Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders of cortisol biosynthesis, which is caused by the loss or severe decrease in the activity of one of the enzymatic steps required for cortisol biosynthesis in the adrenal cortex. The most common form of CAH (95% of all cases) is due to 21-hydroxylase deficiency (21-OHD) resulting from molecular defect in the steroid 21-hydroxylase (CYP21A2) gene, with an overall estimated incidence of 1:10,000 to 1:15,000 live births. The CYP21A2 gene is one of the most polymorphic human genes and more than 100 alleles have been identified in patients with CAH (Human Gene Mutation Database: http://www.hgmd.cf.ac.uk/ac/index.php). The CYP21A2 gene is located on the short arm of chromosome 6, within the region of the major histocompatibility complex (MHC), at a distance of 30 kb from a highly homologous (>95%) pseudogene, designated CYP21A1P. The location of the CYP21A2 gene makes it vulnerable to relatively large genomic recombinations with its homologous gene, CYP21A1P. The proximity of these genes and their location within the HLA region, which has a high rate of recombination, facilitate such events. The molecular defects of CYP21A2 may result from 2 types of recombinations between the CYP21A2 and the CYP21A1P pseudogene: unequal crossing-over during meiosis leading to deletion of CYP21A2 and conversions that result in transfer of altered sequences from CYP21A1P to CYP21A2, where they become detrimental. Genetic defects in the CYP21A2 gene are classified into three categories depending on the residual enzymatic activity and typically correspond to the three types of 21-OHD: salt-wasting (SW), simple virilizing (SV), and non classical (NC) CAH. In vitro studies have shown that mutations resulting in complete inactivation of 21-hydroxylase activity are associated with the SW phenotype, those that reduce 21-hydroxylase activity to approximately 2% are associated with the SV phenotype, whereas those that reduce 21-hydroxylase activity to 10% to 75% are associated with the NC phenotype. In the great majority of cases there is a correlation between genotype and phenotype, although it is not always possible to predict the phenotype on the basis of genotype with accuracy. Most patients are compound heterozygotes, and the severity of the disease is determined by the activity of the less severely affected allele. The incidence of the genetic defects of 21-OHD has been extensively studied and ethnic specific distribution of mutations has been reported. There is no data about the mutations of CYP21A2 gene in our population, which is mainly of Greek origin influenced by a genetic impact of surrounding countries. Studies in neighbouring countries in the Mediterranean region have reported as the most prevalent genetic defects: IVS2-13A/C>G, p.Qln318stop, p.Val281Leu and large lesions of the gene. Therefore, we aimed to analyze the types and frequencies of mutations in the Greek Cypriot patients with 21-OHD and study the correlation between genotype and phenotype. For the purpose of this work we studied 218 patients and 300 healthy controls. The patients were characterized and grouped based on clinical biochemical and molecular findings. The patients were classified in the SW form when clinical and biochemical findings of renal salt wasting were evident and 17-OHP value higher than 75 nmol/L in the first month of their lives. Severe clinical symptoms of CAH without electrolyte imbalance, 17-OHP values higher than 75 nmol/L were characteristic in patients diagnosed with the SV form. Symptoms of hyperandrogenemia in peripubertal years and 17-OHP levels greater than 30 nmol/L after stimulation with ACTH were used to diagnose the patients with the NC form of CAH. The disease-causing mutations were divided into four mutation groups. The null A and B groups contained alleles with mutations resulting in an enzyme with no activity, hence leading to the classical form of CAH, whereas group C contained genotypes composed by a mild mutation on at least one allele resulting in the NC form. In all DNA samples the identification of mutations was performed with DNA sequencing of the PCR products of the amplification of the CYP21A2 gene and with multiplex ligation-dependent probe amplification (MLPA) for detection of deletions and/or conversions. The 3’ untranslated region that is 536 nucleotides downstream the TGA stop codon of the CYP21A2 gene was amplified using specific primer. The statistical analysis was performed by using the SPSS statistical software program and comparisons between the groups were made using the Student’s t-paired test, where p<0.05 was considered as statistically significant. The project was approved by the Cyprus National Ethics Committee.The results of this present work are the following. 1.The most frequent mutations in the total of the 47 non related patients of whom 94 alleles were examined are the p.Val281Leu in 54.8% and IVS2-13A/C>G in 14.9%. 2. In the group of patients with the classic form of CAH, the most common genetic defect was the splice mutation IVS2-13A/C>G in 52.4%.3. In the 64 unrelated alleles of the 37 patients with the NC form , the most frequent point mutations were p.Val281Leu (68.9%) followed by IVS2-13A/C>G (4.05%), p.Qln318stop (4.05%) and p.Val304Met (4.05%).4.The patients with the NC form who carried the combination of mild/severe mutations were more likely to manifest the disease earlier as premature adrenarche and had higher levels of adrenal androgens. 5. The correlation of genotype – phenotype at this stage of the study is almost absolute. All patients who belonged to null, Α and Β groups manifested the classic form (SW and SV) and all patients in group C manifested the NC form of CAH as expected. 6.CYP21A2 genotype analysis in 171 females (girls, adolescents and women) with clinical manifestation of hyperandrogenemia showed: A. 66/171 were found to bear one mutation of the CYP21A2 in heterozygocity with p.Val281Leu being the most frequent and additionally had the highest levels of 17 – OHP. B. The remaining 105 females with clinical hyperandrogenemia in whom no mutation was identified were found to carry the polymorphism p.Asn493Ser in 56.1%. This percentage is considered as unusually high as this particular polymorphism was detected in 19.7% in the group of the 66 CYP21A2 heterozygote females and in 37% in the normal population in Cyprus. C. Extended sequencing of the 3’UTR of the CYP21A2 in 66 females identified with one affected CYP21A2 allele demonstrated that the 29 who carried in heterozygosity the mutation p.V281L to also carry in the 3’UTR in cis the variants *52 T>C, *440 C>T and *443 T>C. Similarly 9 other heterozygote females with the severe p.Qln318stop mutation were found to also carry in the 3’UTR in cis the variants *12 T>C and *52 C>T. Concurrent screening of the CYP21A2 3’UTR in 150 control females with no hyperandrogenic symptoms and no detected mutations in the CYP21A2 gene identified the combination of 3’UTR variants *52 T>C, *440 C>T, *443 T>C in 8% and *12 T>C, *52 C>T in 5.3%. 7.The rare mutations identified in the sample of this study were the point mutations: p.Val304Met, p.Met283Val in combination with the severe IVS2-13A/C and the mild p.Val281Leu, p.Ala391Thr and the unusual combination p.Phe306insT+ p.Val281Leu/ p.Phe306insT+ p.Val281Leu. 8.In a statistically significant sample of 300 (150 men and 150 women) healthy individuals the frequency of CYP21A2 gene mutations carriers was found to be 9.83%. The most cpmmon mutations identified were: p.Val281Leu (4.3%), p.Qln318stop (2.5%), p.Pro453Ser (1.33%), p.Val304Met (0.83%), p.Pro482Ser (0.67%) and p.Met283Val (0.17%). No severe mutations were detected thereby explaining the low incidence of the classic form of CAH in our population. In this study we described the spectrum and frequency of CYP21A2 alleles, as well as the genotype-phenotype relationship in unrelated Greek Cypriot patients with CAH of the 21-OHD form. Additionally we explored the incidence of CYP21A2 heterozygocity in females with hyperandrogenemia and estimated the carrier frequency of CYP21A2 mutations in our population. The most frequent molecular defect in the classical form of CAH, which is rare in Cyprus, is the splice site mutation IVS2-13A/C>G, which is is agreement with most studies reported so far. In the NC form, the prevalent mutation was the p.Val281Leu, as in most populations studied. The overall frequency of p.Val281Leu is however one of the highest found for this mutation both in Greek and other populations in Europe and the Mediterranean area. This mutation is quite common in our population as it was found in 4.3 % in screening of healthy individuals and probably reflects a founder effect. The genotype phenotype correlation is almost absolute and is in agreement with the fact that the phenotypic expression of 21-OHD is primarily related to the type of the molecular defect and correlates with the less severely mutated allele. This questionable milder missense p.Pro30Leu mutation, although known to reduce enzyme activity and generally associated with NC form, is often present in patients with more severe signs of androgen excess. Up to date a large spectrum of mutations in the CYP21A2 gene has been reported and most of these reported mutations affect the coding region of the gene and to a lesser extent the introns and the promoter. Interestingly, variants located in the 3’ untranslated region (UTR) of CYP21A2 which among other regulatory elements contains several micro-RNA-binding sites have not yet been reported to be associated with CAH. As 3'UTR mutations can influence the disease susceptibility by altering protein and microRNA (miRNA) binding regions, the CYP21A2 3'UTR was also screened for mutations in heterozygote hyperandrogenic females and compared with sex-matched healthy controls. A possible involvement of the 3’UTR variants *52 T>C, *440 C>T,*443 T>C and *12 T>C, *52 C>T in the CAH phenotypes of the heterozygous p.V281L and p.Q318stop patients of the present study is highly likable. The CYP21A2 genotypic analysis of a cohort of 300 unrelated asymptomatic individuals detected 59 mutated alleles in a total of 600 alleles, which gives a carrier frequency of 9.83%, similar to Middle European individuals of Austrian and Yugoslav origin. The overall frequency of p.Val281Leu was proven to be the highest, which is probably the result of a founder effect and provides an explanation of its high incidence in in the pool of Cypriot NC-CAH patients. In the present study a carrier frequency of 0.8% was also detected for the rare and mild p.Val304Met mutation, which was previously reported only once in the homozygote state in a female patient of Asian origin and most likely reflects a founder effect as well. None of the 300 subjects tested in the present study were identified with any of the five severe mutations identified in the patients with severe CAH, hence explaining the low incidence of classic CAH in our population. In conclusion, the previously described major mutations are found to dominate the mutation spectrum of Greek Cypriot patients with CAH. The frequency of the underlying genetic defect in patients with NC-CAH is similar to that observed in most Mediterranean populations. There is an excellent genotype – phenotype correlation in all patients, although differences in phenotypic appearance may appear and caused by still undefined factors modifying CYP21A2 gene expression. Although the clinical expression of NC-CAH is not solely depended on the genotype, discrimination between mild and severe alleles should be made. The frequency of the underlying genetic defect in heterozygote female patients with hyperandrogenism is similar to that observed in other populations and probably the carrier status for CYP21A2 can be a factor in the variable phenotype of clinical expression of androgen excess. Finally the frequency of the underlying genetic defects in the Cypriot population is one of highest ever reported and may be considered as a small indication of the carrier frequency of CYP21A2 mutations that exists within the Mediterranean region. Knowing the genetic defect in correlation and its impact on phenotype is of immense help in judicious clinical practice, antenatal diagnosis and appropriate genetic counselling when needed.
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