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0021-972X/98/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1998 by The Endocrine Society Vol. 83, No. 9 Printed in U.S.A. COMMENTS Salt-Wasting Congenital Adrenal Hyperplasia: Detection of Mutations in CYP21B Gene in a Chilean Population* CARLOS E. FARDELLA, HELENA POGGI, PEDRO PINEDA, JULIA SOTO, ISABEL TORREALBA, ANDREÍNA CATTANI, EVELINE OESTREICHER, AND ARNALDO FORADORI Department of Endocrinology (C.E.F., A.C., E.O.) and The Research and Development Unit of the Associated Unit of Clinical Laboratories (H.P., J.S., A.F.), Faculty of Medicine, Catholic University of Chile; and Department of Endocrinology of the Clinical Hospital at University of Chile (P.P.), Endocrinology Service (I.T.), Sotero del Rio and Luis Calvo Mackenna Hospitals, Public Health Services, Santiago, Chile ABSTRACT The steroid 21-hydroxylase deficiency (21OHD) is the most frequent cause of congenital adrenal hyperplasia. We have characterized the disease-causing mutations in the 21-hydroxylase genes of 63 patients with salt-wasting congenital adrenal hyperplasia from a Chilean population of Hispanic origin, a group that has been scarcely evaluated. Using allele-specific PCR, lesions were identified in 97 chromosomes out of 126 tested (77%). The most frequent findings were the gene deletion or large gene conversion (LGC) 5 22.9%, I2 splice 5 19%, R357W 5 12.7%, and Q319X 5 10.5%. We did not find alleles with the mutation F308insT and we found three alleles with the cluster E6. The frequency of the point mutation R357W was at T HE steroid 21-hydroxylase deficiency (21OHD) compromises about 95% of all cases of congenital adrenal hyperplasia (CAH) and has an overall incidence of about 1 in 13,000 live births (1–3). About two thirds of patients have salt loss, making it the most common congenital salt-wasting (SW) disease. Adrenal 21-hydroxylase activity is catalyzed by the cytochrome P450c21, encoded by a gene termed CYP21B, to distinguish it from the duplicated but nonfunctional P450c21A gene (4, 5). The genetics of P450c21 are unusual and complicated. Random deletions and de novo mutations almost never occur, instead, gene conversion accounts for about 85% of all mutant P450c21 alleles. In these gene conversions, all or part of the CYP21B gene is replaced by, or converted to, the sequence of the corresponding sequence of the CYP21A gene (3, 6, 7). Genetic disorders in the P450c21 that reduce more than 99% of the enzyme activity results in deficient synthesis of cortisol and, in the majority of cases, also causes SW and virilization. Clinically, it has been shown that deletion of the Received December 18, 1997. Revision received May 19, 1998. Accepted May 26, 1998. Address all correspondence and requests for reprints to: Carlos E. Fardella, Department of Endocrinology, Pontificia Universidad Catolica de Chile, Lira 44, Santiago, Chile. * This work was supported by Research and Development funds of the Associated Unit of Clinical Laboratories, Catholic University and by Chilean grant Fondecyt 1951094. least two times more frequent than the one found in Caucasians populations, but similar to that communicated in Asian populations; this finding may be explained by the Asian ancestry of our SouthAmerindian population. The frequency of Q319X was also high, similar only to those patients studied in Italy and in a neighboring Argentinian population. In summary, this is a genetic characterization of 21OHD made in an almost pure Hispanic population in Latin America. The high frequency of deletion of CYP21B gene, I2 splice, R357W, and Q319X mutations probably reflects the EuropeanCaucasian-Spanish influence of the conquerors, mixed with Amerindians of Asian ancestry and modulated by other European immigrations. (J Clin Endocrinol Metab 83: 3357–3360, 1998) P450c21 gene and the aberrant splicing in intron 2 are the most frequent cause of the SW form. However, several other mutations also result in a complete inactivation of P450c21 (3, 6, 8). Because it has been demonstrated that ethnic differences may determine changes in the pattern of mutations, we decided to evaluate the frequency of the principal mutations described as causing the SW form in a Chilean population of Hispanic origin, a group that has been scarcely evaluated. The knowledge of the relative frequencies of point mutations might be useful to delineate appropriate strategies for molecular diagnosis and treatment to prevent a birth defect (9 –12). Patients and Methods Patients Sixty three patients with SW CAH (25 males and 38 females) and their parents, when available, were studied. These patients were unrelated and had no known consanguinity. All patients were diagnosed as having SW by onset of hyperkalemia (6 –9 mmol/L), hyponatremia (118 –125 mmol/L), and dehydration in the first month of life that required glucocorticoid and mineralocorticoid treatment. All patients had elevated levels of 17-hydroxyprogesterone (30 –1029 ng/mL), diagnostic for steroid 21OHD. All females were virilized in utero and were born with ambiguous genitalia. Informed consent was obtained from all participants according to the International Guidelines for Biomedical Research Involving Human Subjects, CIOMS, WHO, Geneva, Switzerland, 1982. The protocol was approved by the Research Commission of the School of Medicine at Catholic University of Chile. 3357 3358 JCE & M • 1998 Vol 83 • No 9 COMMENTS Methods Results Genomic DNA was isolated from the citrated blood of 63 unrelated SW CAH patients and their parents as previously described (13). Genotyping was performed by allele-specific PCR as was described by Wedell and Luthman (14). A first round of amplification using specific primers to amplify the CYP21B gene was carried out. The specific primers were synthesized based on the 8-bp deletion in exon 3 present only in the pseudogene (CYP21A). The PCR reactions rendered two fragments, one encompassing exons 1–3 and the other exons 4 –10 of the CYP21B gene. These fragments were used in a second round of amplification to detect the different mutations. For each mutated position, primers specific for the normal and mutant alleles were synthesized. Using this method, we studied the most frequent gene microconversions reported in Caucasian populations with SW CAH (Fig. 1). The presence of deletion or apparent large gene conversion (LGC) was suspected when the above reactions failed to generate the expected fragment and confirmed performing another PCR with specific primers (14). All the samples were studied for each mutation. In all amplifications we used a positive control of each mutation generously provided by Dr. Wedell. The sequence of all primers used and the PCR conditions were extensively described by Wedell and Luthman (14). The parents’ genotype were also analyzed to establish the segregation of the mutated allele. When discrepancies appeared between the children’s and parent’s genotype, a paternity testing was carried out (15). Heterozygous CYP21 deletion or LGC was inferred when the affected child appeared homozygous for a given mutation but only one parent carried the mutation. When the children were homozygous for a given mutation and the parents were not available, we considered one allele an uncertain allele. The frequency of the different mutations was calculated taking into account the number of uncertain alleles involving the mutation (16). We studied 126 chromosomes corresponding to 63 patients with SW CAH and their parents (both parents were available for analysis in 60% of cases). The mutated alleles were identified in 97 chromosomes (77%); 8 of them were uncertain alleles (I2 splice or deletion 5 5, Q319X or deletion 5 2, cluster E6 or deletion 5 1). The most frequent findings were: deletion or LGC 5 22.9%, I2 splice 5 19.0%, and R357W 5 12.7%. We did not find alleles with the mutation F308insT, and we found three alleles with the cluster E6. The frequency of the mutations analyzed in this study, and the frequencies of the same mutations found in other populations are shown in Table 1. The complete genotype was determined in 41/63 patients (65.1%) and one allele in 15/63 patients (23.8%). More than one mutation by allele was found in only 2 patients. The most frequent genotypes corresponded to homozygous deletion (9/63 5 14.3%) and homozygous to I2 splice (7.9%); the other genotypes founded are listed in Table 2. In 7/63 (11.1%) patients, the two alleles remained not characterized, but hemizygosity cannot be excluded with the method used. The parents’ genotypes permitted us to establish the segregation of the mutated allele in every case. However, in one case the patient was a compound heterozygote for Q319X and R357W, the mother being a carrier of Q319X but the father a carrier of normal alleles. Paternity testing by DNA analysis was carried out, demonstrating that the assumed father was not the biological progenitor. Discussion FIG. 1. Diagram of CYP21 B gene showing 10 exons (bars) and localization of different mutations studied. Abbreviations: I2 splice, an A3.G change in second intron that create an aberrant splice acceptor sequence; I173N, an isoleucine to asparagine change at codon 173; R357W, an arginine to triptophan change at codon 357; Q319X, a glutamine to stop codon change at codon 319; F308insT, a T insertion at codon 308; and cluster E6, an isoleucine-valine-methionine to asparagine-glutamine-lysine change at codons 237–238-240. The present study showed that the most common lesions found in our Chilean population with SW 21OHD corresponded to deletion or LGC of CYP21B gene and to the point mutations I2 splice, R357W, and Q319X. These lesions in the CYP21B gene explain more than 70% of all cases of SW 21OHD studied. The frequency of deletion and the aberrant I2 splice found in our study was similar to that previously described in Caucasians (16 –24) or Asian populations (25–27), in which these lesions constituted between 40 –70% of the genetic defects found in the SW form of 21OHD. However, our results differ from those found in a Mexican population, in which the deletion represented less than 1% of the disease alleles (28). The frequency of the point mutation R357W (12.7%) was twice as high as that described in Caucasians populations, TABLE 1. Mutation frequencies on affected alleles and comparison with other populations Study Total (no.) of chromosomes Del/LGC (%) I2 splice (%) R357W (%) Q319X (%) I173N (%) Cluster E6 (%) F308insT (%) Present study Argentine (29)a USA (18)a USA (17)a Italy (20, 21)a Spain (22)a France (23)a Sweden (16)a Taiwan (25)a Japan (27)a 126 48 92 254 90 40 84 186 13 70 22.9 25.0 39.1 30.3 22.4 27.5 36.9 35.3 42.9 ND 19.0 25.0 28.3 46.1 38.9 32.5 22.6 41.3 35.7 32.9 12.7 2.1 7.6 4.3 1.1 5.0 ND 3.8 14.3 18.6 10.5 18.8 6.5 5.1 12.2 5.0 4.8 3.8 0.0 2.9 7.1 0.0 7.6 2.8 3.3 0.0 8.3 17.4 0.0 2.9 2.4 0.0 5.4 ND 0.0 0.0 4.8 1.1 0.0 2.9 0 ND 3.3 0.8 1.1 2.5 1.2 0.5 0.0 0.0 ND, Not determined. a Percentages were recalculated taking into account only patients with SW CAH. Number of reference is in parentheses. COMMENTS 3359 letion reported in the Mexican study was attributed by the authors to the missed detection of salt wasters (28). TABLE 2. Genotypes grouped according to complete or incomplete identification Genotype No. of index patients % Del or LGC/Del or LGC I2 splice/I2 splice or Del or LGCa Del or LGC/R357W Del or LGC/I2 splice I2 splice/Q319X I2 splice/I2 splice R357W/Q319X Del or LGC/Q319X Q319X/Q319X or Del or LGCa R357W/I172N I172N/R357W 1 Q319X I2 splice/R357W Q319X/I172N I2 splice/Cluster E6 I172N/Cluster E6 I2 splice/I172N Cluster E6/Cluster E6 or Del or LGCa I2 splice/ND R357W/ND I172N/ND R357W 1 Q319X/ND Q319X/ND 9 5 4 3 3 2 2 2 2 2 1 1 1 1 1 1 1 5 5 3 1 1 14.3 7.9 6.4 4.8 4.8 3.2 3.2 3.2 3.2 3.2 1.6 1.6 1.6 1.6 1.6 1.6 1.6 7.9 7.9 4.8 1.6 1.6 Del or LGC, CYP21 deletion or apparent large conversion. Homozygosity could not be distinguished from hemizygosity in cases in which parents were unavailable (uncertain alleles). a but similar to that communicated in Asian populations from Japan and Taiwan (Table 1). The frequency of Q319X was also high (10.5%), similar only to those patients studied in Italy and in a neighboring Argentinian population (20, 21, 29). The low frequency of I173N is probably explained by the fact that we did not include patients with the simple virilizant form of classic 21OHD, in which this mutation is more prevalent (3, 6, 8). The lesions F308insT and cluster E6 were extremely uncommon and appear to explain only a small percentage of SW 21OHD in our population. A similar low frequency of these two mutations had been communicated in all the other populations studied (16 –23, 25, 27). In 23% of the chromosomes, none of the five point mutations or a deletion of the 21-hydroxylase gene were found. The allele frequency of the different mutations studied probably reflects the biracial mixture of Chilean population, with Caucasian genes coming from the Spanish conquerors and a gene pool derived from the native Amerindians (Mapuches) (30). Moreover, analysis of the mitochondrial DNA support the idea that Amerindians had an Asian origin and derived from a small number of maternal lineages (31). Thus, we hypothesized that the high frequency of the point mutation R357W found in this study, as well in the Asian population, may be explained by the Asian ancestry of our South-Amerindian population. Similarities in the genotype distributions between Amerindian and Asian populations have also been described for other studies (32, 33). The high frequency of Q319X found in our population, as well as in Argentina and in Italy, probably is the result of the Italian immigration that occurred in these countries. The high frequency of deletion and I2 splice is expected if we consider previous studies done worldwide. The low frequency of de- Acknowledgments We thank pediatric endocrinologists Drs. F. Ugarte, A. Cortı́nez, and M. E. Willshaw. We also thank Dr. Anna Wedell, who provided us with positive controls for genotyping CAH patients. References 1. New MI, White PC, Pang S, Dupont B, Speiser PW. 1989 The adrenal hyperplasias. In: Scriver CR, Beaudet AL, Sly WS, Valle D, eds. The metabolic basis of inherited disease. Vol. II. New York: McGraw-Hill; 1881–1917. 2. Pang S, Clark A. 1993 Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: newborn screening and its relationship to the diagnosis and treatment of the disorder. Screening. 2:105–139. 3. Miller WL. 1994 Genetics, diagnosis and management of 21-hydroxylase deficiency. J Clin Endocrinol Metab. 78:241–246. 4. Higashi Y, Yoshioka H, Yamane M, et al. 1986 Complete sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene. Proc Natl Acad Sci USA. 83:2841–2845. 5. White PC, New MI, Dupont B. 1986 Structure of the human steroid 21hydroxylase genes. Proc Natl Acad Sci USA. 83:5111–5115. 6. 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Endocr Res. 15:277–301. 12. Forest MG, David M, Morel Y. 1993 Prenatal diagnosis and treatment of 21-hydroxylase deficiency. J Steroid Biochem Mol Biol. 45:75– 82. 13. Lahiri D, Nurnberger J. 1991 A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Res. 19:5444. 14. Weddel A, Luthman H. 1993 Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations. Hum Mol Genet. 2:499 –504. 15. Nakamura Y, Leppert M, O’Conell P, et al. 1987 Variable number of tandem repeat (VNTR): markers for human gene mapping. Science. 235:1616 –1622. 16. Wedell A, Thilén A, Ritzén M, Stengler B, Luthman H. 1994 Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation. J Clin Endocrinol Metab. 78:1145–1152. 17. Wilson R, Wei JQ, New M, et al. 1995 Rapid deoxyribonucleic acid analysis by allele-specific polymerase chain reaction for detection of mutations in the steroid 21-hydroxylase gene. J Clin Endocrinol Metab. 80:1635–1640. 18. Speiser PW, Dupont J, Zhu D, et al. 1992 Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest. 90:584 –595. 19. Speiser PW, White PC, Dupont J, Zhu D, Mercado AB, New MI. 1994 Prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency by allele-specific hybridization and southern blot. Hum Genet. 93:424 – 428. 20. Carrera P, Bordone L, Azzani T, et al. 1996 Point mutations in Italian patients with classic, non-classic, and cryptic forms of steroid 21-hydroxylase deficiency. Hum Genet. 98:662– 665. 21. Carrera P, Ferrari M, Beccaro F, et al. 1993 Molecular characterization of 21-hydroxylase deficiency in 70 Italian families. Hum Hered. 43:190 –196. 22. Ezquieta B, Oliver A, Gracia R, Gancedo PG. 1995 Analysis of steroid 21hydroxylase gene mutations in the Spanish population. Hum Genet. 96:198 –204. 23. Barbat B, Bogyo A, Raux-Demay MC, et al. 1995 Screening of CYP21 gene mutations in 129 French patients affected by steroid 21-hydroxylase deficiency. Hum Mutat. 5:126 –130. 24. Schulze E, Scharer G, Rogatzki A, et al. 1995 Divergence between genotype and phenotype in relatives of patients with the intron 2 mutation of steroid 21-hydroxylase. Endocr Res. 21:359 –364. 25. Lee HH, Chao HT, Ng HT, Choo KB. 1996 Direct molecular diagnosis of CYP21 mutations in congenital adrenal hyperplasia. J Med Genet. 33:371–375. 26. Tajima T, Fujeda K, Nakayama K, Fujii-Kuriyama Y. 1993 Molecular analysis of patient and carrier genes with congenital steroid 21-hydroxylase deficiency 3360 COMMENTS by using polymerase chain reaction and single strand conformation polymorphism. J Clin Invest. 92:2182–2190. 27. Higashi Y, Hiromasa T, Tanae A, et al. 1991 Effects of individual mutations in the P-450(c21) activity and their distribution in the patients genomes of congenital steroid 21-hydroxylase deficiency. J Biochem. 109:638 – 644. 28. Tusié-Luna T, Ramirez-Jiménez S, Ordóñez-Sánchez M, et al. 1996 Low frequency of deletion alleles in patients with steroid 21-hydroxylase deficiency in a Mexican population. Hum Genet. 98:376 –379. 29. Dardis A, Bergada I, Rivarola M, Belgorosky A. 1997 Mutations of the steroid 21-hydroxylase gene in an Argentinian population of36 patients with classical congenital adrenal hyperplasia. J Pediatr Endocrinol Metab. 10:55– 61. JCE & M • 1998 Vol 83 • No 9 30. Cruz-Coke R, Moreno RS. 1994 Genetic epidemiology of single gene defects in Chile. J Med Genet. 31:702–706. 31. Shurr TG, Ballinger SW, Gan YY, et al. 1990 Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages. Am J Hum Genet. 46:613– 623. 32. Tokita A, Matsumoto H, Morrison N, et al. 1996 Vitamin D receptor alleles, bone mineral density and turnover in premenopausal Japanese women. J Bone Miner Res. 11:1003–1009. 33. Lim SK, Park YS, Park JM, et al. 1995 Lack of association between vitamin D receptor genotypes and osteoporosis in Koreans. J Clin Endocrinol Metab. 80:3677–3681. 6th Multidisciplinary Conference: Osteoporosis The Greenbrier, White Sulphur Springs, West Virginia October 22–25, 1998 Presented by The Division of Radiologic Sciences, Wake Forest University School of Medicine, this conference has been designed for all physicians with an interest in diagnosis and treatment of osteoporosis. Family Medicine physicians, gynecologists, endocrinologists, orthopedic surgeons, radiologists, and rheumatologists will benefit from this multidisciplinary approach to osteoporosis. Problems in patient management will be discussed, with an emphasis on integration of key considerations from each contributing discipline. Specific topics to be addressed are pathophysiology of osteoporosis, risk factors and screening, clinical utility of densitometry, laboratory evaluation in osteoporosis, corticosteroid-induced osteoporosis, osteoporosis in athletes, acquisition of postmenopausal osteoporosis, treatment of osteoporotic fractures, and prevention of osteoporosis. Program director: Leon Lenchik, M.D. Faculty: Manuel Jayo, D.V.M., Ph.D., Douglas Linville, M.D., Gaetano Monteleone, Jr., M.D., Michael Sollenberger, M.D., Thomas Snyder, M.D., and Paul Sutej, M.D. Fee: $425. Credit available: 15 hours AMA category 1; 15 hours AAFP; 15 hours ACOG. For further information, contact Pat Rice, Department of Radiology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157-1088. Telephone: 336-716-2470; or (physicians’ access line) 800-277-7654.