In march Alkor Bio’s Laboratory of Molecular Diagnostics has finished development of the kits for mutations determination in the CYP21A2 gene. Approximately 90% of the classic forms of CAH, salt-wasting and simple virilizing, are due to mutations in the CYP21A2 gene causing the disorder in functioning of the 21-hudroxylasa enzyme.
Development of the kits for revealing of mutations in the CYP21A2 gene was ordered by Gene-technology Company from Samara city (Russia). The approbation of the kits was carried out in the Samara Regional Center of Family planning and Reproduction. After successful completion of clinic trials Gene-technology Company started to use new kits for diagnostic purposes.
Congenital deficiency of the enzyme 21-hydroxylase is usually negatively affects on reproductive function, so these tests have a greatest demand in medical institutes specializing in infertility problems.
The Head of PCR-Diagnostic’s Group of Alkor Bio’s Laboratory of Molecular Diagnostics Vitaly Vedernikov said:
“We’ve developed 5 kits for revealing mutations in the 21-hydroxylasa gene: CYP21A2*8, CYP21A2*9, CYP21A2*10, CYP21A2*17, CYP21A2*19. Besides, we’ve developed two additional kits needed in the preliminary stages of analysis: the kit for quantification of human DNA and the kit for the preliminary amplification of the gene in presence of the pseudogene. Detection of polymorphisms in the gene 21-hydroxylase consists of 5 stages: DNA Extraction from clinical material; quantification of DNA concentration; DNA dilution to target concentration; preliminary amplification of the CYP21A2 gene and further 1000 fold dilution of PCR-products; duplex analysis for identification of polymorphisms in the CYP21A2 gene. On the 1st stage the DNA extraction could be proceeded by any kit for this purposes. Quantitive analysis of extracted DNA with further DNA dilution to target concentrations is needed to have correct amplification of the CYP21A2 gene on the 4th stage in the presence of the pseudogene (2nd and 3rd stages respectively). 2nd and 3rd stages can be excluded in case of using "Extra DNA Bio" (Alkor Bio production) for DNA extraction from whole blood, due to getting target DNA concentration with this kit without additional manipulations. Preliminary amplification of the 21-hydroxylase gene is performed on the 4th stage. It needs to exclude false-positive results associated with detecting polymorphisms in pseudogene (don’t cause development of the pathology). CYP21A2 gene and it pseudogene have 95% homology in nucleotide sequence. So, we’ve developed the kit allowing specifically amplify only the CYP21A2 gene in presence of it pseudogene including recombinant forms. The 5th stage of the analysis is determination of the mutations. On this stage must be used amplicons obtained on the preliminary amplification stage of the CYP21A2 gene as DNA template. For polimorphisms’s determination we used an approach based on the melting of duplexes which allows determining mutations with high accuracy. We are going to develop kits to other polymorphisms in the CYP21A2 gene”.
To create new kits Alkor Bio Group’s developers used reagents of their own production: Taq M polymerase, probes and primers. Let us remind that in the begging of 2015 Alkor Bio Group announced about the market launch of a new service – the development and production of PCR-test-systems for other companies. PCR-test-systems are widely used for determination and quantitative analysis of microorganisms, determination of genetic polymorphisms (changes in genome-structure) and other research. It could be interesting first of all for Centers of Medical Genetics, Diagnostic laboratories and Research institutes.
The frequency of incidence congenital adrenal hyperplasia (base form) in the world is 1:15000-16000 of newborns, in Russia – 1:8662. Adrenogenital syndrome and congenital adrenal hyperplasia are the human hereditary diseases with an autosomal recessive nature of inheritance, which cause the disorders in steroid hormones biosynthesis in adrenal cortex. Over the 90-95% cases of congenital adrenal hyperplasia are caused of congenital deficiency of steroid 21-hydroxylase. The analysis on mutations in the CYP21A2 gene is very important for predicting disease severity during prenatal diagnostic and timely prenatal or early postnatal therapy. Timely prenatal therapy helps to prevent complications associated with disorders in steroid genesis in utero, such as the damage of reproductive function, stunting, sex transformation, water and electrolyte imbalance in the case of the most severe salt-wasting form of the disease.