Thalassaemia and Aberrations of Growth and Puberty

Endocrine dysfunction in Thalassaemia major (TM) is a common and disturbing complication, which requires prompt recognition and treatment. The contribution of the underlying molecular defect in TM to the development of endocrinopathies is significant because the patients with the more severe genetic defects have a greater rate of iron loading through higher red cell consumption. TM patients frequently present delay of growth and puberty with reduction of final height. The pathogenesis of growth failure is multifactorial and is mainly due to chronic anemia and hypoxia, chronic liver disease, zinc and folic acid deficiency, iron overload, intensive use of chelating agents, emotional factors, and endocrinopathies (hypogonadism, delayed puberty, hypothyroidism) and GH-IGF-1 axis dysregulation. Although appropriate iron chelation therapy can improve growth and development, TM children and adolescents treated intensively with desferrioxamine remain short as well, showing body disproportion between the upper and lower body segment. Body disproportion is independent of pubertal or prepubertal period of greater height gain. Treatment with recombinant GH (rhGH) is recommended when GH deficiency is established, and even so, the therapeutic response is often non satisfactory. Growth acceleration is mostly promoted with sex steroids in children with associated pubertal delay. Sexual complications in TM, which include Delayed Puberty, Arrested Puberty and Hypogonadism, present the commonest endocrine complication. Iron deposition on gonadotroph cells of the pituitary leads to disruption of gonadotrophin production which is proven by the poor response of FSH and LH to GnRH stimulation. In the majority of patients gonadal function is normal as most women with Amenorrhea are capable of achieving pregnancy with hormonal treatment and similarly men with azoospermia become fathers. Secondary Hypogonadism appears later in life, and is manifested in women as Secondary Amenorrhea and in men as decline in sexual drive and azzoospermia. The damage to the hypothalamus and pituitary is progressive, even when intensive chelating therapy is given and the appearance of Hypogonadism in both sexes is often unavoidable. Close follow up and proper management is crucial for every patient with TM. Early recognition of growth disturbance and prevention of hypogonadism by early and judicious chelation therapy is mandatory for the improvement of their quality of life. Patients with TM can now live a better life due to modern advances in their medical care and our better understanding in the pathogenesis, manifestation and prevention of endocrine complications

Osteoporosis Syndrome in Thalassaemia Major: An Overview

Osteoporosis in thalassaemia major (TM) represents a prominent cause of morbidity. The mechanism of pathogenesis of bone disease (BD) in TM is multifactorial and complicated. Peak bone mass is achieved shortly after completion of puberty and normally remains stable until the third decade of life when age-related bone mass begins. Growth hormone (GH) and sex steroids play a crucial role in bone remodeling and in the maintenance of skeletal architecture during adult life. GH and insulin growth factors (IGFs) have anabolic effect in bone formation. Sex steroids act probably by increasing the expression of RANKL by osteoblastic cells and alterations in the RANK/RANKL/OPG system in favor of osteoclasts. Impaired GH secretion and lack of sex steroids in thalassemic patients due to pituitary damage, contribute to failure of achieving optimal peak bone mass. Other endocrine complications such as hypoparathyroidism and vitamin D deficiency have also a detrimental role on bones in TM. It is still questionable whether the international criteria for defining osteopenia and osteoporosis are relevant to patients with TM; also a question arises for the diagnostic methods such as DEXA scan and management of osteoporosis with known treatment protocols, in the thalassaemic patient

The pathogenic p.Gln319Ter variant is not causing congenital adrenal hyperplasia when inherited in one of the duplicated CYP21A2 genes

ObjectiveThe study aimed to identify the pathogenic status of p.Gln319Ter (NM_000500.7: c.955C>T) variant when inherited in a single CYP21A2 gene (bimodular RCCX haplotype) and to discriminate between a non-causing congenital adrenal hyperplasia (CAH) allele when inherited in a duplicated and functional CYP21A2 gene context (trimodular RCCX haplotype).Methods38 females and 8 males with hyperandrogenemia, previously screened by sequencing and identified as carriers for the pathogenic p.Gln319Ter, were herein tested by multiplex ligation-dependent probe amplification (MLPA) and a real-time PCR Copy number Variation (CNV) assay.ResultsBoth MLPA and real-time PCR CNV analyses confirmed a bimodular and pathogenic RCCX haplotype with a single CYP21A2 in 19/46 (41.30%) p.Gln319Ter carriers and who in parallel all shared elevated 17-OHP levels. The remaining 27 individuals that also carried the p.Gln319Ter exhibited low 17-OHP levels as a result of their carriership of a duplicated CYP21A2 with a trimodular RCCX haplotype. Interestingly, all of these individuals also carried in linkage disequilibrium with p.Gln319Ter two single nucleotide polymorphisms, the c.293-79G>A (rs114414746) in intron 2 and the c.*12C>T (rs150697472) in the 3’-UTR. Therefore, these variants can be used to distinguish between pathogenic and non-pathogenic genomic contexts of the c.955T (p.Gln319) in the genetic diagnosis of congenital adrenal hyperplasia (CAH).ConclusionThe employed methodologies identified a considerable number of individuals with non-pathogenic p.Gln319Ter from the individuals that typically carry the pathogenic p.Gln319Ter in a single CYP21A2. Therefore, it is extremely important the detection of such haplotypes for the prenatal diagnosis, treatment and genetic counseling in patients with CAH

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Osteoporosis in thalassaemia major (TM) represents a prominent cause of morbidity. The mechanism of pathogenesis of bone disease (BD) in TM is multifactorial and complicated. Peak bone mass is achieved shortly after completion of puberty and normally remains stable until the third decade of life when age-related bone mass begins. Growth hormone (GH) and sex steroids play a crucial role in bone remodeling and in the maintenance of skeletal architecture during adult life. GH and insulin growth factors (IGFs) have anabolic effect in bone formation. Sex steroids act probably by increasing the expression of RANKL by osteoblastic cells and alterations in the RANK/RANKL/OPG system in favor of osteoclasts. Impaired GH secretion and lack of sex steroids in thalassemic patients due to pituitary damage, contribute to failure of achieving optimal peak bone mass. Other endocrine complications such as hypoparathyroidism and vitamin D deficiency have also a detrimental role on bones in TM. It is still questionable whether the international criteria for defining osteopenia and osteoporosis are relevant to patients with TM; also a question arises for the diagnostic methods such as DEXA scan and management of osteoporosis with known treatment protocols, in the thalassaemic patient

Genotype Is Associated to the Degree of Virilization in Patients With Classic Congenital Adrenal Hyperplasia

Background: Molecular defects of CYP21A2 consistently decrease 21-hydroxylase activity and result in a variable expression of disease severity in patients with congenital adrenal hyperplasia (CAH).Aim: The genotype and biochemical findings were examined in an attempt to reveal any association to the degree of virilization in classic CAH patients.Methods: The study included 18 CAH patients with complete characterization of CYP21A2 mutations and were sorted based on the severity of the inherited mutations and the expected percentage of 21-hydroxylase enzyme activity.Results: Eleven out of the 18 patients manifested the SW form with the remaining seven exhibiting the SV form. The most frequent genetic defect in the classic salt-wasting (SW) and simple virilising (SV) forms was the IVS2-13A/C>G (36.1%) mutation, followed by delEX1-3 (19.4%) and p.Ile172Asn (19.4%). Four patients, who shared a combination of two mutations belonging to the most severe type, manifested only the SW form. Four out of five patients who shared homozygosity in the IVS2-13A/C>G mutation, demonstrated the SW form and only one demonstrated the SV form. All four patients who shared the p.Ile172Asn mutation, either in the homozygous or compound heterozygous state, manifested the SV form. Interestingly, a female neonate with SW, bearing the IVS2-13A/C>G/Large del, exhibited complete male virilisation (Prader 5). The remaining four affected female new-borns also exhibited the SW form, with two of them virilised as Prader 3 and the other two as Prader 4. Virilisation with clitoromegaly was also observed in one female, who presented premature adrenarche and carried the least severe p.Pro30Leu mutation.Conclusion: The frequency of the underlying mutations in our patients, with the classic form of CAH, varies but were quite similar to the ones reported in the Mediterranean region. Therefore, the identification of severe CYP21A2 defects in Cypriot patients and their comparison with the incidence and severity in different populations, will create a valuable diagnostic tool for genetic counseling in the classic form of CAH

Pathogenic and low-frequency variants in children with central precocious puberty

Background: Central precocious puberty (CPP) due to premature activation of GnRH secretion results in early epiphyseal fusion and to a significant compromise in the achieved final adult height. Currently, few genetic determinants of children with CPP have been described. In this translational study, rare sequence variants in MKRN3, DLK1, KISS1, and KISS1R genes were investigated in patients with CPP. Methods: Fifty-four index girls and two index boys with CPP were first tested by Sanger sequencing for the MKRN3 gene. All children found negative (n = 44) for the MKRN3 gene were further investigated by whole exome sequencing (WES). In the latter analysis, the status of variants in genes known to be related with pubertal timing was compared with an in-house Cypriot control cohort (n = 43). The identified rare variants were initially examined by in silico computational algorithms and confirmed by Sanger sequencing. Additionally, a genetic network for the MKRN3 gene, mimicking a holistic regulatory depiction of the crosstalk between MKRN3 and other genes was designed. Results: Three previously described pathogenic MKRN3 variants located in the coding region of the gene were identified in 12 index girls with CPP. The most prevalent pathogenic MKRN3 variant p.Gly312Asp was exclusively found among the Cypriot CPP cohort, indicating a founder effect phenomenon. Seven other CPP girls harbored rare likely pathogenic upstream variants in the MKRN3. Among the 44 CPP patients submitted to WES, nine rare DLK1 variants were identified in 11 girls, two rare KISS1 variants in six girls, and two rare MAGEL2 variants in five girls. Interestingly, the frequent variant rs10407968 (p.Gly8Ter) of the KISS1R gene appeared to be less frequent in the cohort of patients with CPP. Conclusion: The results of the present study confirm the importance of the MKRN3-imprinted gene in genetics of CPP and its key role in pubertal timing. Overall, the results of the present study have emphasized the importance of an approach that aligns genetics and clinical aspects, which is necessary for the management and treatment of CPP

Gonadal function and fertility issues in Thalassaemia

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Molecular defects of the CYP21A2 gene in greek cypriot patients with congenital adrenal hyperplasia

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 pG 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.Η Συγγενής Υπερπλασία Επινεφριδίων (ΣΥΕ) αποτελεί μία ομάδα διαταραχών, οι οποίες κληρονομούνται με αυτόσωμο υπολειπόμενο χαρακτήρα και χαρακτηρίζονται από ελλειμματική βιοσύνθεση της Κορτιζόλης, λόγω ένδειας ενός εκ των ενζύμων, τα οποία απαιτούνται για την βιοσύνθεση της στον φλοιό των επινεφριδίων. Η συχνότερη μορφή της ΣΥΕ (95% των περιπτώσεων) οφείλεται σε έλλειψη της 21-υδροξυλάσης, ως αποτέλεσμα γενετικών λαθών στο γονίδιο CYP21Α2, το οποίο αποτελεί ένα από τα πλέον πολυμορφικά γονίδια.Τα γενετικά λάθη του γονιδίου CYP21Α2 προέρχονται από δύο τύπους ανασυνδυασμών ανάμεσα στο CYP21Α2 και στο ψευδογονίδιο CYP21Α1P: άνισες διασταυρώσεις κατά τη διάρκεια της μείωσης, οι οποίες οδηγούν σε απαλείψεις CYP21Α2 και μετατροπές, με αποτέλεσμα την μεταφορά αλλοιωμένων αλληλουχιών από το CYP21A1P στο CYP21Α2, όπου καθίστανται επιζήμιες. Τα γενετικά λάθη του γονιδίου CYP21Α2 ταξινομούνται σε τρείς κατηγορίες ανάλογα με τον βαθμό της ενζυματικής λειτουργίας και τυπικά ανταποκρίνονται στις τρεις κλινικά μορφές της έλλειψης της 21- Υδροξυλάσης: με απώλεια άλατος - salt-wasting (SW), απλή αρρενοποιητική - simple virilizing (SV), και όψιμης εκδήλωσης- nonclassical (NC) ΣΥΕ. Στην πλειονότητα των περιπτώσεων παρατηρείται συμφωνία μεταξύ γονοτύπου και φαινοτύπου, αν και δεν είναι πάντα εφικτό να γίνει επακριβώς η πρόβλεψη της κλινικής έκφρασης βάσει της υποκείμενης γενετικής διαταραχής. Οι περισσότεροι ασθενείς ανευρίσκονται να είναι σύνθετοι ετεροζυγώτες, όπου η σοβαρότητα της νόσου καθορίζεται από την δραστηριότητα του λιγότερο επηρεαζόμενου αλληλίου. Ο σκοπός της παρούσας διατριβής είναι η μελέτη του φάσματος των μεταλλάξεων που αναγνωρίστηκαν σε Ελληνοκύπριους ασθενείς με ΣΥΕ και η εξακρίβωση της επίδρασης του υποκείμενου γονότυπου στη κλινική έκφραση της πάθησης. Το υλικό της μελέτης απετέλεσαν 218 ασθενείς και 300 υγιείς μάρτυρες, στους οποίους έγινε μοριακή ανάλυση του γονιδίου CYP21Α2 με προσδιορισμό της αλληλουχίας προϊόντων της PCR σε αυτόματο αναλυτή (ABI Sequencer) και με τη μέθοδο MLPA για ανίχνευση διπλασιασμών και μεγάλων απαλείψεων. Συνοπτικά τα αποτελέσματα έχουν ως εξής. 1. Οι πιο συχνές μεταλλάξεις συνολικά, που αναγνωρίστηκαν στους 47 μη συσχετιζόμενους ασθενείς, των οποίων μελετηθήκανε 94 αλλήλια είναι οι p.Val281Leu (54.8 %) και IVS2-13A/C>G (14.9%). 2. Στην ομάδα των 10 ασθενών, οι οποίοι είχαν την κλασσική μορφή της ΣΥΕ η πιο συχνή σημειακή μετάλλαξη ήτανε η IVS2-13A/C>G (52.4%).3. Τα πιο συχνά μοριακά λάθη, που αναγνωρίστηκαν στην ομάδα των 37 ασθενών με την μη κλασσική μορφή ΣΥΕ, είναι η μετάλλαξη p.Val281Leu σε ποσοστό 68.9%, η p.Pro453Ser (6.75%) και οι μεταλλάξεις IVS2-13A/C (4.05%), p.Qln318stop (4.05%) και p.Val304Met (4.05%). 4.Οι ασθενείς με τη μη κλασσική μορφή, που έφεραν συνδυασμό ήπιας/σοβαρής μετάλλαξης σε μεγαλύτερο ποσοστό εκδήλωσαν τη νόσο πριν από την εφηβεία και είχαν υψηλότερα επίπεδα επινεφριδιακών ανδρογόνων. 5. Η συνολική συσχέτιση γονότυπου – φαινότυπου στην παρούσα φάση της μελέτης ήτανε 100%. Όλοι οι ασθενείς των ομάδων null, Α και Β είχαν την κλασσική (απώλεια άλατος ή απλή αρρενοποιητική) μορφή ΣΥΕ. Όλοι οι ασθενείς της ομάδας C εκδήλωσαν την μη κλασσική μορφή, όπως ήταν αναμενόμενο. 6.Ανάλυση του γονότυπου CYP21A2 σε 171 θήλεις με κλινική εικόνα υπερανδρογοναιμίας εδειξε τα ακολουθα: Α. 66 θήλεις βρέθηκαν να έχουν μία μετάλλαξη του γονιδίου CYP21A2 με την p.Val281Leu να είναι η πιο συχνή, οι οποίες είχαν και τις υψηλότερες τιμές 17-OH Προγεστερόνης.Β. Οι υπόλοιπες 105 θήλεις με κλινικά σημεία υπερανδρογοναιμίας, στις οποίες δεν αναγνωρίστηκε καμία μετάλλαξη στο γονίδιο CYP21A2 είχαν σε ασυνήθιστα υψηλή συχνότητα 56.1% τον πολυμορφισμό p.Asn493Ser, ο οποίος βρέθηκε να είναι 19.7% στην ομάδα των 66 CYP21A2 ετεροζυγωτών θηλέων. Επιπρόσθετα σε δείγμα 300 υγιών Κυπρίων μαρτύρων η συχνότητα των φορέων της παραλλαγής p.Asn493Ser εκτιμάται να είναι 37%. Γ. Η αλληλούχιση της μη μεταφραζόμενης περιοχής 3’UTR του γονιδίου CYP21A2 στις 66 θήλεις με ένα επηρεασμένο αλλήλιο CYP21A2 έδειξε ότι όσες έφεραν σε ετεροζυγωτία την παρανοηματική μετάλλαξη p.Val281Leu έφεραν επίσης στην περιοχή 3’UTR in cis τους πολυμορφισμούς *52 T>C, *440 C>T και *443 T>C και όσες έφεραν την σοβαρή μετάλλαξη p.Qln318stop είχαν επίσης in cis τους πολυμορφισμούς *12 T>C και *52 C>T στην ίδια περιοχή του 3’UTR. Αντιθέτως η κατανομή και των δύο ανωτέρω πολυμορφισμών δεν παρατηρήθηκε σε καμία από τις υπόλοιπες CYP21A2 ετερόζυγες θήλεις. 7.Οι σπάνιες γενετικές βλάβες που αναγνωρίστηκαν ήταν οι σημειακές μεταλλάξεις p.Val304Met, p.Met283Va, p.Ala391Thr καθώς και ο ασυνήθης συνδυασμός p.Phe306insT+ p.Val281Leu/ p.Phe306insT+ p.Val281Leu. 8.Σε δείγμα 300 κλινικά ασυμπτωματικών ατόμων (150 άνδρες και 150 γυναικών) η συχνότητα φορέων μεταλλάξεων του γονιδίου CYP21A2 στον Κυπριακό πληθυσμό είναι 9.83%. Οι πιο συχνές μεταλλάξεις που αναγνωρίστηκαν ήταν η ήπια p.Val281Leu (4.3%), ακολουθούμενη από τις p.Qln318stop (2.5%), p.Pro453Ser (1.33%), p.Val304Met (0.83%), p.Pro482Ser (0.67%) και p.Met283Val (0.17%). Οι σοβαρές πέντε μεταλλάξεις, οι οποίες αναγνωρίστηκαν στους ασθενείς της παρούσας μελέτης δεν ανιχνεύτηκαν σε κανένα από τους υγιείς μάρτυρες, έτσι που να ερμηνεύεται η σπανιότητα της κλασσικής μορφής στον πληθυσμό μας. Από τα προκαταρτικά αυτά αποτελέσματα φαίνεται ότι το φάσμα και η συχνότητα των μεταλλάξεων του γονιδίου CYP21A2 στους Ελληνοκύπριους ασθενείς με ΣΥΕ προσομοιάζει με τις πλέον συχνά ανευρεθείσες γενετικές βλάβες. Παρατηρείται μία εξαιρετική συσχέτιση του γονότυπου με τον κλινικό φαινότυπο, άνκαι μερική διαφωνία είναι λογικά αναμενόμενη, λόγω διαφοράς στην έκφραση του γονιδίου της 21 – Υδροξυλάσης. Μέχρι τώρα η πραγματική εικόνα της συχνότητας των μεταλλάγμένων αλλήλίων και συνεπώς φορέων ΣΥΕ λόγω έλλειψης της 21 – Υδροξυλάσης δεν έχει μελετηθεί με εμπεριστατωμένη μοριακή ανάλυση του γονιδίου CYP21A2 σε συγκεκριμένο Ευρωπαϊκό πληθυσμό. Στην παρούσα μελέτη έγινε ανίχνευση για μεταλλάξεις του γονιδίου CYP21A2 σε στατιστικά αξιόπιστο για τον πληθυσμό της χώρας με αλληλούχιση DNA και ανάλυση MLPA για μεταλλάξεις στο γονίδιο CYP21A2 και στον προμότορά του. Η γνώση της υποκείμενης γενετικής βλάβης είναι αναγκαίο εργαλείο τόσο για την ακριβή διάγνωση και ταυτοποίηση των ασθενών όσο και για την παροχή ορθολογιστικής γενετικής συμβουλής, όταν καθίσταται αναγκαία