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0021-972X/04/$15.00/0 Printed in U.S.A. The Journal of Clinical Endocrinology & Metabolism 89(1):368 –374 Copyright © 2004 by The Endocrine Society doi: 10.1210/jc.2003-031056 Molecular Genetic Analysis of Tunisian Patients with a Classic Form of 21-Hydroxylase Deficiency: Identification of Four Novel Mutations and High Prevalence of Q318X Mutation MAHER KHARRAT, VÉRONIQUE TARDY, RIDHA M’RAD, FAOUZI MAAZOUL, LAMIA BEN JEMAA, MOHAMED REFAÏ, YVES MOREL, AND HABIBA CHAABOUNI Laboratoire de Génétique Humaine (M.K., R.M., F.M., L.B.J., M.R., H.C.), Faculté de Médecine de Tunis, 1006, Tunisie; and Laboratoire de Biochimie Endocrinienne (V.T., Y.M.), Institut National de la Santé et de la Recherche Médicale unité 329, Université de Lyon et Hôpital Debrousse, 69322 Lyon Cedex 05, France Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders mainly due to defects in the steroid 21hydroxylase (CYP21) gene. To determine the mutational spectrum in the Tunisian CAH population, the CYP21 active gene was analyzed in 51 unrelated patients using our cascade strategy (digestion by restriction enzyme, sequencing). All patients had a classical form of 21-hydroxylase deficiency. Mutations were detected in over 94% of the chromosomes examined. The most frequent mutation in the Tunisian CAH population was found to be Q318X, with large prevalence (35.3%), in contrast to 0.5–13.8% described in other series. Incidence of other mutations does not differ, as previously described: large dele- tions (19.6%), mutation in intron 2 (17.6%), and I172N (10.8%). Four novel mutations were found in four patients with the salt-wasting form. These four novel mutations include three point mutations that have not been reported to occur in the CYP21P pseudogene: R483W, W19X, 2669insC, and one small conversion of DNA sequence from exon 5 to exon 8. Our results have shown a good genotype/phenotype correlation in the case of most mutations. This is the first report of screening for mutations of 21-hydroxylase gene in the Tunisian population and even in the Arab population. (J Clin Endocrinol Metab 89: 368 –374, 2004) C ONGENITAL ADRENAL HYPERPLASIA (CAH) is an autosomal recessive disease. The most common form (95%) is due to 21-hydroxylase deficiency (21-OHD) resulting from molecular defect in the steroid 21-hydroxylase (CYP21) gene (1, 2). There are three major disease phenotypes depending on the specific mutation in the gene coding for 21-hydroxylase, CYP21. In the classic salt-wasting (SW) form, the most severe form, patients suffer from renal salt loss due to the lack of aldosterone as well as virilization due to accumulated adrenal androgen. In the classic simple virilizing (SV) form, patients also undergo virilization. In the nonclassic form, patients lack the neonatal symptoms and present with late-onset androgen excess, such as pseudoprecocious puberty and hirsutism. The incidence of the classic forms of 21-OHD is 1/14,000 in the Caucasian population (3), thereby being one of the most frequent autosomal recessive disorders, and three fourths of classical cases are SW (4). The gene encoding 21-hydroxylase, CYP21 is located in a locus with a complicated structure. It is found on the short arm of chromosome 6 (band 6p21.3) together with a highly homologous inactive pseudogene, CYP21P. The two genes are located in tandem repeats with the genes encoding the fourth component of serum complement (C4A and C4B) (5, 6). The CYP21 and CYP21P genes consist of 10 exons and show a high homology with a nucleotide identity of 98% in their exon and 96% in their intron sequences (7, 8). The proximity and the high degree of homology between the two genes are believed to be the main reason for unequal crossover and gene conversion-like events, which give rise to mutations in CYP21 (9, 10). Approximately 95% of all disease-causing mutations in CYP21 are either deletion/ conversion (large deletions) or any of nine point mutations that have been transferred from CYP21P into the active CYP21 (11, 12). The remaining 5% of the disease-causing mutations are rare and unique for single families or are considered as population specific. These uncommon alleles do not originate from the pseudogene. Large deletions are characterized by a single nonfunctional chimeric gene (CYP21P/CYP21) having CYP21P sequences at the 5⬘ end and CYP21 sequences at the 3⬘ end (13). The incidence of the CYP21 mutations in 21-OHD has been extensively studied in the last years. No significant difference has been observed in the Caucasian population. Large deletions account for 25% of 21-OHD alleles, depending on ethnic group, and about 75% of chromosomes encoding CAH seem to carry point mutations. The most frequently reported mutation found in patients with classic forms is an A or C3 G change in the second intron affecting pre-mRNA splicing (IVS2–13A/C3 G), this mutation has been detected in patients affected with either the SW or SV form of the disease (14). Abbreviations: CAH, Congenital adrenal hyperplasia; CYP21, steroid 21-hydroxylase gene; 21-OHD, 21-hydroxylase deficiency; RFLP, restriction fragment length polymorphism; SV, simple virilizing; SW, salt wasting. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community. 368 Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients J Clin Endocrinol Metab, January 2004, 89(1):368 –374 369 In this report, we study the genetic aspect of the classical form of CAH in a Tunisian population. Our aims are to identify mutations in the CYP21 gene, determine their frequency, and correlate genotype with phenotype. Patients and Methods Patients Informed consent for mutation analysis was obtained from all patients and family members. Molecular analysis concerned 51 unrelated Tunisian CAH patients that were referred to the Department of Congenital and Hereditary Diseases of the Charles Nicolle Hospital in Tunis. All patients (10 males and 41 females) had a classical form of 21-OHD (44 patients with the SW form and 7 with the SV form) (Table 1). CAH with SW was characterized by onset of hyperkaliemia, hyponatremia, and dehydration in the first month of life. All females had ambiguous genitalia, grade III–IV on the scale of Prader (15). All patients with the SV form were females with ambiguous genitalia (from posterior fusion of the labioscrotal folds to stage IV of Prader) and with no evidence of sodium depletion. Consanguinity was present in 31 families (60.8%), absent in 15 families, and unknown in five families. Consanguineous families TABLE 1. Genotype and phenotype of 51 Tunisian affected patients Patient no. Genotype mutations (Fc/Met) Phenotype Sex Prader Stage 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/Q318X Q318X/In2 R356W/Q318X P30L⫹In2/Q318X LD/Q318X LD/LD LD/LD LD/LD LD/LD LD/LD LD/LD LD/LD LD/LD LD/LD LD/In2 In2/In2 In2/In2 In2/In2 In2/In2 In2/In2 In2/In2 In2/In2 R356W/R356W Q318X⫹In2/Q318X⫹In2 2669insC/In2 R483W/R483W Small conv/Q318X Q318X/ND Q318X/ND ND/Q318X ND/W19X ND/ND I172N/I172N I172N/I172N I172N/I172N I172N/I172N I172N/I172N Q318X/In2 I172N/Q318X SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SW SV SV SV SV SV SV SV F M F F F F M F F M F F F F F F F M M F F M F F F F F F M F M F F F F M F F F F F F F M F F F F F F F IV IV IV IV IV IV IV IV III IV III–IV ? III IV IV ? 17OHP (ng/ml) Nf Nf 12.7a 32a 952 ⬎125 Inc 440 Nf Inc Nf ⬎275 Nf Nf Nf Inc Inc ⬎125 156 Nf Nf a IV IV ? ? III III III IV IV ? IV IV ? IV IV IV IV III II II II II III III–IV ? 440 Nf Nf Inc Inc 121 Inc ⬎25 Inc 202 Nf Inc Inc ⬎275 36a 88 411 Nf 12.7a 105 Nf 230 ⬎25 ⬎25 75 ⬎125 Nf Inc 104 Consanguinity Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No ? No No No No Yes Yes Yes Yes Yes No No No ? No Yes Yes Yes Yes Yes ? ? Yes Yes No Yes Yes No Yes No No Yes Yes Yes Yes Yes Yes No ? In2, IVS2-13A/C3 G mutation; Inc, increased; LD, large deletions or large gene conversions; ND, no mutation detected; Nf, 17OHP not furnish; Fc, father’s chromosome; Mc, mother’s chromosome; F, female; M, male. a 17OHP under treatment with hydrocortisone. 370 J Clin Endocrinol Metab, January 2004, 89(1):368 –374 were unrelated and originated from different regions of the country: 41.9% from Southern, 22.6% from Central, and 35.5% from Northern Tunisia. Two patients died at very early life, during the first month. Blood samples were obtained from all patients and from their parents if available. Molecular genetic studies In this study, CYP21 was amplified with gene-specific PCR primers. Restriction enzyme digestion of PCR-amplified DNA was used to detect the presence of six more common point mutations. Rare mutations were detected by direct sequencing of all the exons of the CYP21 gene. Large deletions and the 8-bp-deletion located in exon 3 were detected by PCR method. PCR amplification Genomic DNA was prepared from peripheral blood leukocytes by standard procedures (16). Amplifications were performed in a vol of 100 l containing approximately 0.7 g of genomic DNA, 150 ng of each nucleotide primer, 0.2 mm of each deoxynucleotide triphosphate, 1.5 mm of MgCl2, 1⫻ PCR buffer (Eurobio, Paris, France), and 2.5 U of Taq DNA polymerase (Eurobio). Thirty cycles of amplification were used, each consisting of a denaturation step for 60 sec at 95 C, annealing step for 60 sec at 58 C, and extension step for 60 sec at 72 C. The amplification products were analyzed by 1% agarose gel electrophoresis with presence of ethidium bromide stain. Detection of six point mutations Because they were the more common within different reported populations, we studied the following mutations: the P30L in exon 1, the splice site mutation in intron 2 (IVS2–13A/C3 G), the I172N in exon 4, the cluster of three mutations (I236N, V237E, M239K) in exon 6 designed as cluster E6, the V281L in exon 7, and the Q318X stop codon in exon 8. For analysis of these mutations, four different specific amplifications using CYP21 gene-specific primers were carried out on genomic DNAs followed by digestion with appropriate restriction enzymes according to the manufacturer’s protocol (Table 2). To obtain selective CYP21 amplifications, we used specific primers that are unable to amplify pseudogene. A list of PCR primers is reported in Table 3. The primers were used to amplify the four different regions shown on Fig. 1. Detection of large deletions and 8-bp-deletion The presence of an 8-bp-deletion in exon 3 or large deletions in homozygous state were suspected when the above reactions failed to generate the expected fragment. These genetic defects were confirmed by nonselective amplification of exon 3 of CYP21 and CYP21P genes with primers P7 and P8 (Table 3). The result of PCR gives a single band of 56-bp with absence of the band of 64-bp normally present, as described (17). Then, we realized a second PCR (from intron 2 to exon 6) to differentiate between large deletions and the 8-bp-deletion using selective amplification of CYP21 gene with specific primers P3 and P9. The absence of a 789-bp band confirms a large lesion. Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients Detection of rare point mutations by direct sequencing If no mutation was detected on at least 1 allele, direct sequencing of two independent PCR amplifications of the CYP21 gene, using selective primers (Fig. 1 and Table 3), was performed with a 373A model automatic sequencer (PE Applied Biosystems, Forest City, CA) using the dideoxynucleotide terminator methodology, as previously described (18, 19). Once a deletion or point mutation was identified for a patient, segregation of the corresponding mutation was studied in both parents, every time available. Results We screened for six point mutations, large deletions, and noncommon mutations using restriction fragment length polymorphism (RFLP) methods, PCR, and sequencing of CYP21 gene, respectively. Mutations were found in 94.1% (96/102) of the disease chromosomes studied corresponding to 51 unrelated Tunisian patients clinically diagnosed as having a classical form of 21-OHD. The frequency of the mutations analyzed in this study and the frequencies of the same mutations found in other populations are shown in Table 4. Chromosomes were affected by one of these mutations in 36 patients (70.6%), nine patients (17.6%) were compound heterozygous with different mutations on each chromosome, one patient (2%) was homozygous for two different mutations, four patients (7.8%) had only one copy of one mutation, and one patient (2%) harbored none of the tested mutations. The molecular defects detected were distributed as follows: Q318X in exon 8 (35.3%), large lesion of the CYP21 gene (19.6%), IVS2–13A/C3 G in intron 2 (17.6%), I172N in exon 4 (10.8%), and R356W in exon 8 (2.9%). Del 8-bp and cluster E6 were not detected in this study. TABLE 3. PCR primers used for amplification of CYP21 gene Primers Sequence (5⬘–3⬘) P1 P2 P3 P4 P5 P6 P7 P8 P9 P10 GGACCTGTCCTTGGGAGACTAC GCCGTGTGGTGCGGTGGGGCAAGGCTA AGGTCAGGCCCTCAGCTGCCTTCA GGCTTTCCAGAGCAGGGAGTAGTC TCTCCGAAGGTGAGGTACCAG TCGGTGGGAGGGTACCTGAA GTCTAGGAACTACCCGGACCTGTC CTTCTTGTGGGCTTTCCAGAGCAG AGCTGCATCTCCACGATGTGA CTGAGGTACCCGGCTGGCATCGGT Underlined primers are reverse primers. Underlined bases confer CYP21 specificity to the primer. The sequences selected for specificity were: the normal counterparts of the 8-bp deletion in exon 3, codons 236 –239 in exon 6 that differentiate CYP21 from CYP21A by four deoxynucleotides, or nucleotides 620 – 630 in intron 2 that differentiate CYP21 from CYP21A by four deoxynucleotides. TABLE 2. PCR fragments and appropriate restriction enzymes used for the detection of six point mutations PCR Forward primers Reverse primers Mutations Enzymes Fragment I (1460 bp) P1 at exon 3 P2 at exon 8 V281L Cluster E6 Q318X BsiHKA I Nde II Pst I Fragment II (128 bp) Fragment III (330 bp) Fragment IV (854 bp) P3 at intron 2 P1 at exon 3 P6 at promoter P4 at exon 3 P5 at exon 4 P4 at exon 3 IVS2-13 A/C3 G I172N P30L Alu I Bsr I Aci I Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients J Clin Endocrinol Metab, January 2004, 89(1):368 –374 371 FIG. 1. Schematic diagram of primer positions in the CYP21 gene (see Table 3) and localization of four new mutations. The numbered boxes represent the exons. A, Amplification strategy for PCR-RFLP analysis. B, Amplification strategy for PCR-direct sequence. TABLE 4. Distribution of mutations obtained in 51 Tunisian patients with classic form and comparative frequencies with previous reports (Refs. 22, 24, 26, 27, 28, and 29) Frequency found (%) Reported frequency (%) 36 20 18 11 3 2 1 1 0 0 0 0 4 6 35.3 19.6 17.6 10.8 2.9 2.0 1.0 1.0 0 0 0 0 3.9 5.9 3–13.8 12–33 20 –31 2–12 4 –17 96 102 94.1 Mutations SW SV Total Q318X Large deletions IVS-13A/C3 G I172N R356W R483W W19X 2669insC del 8-bp E6 cluster P30L V281L Complex alleles Not detected 34 20 17 0 3 2 1 1 0 0 0 0 4 6 2 0 1 11 0 0 0 0 0 0 0 0 0 0 Total detected Total 82 88 14 14 0–6 0 –5 0–6 0 –2 Direct sequencing revealed three novel point mutations never described: the first is W19X in exon 1 (1 allele) that is a non-sense mutation, the second is a frame shift mutation due to insertion of C in 2669 position in exon 10 (1 allele), and the third one is R483W in exon 10 (2 alleles) that is a missense mutation. In addition we revealed one allele with a novel small conversion DNA sequence from exon 5 to exon 8, generated by transfer of deleterious mutations from the CYP21P to the functional CYP21 gene, confirmed by direct sequencing. The patient carrying the small conversion is a compound heterozygous having the Q318X mutation on her other allele (data not shown). Two families in this series had one allele with more than one mutation. Patient 36 had the Q318X and IVS2–13A/ C3 G mutations both on his maternal and paternal allele, and patient 16 carried the IVS2–13A/C3 G and P30L on her paternal allele. These complex alleles probably resulted from large conversions or multiple mutations events. The distribution of mutation frequencies in the Tunisian population is significantly different from those previously reported in all parts of the word (20). The Q318X is very frequent (35.3%), whereas R356W (2.9%) is lower than in Western countries and other populations. Thirteen of the 36 alleles (83.3%) with single Q318X mutation were linked to a polymorphism (601 C3 G) in intron 2, revealed by AciI enzyme that we use for screening P30L mutation. This polymorphism was detected only on chromosomes carrying Q318X mutation. SW forms were predominant, representing more than 86% of reported classical forms. Q318X was present in 21 of 44 (47.7%) SW cases. Homozygoty for this mutation (13/44 ⫽ 29.5%) was the most frequent genotype. In the SV form, all the patients carry at least one I172N mutation. Five of seven affected children were homozygous for this mutation; I172N was not observed in SW cases. All remaining mutations were associated with the SW phenotype. Discussion Molecular analysis of CYP21 gene was performed to determine genetic aspects of 51 unrelated Tunisian patients with a classical form of 21-OHD. This study is the first report about distribution of mutations causing 21OHD in the Tunisian population and even in the Arab population. The rate of consanguinity is 60.8%, no other population reported in literature has so high a rate. Consanguineous families were unrelated and originated from different regions of Tunisia. In this study, we screened for six of the most common mutations, large deletions, and rare mutations using RFLP, PCR, and direct sequencing methods, respectively. Identifying these mutations, we were able to characterize 94.1% of chromosomes. Our results indicate that four mutations (Q318X, IVS2–13A/ C3 G, I172N, and large deletions) represent about 87% of 372 J Clin Endocrinol Metab, January 2004, 89(1):368 –374 Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients TABLE 5. Frequency of Q318X mutation in patients with SW forms in different populations a Country Chromosomes studied Affected chromosomes with Q318X % of alleles Ref. Tunisia Argentina Japan Lebanon Chile Mexico Spain Italy Hungary Southern Germany Brazil USA Finland Sweden 88 48 52 28 126 42 120 90 101 184 58 254 72 186 34 9 6 3 13 4 11 8 8 to 13a 14 3 13 2 1 38.6 18.8 11.5 10.7 10.5 9.5 9.2 8.9 8.0 to 13.0 7.6 5.2 5.1 2.8 0.5 Present study 27 28 30 31 32 33 22 34 35 36 37 38 39 Parents of patients were not studied. FIG. 3. DNA sequence of CYP21 gene of patient 17 showing the insertion of C in 2669 position at exon 10, causing a frame shift mutation in heterozygous form. FIG. 2. DNA sequence of CYP21 gene of patient 16 showing the Cto-T transition at nucleotide 2668 in homozygous form, which results in the arginine-to-tryptophan mutation at codon 483. those causing severe forms of 21-OHD in the Tunisian population. The most frequent mutation in Tunisian classical forms was Q318X, with 35.3%, in contrast to 0.5–13.8% described in other series (Table 4). This lesion constituted 38.6% of CYP21 gene defects in the SW form. However, our results differ from all those reported in other populations (Table 5). The reason for the high frequency of the Q318X mutation is still unknown. However, a founder effect could be partly responsible for the observed distribution of this mutation in our population. Indeed, we have detected linkage desequilibrium between this mutation and a CYP21 gene polymorphism (601 C3 G in intron 2) in 83.3% of alleles, a loci probably due to the antiquity of the founder chromosomes. It would be interesting to search for this mutation in other Arab populations, to clarify the history of this particular founder chromosome. The subsequent most frequent mutation was large deletions (19.6%), followed by IVS-13A/C3 G (17.6%) and I172N (10.8%). The R356W mutation (2.9%) was less frequent than other reports (Table 4). Some alleles harbor more than one mutation; we found such alleles (Q318X⫹IVS2–13A/C3 G and IVS2–13A/C3 G⫹P30L). Cluster E6 and del 8-bp, which are two mutations associated with the SW form (21, 22), were absent in our study. Absence of V281L mutation may be FIG. 4. DNA sequence of CYP21 gene of patient 28 showing the Gto-A transition at nucleotide 56 in the heterozygous form, which results in the tryptophan-to-stop codon at position 19. explained by the fact that we did not include patients with the nonclassic form. Four novel mutations were revealed; they have never been previously reported elsewhere. In patient 38, we detected a novel C-to-T transition in codon 483 that results in substitution of the amino acid arginine (CGG) by the amino acid tryptophan (TGG) (Fig. 2). The R483W mutation was found in homozygous state and was carried by both parents. The amino acid R483 is positioned in a conserved C-terminal region of the 21-OH protein, and it has been shown that another mutation in this position, R483P, Kharrat et al. • Genetic Analysis of Tunisian 21-OHD Patients results in an increased degradation pattern of the enzyme (23). In patient 37, we detected a novel insertion of C in 2669 position (2669insC) at exon 10, causing a frame shift mutation (Fig. 3). This mutation, found in heterozygous state, was inherited from the father. In patient 43, we detected a novel G-to-A transition, changing codon 19 from tryptophan (TGG) to the stop codon (TAG), which results in mutation of W19X (Fig. 4). This mutation was found in heterozygous state and was inherited from the mother. Neither of these three point mutations has been observed in CYP21P pseudogene; and thus, the presence of each in the patients mutant CYP21 gene does not appear to be the result of gene conversion. However, in patient 39, we detected a novel small conversion from exon 5 to exon 8. This patient, who carried the Q318X mutation inherited from his mother, had a transfer of deleterious mutations from CYP21P to CYP21 gene allele by gene conversion on her paternal chromosome. All novel mutations were found in patients with the SW form; and therefore, they should confer no enzymatic activity. Generally the severity of the phenotype depends on mutations present on both alleles. Large deletions, Q318X and R356W mutations found in our series, were always associated with the SW form, whereas the IVS2–13A/C3 G mutation was found in either SW or SV, and I172N mutation was associated with SV (24, 25). In conclusion, the methods described in this study are suitable for genetic screening, including the prenatal diagnosis of disease. It is the first time that so large a prevalence of Q318X mutation has been reported. Therefore, a screening for this mutation in the Arab population should be evaluated. Acknowledgments Received June 19, 2003. Accepted September 16, 2003. Address all correspondence and requests for reprints to: Pr. Habiba Chaabouni, Laboratoire de Génétique Humaine, Faculté de Médecine de Tunis, 1006, Tunisie. E-mail: [email protected]. M.K. and V.T. have contributed equally to this work and could be considered as first authors. References 1. White PC, New MI, Dupont B 1987 Congenital adrenal hyperplasia. N Engl J Med 316:1519 –1524 2. Miller WL, Levine LS 1987 Molecular and clinical advances in congenital adrenal hyperplasia. J Pediatr 111:1–17 3. Pang SY, Wallace MA, Hofman L, Thuline HC, Dorche C, Lyon IC, Dobbins RH, Kling S, Fujieda K, Suwa S 1988 Worldwide experience in newborn screening for classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Pediatrics 81:866 – 874 4. New MI, White PC 1995 Genetic disorders of steroid metabolism. In: Thakker RV, ed. 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