Prenatal molecular genetic diagnosis technique for congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Aim: to present a technique for prenatal molecular genetic diagnosis (PD) of congenital adrenal hyperplasia (CAH), including test of common pathogenic CYP21A2 variants, linkage microsatellite markers and sex markers analysis. Methods. DNA samples were isolated from whole blood samples and chorionic villi sample. The research based on methods from literature and own developments. The assay included the analysis of common mutations in the CYP21A2 gene (P30L, IVS2AS A/C-G,-13, 8 bp del, I172N, V281L, Q318X, R356W, P453S), gene-pseudogene rearrangement (deletion of 30 kb), STR markers (TNFa, D6S273, D6S2781, LH1, D6S1666, D6S2443) and sex markers (SRY and AMELX/AMELY). PCR was baseline method for all steps. We performed a nested PCR approach and subsequent restriction analysis for point mutations of the CYP21A2 gene. Methods of gel electrophoresis and fragment analysis on a genetic analyzer were used for detection. Results. Our study describes the detailed approach of prenatal molecular genetic investigation of CAH. Preliminary molecular genetic examination of a family with a salt-wasting form of CAH child was carried out. A compound genotype with pathogenic maternal variants IVS2AS A/C-G, -13 and a complex mutant paternal allele P30L + 8 bp del + IVS2AS A/C-G, -13 was revealed in the affected child. In the fetal sample we identified a heterozygous genotype with a pathogenic maternal allele of the 2nd intron. The polymorphic microsatellite markers flanking the CYP21A2 gene confirmed the normal paternal and pathogenic maternal haplotypes in the fetus. Additionally STR data indicated that there was no contamination of maternal DNA fetal material. The mother was uninformative (homozygous) with two microsatellite markers, the father was informative with all six. Conclusions. We devised an improved stepwise molecular diagnostic strategy includes the direct analysis of pathogenic variants in combination with indirect diagnostics by STR markers and sex analysis for reliable PD of CAH.

Congenital adrenal hyperplasia, CAH, Prenatal diagnosis, CYP21A2 gene, STR

1. OMIM #201910: Adrenal Hyperplasia, Congenital, due to 21-Hydroxylase Deficiency. https://omim.org/entry/201910.

2. Nimkarn S, Gangishetti PK, Yau M, et al. 21-Hydroxylase-Deficient Congenital Adrenal Hyperplasia. 2002 Feb 26 [Updated 2016 Feb 4]. In: Adam MP, Mirzaa GM, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2022. Available from: https://www.ncbi.nlm.nih.gov/books/NBK1171/.

3. Merke D.P., Auchus R.J. Congenital Adrenal Hyperplasia Due to 21-Hydroxylase Deficiency. N Engl J Med. 2020;383(13):1248-1261. doi:10.1056/NEJMra1909786.

4. Speiser P.W., White P.C. Congenital Adrenal Hyperplasia. N Engl J Med. 2003;349(8):776-788. doi:10.1056/NEJMra021561.

5. Мокрышева НГ, Мельниченко ГА, Адамян ЛВ и др. Клинические рекомендации «Врожденная дисфункция коры надпочечников (адреногенитальный синдром)». Ожирение и метаболизм. 2021;18(3):345-382. https://doi.org/10.14341/omet12787.

6. Карева МА. Врожденная дисфункция коры надпочечников у детей: эпидемиология, генетическая основа, персонализированный подход к диагностике и лечению, мониторинг соматического и репродуктивного здоровья. Автореферат дисс. на соискание ученой степени д.м.н. М., 2018. 46 с. https://www.dissercat.com/content/vrozhdennaya-disfunktsiya-kory-nadpochechnikov-u-detei-epidemiologiya-geneticheskaya-osnova.

7. Yau M., Khattab A., New M.I. Prenatal Diagnosis of Congenital Adrenal Hyperplasia. Endocrinol Metab Clin North Am. 2016;45(2):267-81. doi:10.1016/j.ecl.2016.01.001.

8. Narasimhan M.L., Khattab A. Genetics of congenital adrenal hyperplasia and genotype-phenotype correlation. Fertility and Sterility. 2019;111(1):24-29. https://doi.org/10.1016/j.fertnstert.2018.11.007.

9. New M.I., Abraham M., Gonzalez B. et al. Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. PNAS. 2013;12,110(7): 2611-2616. www.pnas.org/cgi/doi/10.1073/pnas.1300057110.

10. Осиновская Н.С., Султанов И.Ю. Идентификации двух мутаций в транс-положении в гене CYP21A2 у плода без врожденной дисфункции коры надпочечников (ВДКН). Случай пренатальной диагностики. Медицинская генетика 2017;16(5):39-41.

11. Oh B.H., Park J.K., Choi Y.M. et al. Prenatal Diagnosis of Heterozygote of Salt Wasting Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency by Genetic Linkage Analysis. Journal of Korean Medical Science. 1988; 3(2):73-77.

12. Mao R., Nelson L., Kates R. et al. Prenatal diagnosis of 21-hydroxylase deficiency caused by gene conversion and rearrangements: pitfalls and molecular diagnostic solutions. Prenatal Diagnosis. 2002; 22(13): 1171-1176. doi: 10.1002/pd.467.

13. Kazmi D., Bailey J., Yau M. et al. New Developments in Prenatal Diagnosis of Congenital Adrenal Hyperplasia. Journal of Steroid Biochemistry and Molecular Biology. 2017;165(Pt A):121-123. doi: 10.1016/j.jsbmb.2016.06.016.

14. Vermeulen С., Geeven G., de Wit E. et al. Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping. The American Journal of Human Genetics. 2017;101(7):326-339. http://dx.doi.org/10.1016/j.ajhg.2017.07.012.

15. Oriola J., Plensa I., Machuca I. et al. Rapid screening method for detecting mutations in the 21-hydroxylase gene. Clinical Chemistry. 1997; 43(4): 557-561.

16. Keen-Kim D., Redman J.B., Alanes R.U. Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency). Journal of Molecular Diagnostics. 2005;7(2):236-246. doi: 10.1016/S1525-1578(10)60550-8.

17. Sullivan K.M., Mannucci A., Kimpton C.P., Gill P. A rapid and quantitative DNA sex test: fluorescence-based PCR analysis of X-Y homologous gene amelogenin. BioTechniques. 1993;15: 637-641.

18. OMIM *613815 Cytochrome P450, Family 21, Subfamily A, Polypeptide 2; CYP21A2. https://omim.org/entry/613815.

19. ClinVar. https://www.ncbi.nlm.nih.gov/clinvar/.

20. Reyes T.M.E., Mesa T.C., Cruz P.A.L. et al. Molecular diagnosis of patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. BME Endocrine Disoders. 2020;20:165 https://doi.org/10.1186/s12902-020-00643-z.