy.

An additional challenge in the molecular evaluation of CYP21A2 results from the proximity and homology (98% in exons and 96% in intron of a pseudogene,CYP21A1P. Impure PCR products can be potentially generated by amplifying the pseudogene, with detection of pseudogene mutations rather than functional gene mutations. To address this issue,several methods have been developed in the past three decades, such as high-resolution melting curve, locus-specific PCR, denaturing high-performance liquid chromatography analysis, and multiplex minisequencing. These methods adopt a common core strategy based on the known mutations and have some limitations with the maximum overall sensitivity of mutation detection achieved, which is reported to range from 90% to 95%."