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Three Novel Mutations in CYP21 Gene in Brazilian Patients with the Classical Form of 21-Hydroxylase Deficiency Due to a Founder Effect

Unidade de Endocrinologia do Desenvolvimento e Laboratório de Hormônios e Genética Molecular—Laboratório de Investigação Médica 42, Disciplina de Endocrinologia, Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, São Paulo 01060-970, Brazil

Address all correspondence and requests for reprints to: Ana Elisa C. Billerbeck, Ph.D., Hospital das Clínicas, Faculdade de Medicina da Universidade de São Paulo, Disciplina de Endocrinologia, Caixa Postal 3671, São Paulo, 01060-970, Brazil. E-mail: . aecbil{at}usp.br

Abstract

Three different new mutations were found after CYP21 gene sequencing in three unrelated patients with the classical form of the 21-hydroxylase deficiency. These mutations were also screened in their affected relatives. In one patient and her brother, both affected with the simple virilizing form and in their aunt, with the nonclassical form, an AG>GG transition was found in the acceptor site of intron 2. In another patient with the salt wasting form, we found a 1003^1004 insA, in exon 4, that altered the reading frame and created a stop codon in codon 297. In the third patient and his sister, we found a C>T transition in codon 408. This transition led to the substitution of arginine by cysteine (R408C) in a conserved region where arginine is conserved in at least four different species. These siblings with the R408C mutation, both affected with the salt wasting form, have the IVS2–13A/C>G mutation in the other allele, suggesting that the R408C should lead to complete impairment of enzymatic activity. To rule out the possibility of polymorphism, R408C was screened through allele specific PCR, and it was not found in 100 normal alleles. The screening of these three new mutations by allele-specific PCR or enzymatic restriction in 212 CAH patients disclosed their presence in 2.3% (9/387) of the alleles. All three new mutations were found in compound heterozygous state with previously known mutations. Microsatellite studies, using markers flanking CYP21 gene, revealed that each new mutation presents the same haplotype, suggesting a gene founder effect, similar to what was previously observed with the G424S mutation also described in our population. Although microconversion events are the main cause of mutations in the CYP21 gene, random mutations with a common origin can also be the cause of 21-hydroxylase deficiency.

CONGENITAL ADRENAL HYPERPLASIA (CAH) due to 21-hydroxylase (21OH) deficiency is an autosomal recessive disorder that accounts for 95% of CAH cases (1). It has a wide spectrum of clinical manifestations due to the degree of impairment of enzyme activity caused by different mutations in the CYP21 gene. Population studies showed that these mutations can be large or point mutations (2, 3, 4, 5, 6). Basically, 95% of the mutated alleles in patients with CAH-21OH are due to the conversion of DNA sequences from CYP21P pseudogene to the active CYP21 gene; the remaining alleles have new mutations due to random events (3, 7).

The nine most common CYP21P-derived mutations can be rapidly diagnosed by allele-specific PCR or dot blot hybridization, and the other mutations need to be studied through direct CYP21 sequencing. Several population studies, using techniques for the identification of large mutations and 7- to 15-point mutations, identified mutations in 73–95% of the affected alleles (2, 3, 4, 5, 8). Our previous studies of 18 mutations in Brazilian patients with CAH-21OH, using allele-specific PCR, Southern blotting techniques, and CYP21 sequencing, identified mutations in 82% of the alleles, and 98% of these mutations were originated from conversion events involving pseudogene (6, 9, 10).

Genetic linkage disequilibrium between specific HLA haplotypes and CYP21 mutations has been described by several authors: CYP21 gene deletion and HLA BW47,DR7 in classical form (11, 12, 13) and V281L mutation and HLA B14, DR1 in nonclassical (NC) form (14, 15, 16). Most of the recent studies involving CYP21 mutations do not provide data on haplotypes, and founder effect has not often been described for mutations in the CYP21 gene.

In this paper, we report three new mutations found after CYP21 sequencing, not originated from microconversion events, in 12 patients with CAH-21OH. The study of two microsatellites (D6S273 and TAP 1) flanking the CYP21 gene showed that patients with each of the new mutations share the same haplotype, suggesting a gene founder effect.

Subjects and Methods

Informed consent for the study was obtained from all patients’ parents or tutors, and the study was approved by the Ethical Committee of our Institution.

We studied the CYP21 gene of three unrelated patients with the classical form of CAH-21OH in whom only one mutation was found after screening of 18 mutations previously reported (6, 9, 10). The whole cohort of 212 patients with CAH was screened for the new mutations, and a total of 12 patients harbored one of the new mutations (Table 1).

DNA samples from patients and their parents were obtained from peripheral blood leukocytes by SDS-proteinase-K-salting-out procedures. Genomic DNA (100–500 ng) was submitted to PCR for the amplification of CYP21 functional gene in two different fragments. The specificity was achieved by using a primer located at exon 3 that does not contain the 8-bp deletion present in the pseudogene. The primers used were 1–48 and 4–55 that amplify fragments of 1.2 and 2.3 kb, respectively (17). These amplified fragments were submitted to direct sequencing with different specific primers (17) through the dideoxynucleotide terminator methodology using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Perkin-Elmer Corp., Foster City, CA) and were analyzed in the ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems). The numbering of the nucleotides was according to White et al. (18).

The new mutations found were screened in 212 patients with CAH-21OH and in 100 CYP21P alleles using either digestion of PCR product or allele-specific PCR. For the screening of these mutations in pseudogene, primers P55 and P48 were substituted for primers P56 and P49, which contain the 8-bp deletion in exon 3 (17).

Microsatellite studies. Two microsatellite loci (D6S273 and TAP 1) flanking the CYP21 gene, located less than 3 cM apart, 5' and 3' to the CYP21 gene, were studied in all patients and relatives who carry these new mutations. In brief, 2 ng of genomic DNA was submitted to PCR with 15 pmol of each primer, 1.5 mM MgCl₂, 2 U of Taq polymerase (Amersham Pharmacia Biotech, Uppsala, Sweden), 200 µM of deoxy-NTP in 50-µl reaction in the GeneAmp PCR system 9700 thermo-cycler (PE Applied Biosystems). The primer sequences were obtained from Génèthon (France; D6S273) and Martin et al. (Ref. 19 , see Table 3; TAP 1), and the 3' primer from both markers was labeled with fluorescence. The samples were submitted to 28 amplification cycles of 1.5 min at 95 C, 1 min at 55 C, and 1.5 min at 72 C followed by a final extension step of 45 min at 72 C. Two microliters of the amplified product were mixed with 24 µl of formamide (Hi-DI formamide; PE Applied Biosystems) and 1 µl of the size standard marker TAMRA 350, submitted to capilar electrophoresis on the ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems) and analyzed by GeneScan.

The mutations and the microsatellites found in the index cases were segregated in the DNA of their parents.

Fisher exact test was applied to verify the association of these mutations with respective haplotype.

Results

The sequencing of the 2.3-kb fragment (P4-P55 PCR product) from a girl (patient 1) identified in exon 4 an insertion of an adenine between nucleotides 1003 and 1004 (1003^1004 insA) in heterozygous state, resulting in a frameshift mutation that creates a stop codon in exon 7 (codon 297). This patient had the IVS2–13 A/C>G mutation in the other allele, and this genotype is in accordance to her SW phenotype. This insertion abolishes a PstI restriction site and therefore could be easily screened after a nested PCR (P55-P16 PCR product) (17). The restriction of this fragment in normal individuals results in two fragments: 209 and 292 bp. Digestion of P55-P16 PCR product of patient 1 originated three fragments of 209, 292, and 501 bp. The 501-bp fragment resulted from the loss of the one PstI restriction site between fragments of 209 and 292 bp. The screening of 1003^1004 insA mutation by PstI digestion in our CAH-21OH cohort, identified this mutation in heterozygous state in three other patients: a girl and a boy with the SW form (patients 2 and 4) in heterozygous state with Ins T in exon 7 and R408C mutations, respectively, and a girl with the SV form (patient 3) in heterozygous state with IVS2–13 A/C>G. This latter phenotype is in accordance with the presence of IVS2–13 A/C>G mutation in the other allele. Microsatellite study showed the same haplotype in the patients and their relatives who carry this mutation (allele 3 to the marker D6S273, allele 6 to the marker TAP 1, Table 1). This association was statistically significant (P = 0.0064).

A heterozygous C>T transition in codon 408 leading to substitution of arginine by cysteine (R408C) was found in a boy (patient 6) and his sister (patient 7), both affected with SW form. In these patients R408C mutation was in compound heterozygous state with IVS2–13 A/C>G, suggesting that R408C must confer severe impairment of the enzyme activity. The screening of this mutation in our CAH-21OH cohort was done by allele-specific PCR based on the methodology of Wedell and Luthman (17). The fragment amplified with the primers P4 and P55 was submitted to a second round of PCR using the primers P11, P45 and the allele-specific primers: R408 GCCTGGCTCCAGGAAGCG and C408 TGCCTGGCTCCAGGAAGCA. This mutation was found in three other patients: in heterozygous state with 1003^1004 insA in patient 4 affected by SW form, with I172N mutation in patient 5 affected by SV form, and with IVS2–13 A/C>G in patient 8 affected by SW form. One hundred normal alleles were screened for this mutation and did not reveal its presence. Microsatellite studies showed the same haplotype in the patients and their relatives who carry this mutation (allele 5 to the D6S273, allele 5 to the TAP 1, Table 1). This association was statistically significant (P = 0.0103).

The sequencing of the 1.2-kb fragment (P1-P48 PCR product) of a girl (patient 9), her brother (patient 10), and their aunt (patient 11) revealed a heterozygous AG>GG transition in the acceptor site of intron 2. The two children with the SV form had in the other allele the I172N mutation, which confers 3–7% of enzyme activity (20). Their aunt, however, had a P30L mutation in the other allele, which confers more than 30% of enzyme activity, explaining why she presented the NC form of the disease (21). This mutation abolishes a PstI restriction site in the P1-P48 PCR product. The restriction of this fragment in normal individuals results in three fragments of 59, 538, and 544 bp. Digestion of P1-P48 PCR product of these three patients originated four fragments of 59, 538, 544, and 603 bp. The 603-bp fragment resulted from the loss of one PstI restriction site between fragments of 544 and 59 bp in one allele. The screening of this mutation by PstI digestion in our cohort with CAH-21OH identified its presence in compound heterozygous state with IVS2–13A/C>G in another girl (patient 12) with the SW form. Microsatellite studies showed the same haplotype in the patients and their relatives who carry this mutation (allele 3 to the D6S273, allele 1 to the TAP 1, Table 1). This association was statistically significant (P = 0.0002). These three new mutations were not found in 80 pseudogenes.

Discussion

We had previously screened for the presence of 18 mutations in our cohort of Brazilian patients with CAH-21OH and did not identify mutations in 18% of the alleles. In an attempt to identify these alleles, we have sequenced the entire CYP21 gene of three unrelated patients with CAH-21OH. Three new mutations were found, and the whole cohort of CAH-21OH was screened for these mutations.

Four patients presented the 1003^1004 insA mutation in a compound heterozygous state. All these patients, but one, have the SW form of the disease, which suggests that 1003^1004 insA confers severe impairment of enzyme activity. Frameshift mutations alter the open reading frame and together with a stop codon are responsible for altered and truncated proteins and normally are related to the SW form of the disease (22). The patient with SV form of the disease has in the other allele the IVS2–13A/C>G, which was observed in different population studies mainly in patients with SW form, although its presence in cases with SV form has also been described (23).

The R408C mutation in exon 9 of CYP21 gene is located in a region that is conserved in the human CYP21 gene (18) and in at least four different species: Mus musculus, Bos taurus, Rattus norvegicus, and Sus scrofa (24, 25, 26, 27). In the 3D model of human P450c21 constructed by Mornet and Gibrat (28), R408 is conserved in the meander (a portion of chain between helix K' and the cys pocket whose 3D structure is highly conserved in all P450s), and therefore this mutation should alter the tertiary structure of the protein. This transition was not identified in 100 alleles of normal subjects, ruling out the possibility of polymorphism. This mutation was found in four patients, three of them presented the SW form and the other the SV form, in which the phenotype is in accordance with the I172N mutation presented in the other allele (20).

The AG sequence in the splicing acceptor site of the gene is highly conserved in all species, and therefore a mutation in this site is responsible for altering gene splicing. In 21-hydroxylase deficiency, mutations that create or destroy the splicing site are correlated with SW form of the disease (3, 4, 29). An AGGG transition in the acceptor site of intron 2 was found in heterozygous state in two siblings (a girl and her brother), both with the SV form, and in their aunt with the NC form of the disease. Although this mutation should confer very low enzyme activity, these two different clinical forms are in accordance with the mutation present in the other allele. This mutation was also found in a compound heterozygous state with IVS2–13 A/C>G mutation in another patient with SW form, confirming that this mutation should confer severe impairment of enzyme activity.

Different from other genetic diseases, in CAH-21OH, the majority of the mutations are due to intergenic recombination between active CYP21 gene and its 98% homologous pseudogene. However, mutations that are not present in the pseudogene have also been described (7, 22). These mutations result from casual events, and most of them were found as isolated cases.

The three new mutations described here were not found in the pseudogene, suggesting that they were not originated from microconversion events, but probably represent random events. These new mutations correspond to 2.3% of mutant alleles in our series of 212 patients. The presence of the same random mutagenic event in patients from nonrelated families suggests the possibility of a common origin of the mutation.

Founder effect was not a very often described event in CAH-21OH. The majority of the studies that have focused on CYP21 mutations have not provided data on haplotypes. Some strong associations have been previously observed in well-defined populations. Dupont et al. (30) described close linkage of 21OH deficiency and the HLA complex. Associations were described: high frequency of HLA-Bw47; DR7 has been observed in patients with classical form (11, 12) and high frequency of HLA-B14; DR1 in patients with NC form, mainly in Ashkenazi Jewish, Hispanic, and Italians populations (12, 14, 15). This latter haplotype was also associated to C4B and CYP21P gene duplications (31, 32). Subsequently, it was demonstrated that these haplotypes bore specific CYP21 mutations, CYP21 deletion in classical form (13), and V281L mutation in NC form (16). This suggests a common origin or founder effect of these mutations. Lately, strong associations have been observed in Finnish population, where they observed multiple founder mutation-haplotypes combinations (33). Strong linkage disequilibrium has not been observed between MHC haplotypes or polymorphism and CAH-21OH in mixed populations, suggesting that most mutations are of various independent origins (33).

Microsatellite markers flanking the CYP21 gene have been used in CAH-21OH deficiency for different purposes such as prenatal studies, segregation of disease-causing chromosomes, to detect allele drop-out and adjunct to molecular studies in newborn screening (34, 35, 36). More recently, Fitness et al. (36) described linkage disequilibrium between two CYP21 mutations, Q318X and V281L, with microsatellite markers in a New Zealand population.

In this paper, we suggest a common origin for these three new mutations. The alleles that harbored each of the three new mutations disclosed the same microsatellite haplotype, suggesting a founder effect for each one, as observed with the G424S mutation described earlier in seven patients of our series (10). The G424S mutation was first described in a Mullatto patient with SV form in linkage disequilibrium with HLA-DR17 and the deletion of both the CYP21 pseudogene and C4 gene.

Until now, these four mutations are the only ones described in Brazilian population that presented a gene founder effect. The search for these mutations will reveal if they are restricted to our population or if they have a wider ethnic distribution. We believe that these mutations might be present in other populations mainly because our Brazilian population has a greatly diverse origin.

Although microconversion events are the main cause of mutations in the CYP21 gene, casual events that can have a founder effect can also cause 21-hydroxylase deficiency.

Acknowledgments

We thank the staff of Laboratório de Hormônios e Genética Molecular LIM/42 and Mrs. Mayra Davi Lima for technical assistance and Professor Paulo Otto for microsatellite statistical analysis.

Footnotes

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 95/8325-6, 99/06468-5, 99/12107-5, and 98/00243–9 (to T.A.S.S.B.).

Abbreviations: CAH, Congenital adrenal hyperplasia; NC, non-classical; 21OH, 21-hydroxylase; SW, salt wasting; SV, simple virilizing.

Received December 6, 2001.

Accepted June 4, 2002.

References

1. Morel Y, Miller WL 1991 Clinical and molecular genetics of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Adv Hum Genet 20:1–68[Medline]
2. Mornet E, Crété P, Kuttenn F, Raux-Demay MC, Boue J, White PC, Boue A 1991 Distribution of deletions and seven point mutations on CYP21B genes in three clinical forms of steroid 21-hydroxylase deficiency. Am J Hum Genet 48:79–88[Medline]
3. Speiser PW, Dupont J, Zhu D, Serrat J, Buegeleisen M, Tusie-Luna MT, Lesser M, New MI, White PC 1992 Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest 90:584–595
4. Wedell A, Thilén A, Ritzén EM, Stengler B 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[Abstract]
5. Wilson RC, Wei JQ, Cheng KC, Mercado AB, New MI 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[Abstract/Free Full Text]
6. Bachega TASS, Billerbeck AEC, Madureira G, Marcondes JA, Longui CA, Leite MV, Arnhold IJ, Mendonca BB 1998 Molecular genotyping in Brazilian patients with the classical and nonclassical forms of 21-hydroxylase deficiency. J Clin Endocrinol Metab 83:4416–4419[Abstract/Free Full Text]
7. Lee HH, Chao HT, Lee YJ, Shu SG, Chao MC, Kuo JM, Chung BC 1998 Identification of four novel mutations in the CYP21 gene in congenital adrenal hyperplasia in the Chinese. Hum Genet 103:304–310[CrossRef][Medline]
8. Dardis A, Bergada I, Bergada C, Rivarola M, Belgorosky A 1997 Mutations of the steroid 21-hydroxylase gene in an Argentinian population of 36 patients with classical congenital adrenal hyperplasia. J Pediat Endocrinol Metab 10:55–61[Medline]
9. Bachega TASS, Billerbeck AEC, Madureira G, Arnhold IJ, Medeiros MA, Marcondes JA, Longui CA, Nicolau W, Bloise W, Mendonca BB 1999 Low frequency of CYP21B deletion in Brazilian patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Hum Hered 49:9–14[CrossRef][Medline]
10. Billerbeck AEC, Bachega TASS, Frazzatto ET, Nishi MY, Goldberg AC, Marin MCC, Madureira G, Monte O, Arnhold IJP, Mendonca BB 1999 A novel missense mutation Gly424Ser in Brazilian patients with 21-hydroxylase deficiency. J Clin Endocrinol Metab 84:2870–2872[Abstract/Free Full Text]
11. Klouda PT, Harris R, Price DA 1978 HLA and congenital adrenal hyperplasia. Lancet 2:1046
12. Pollack MS, Levine LS, Zachmann M, Prader A, New M, Oberfield F, Dupont B 1979 Possible genetic linkage disequilibrium between HLA and the 21-hydroxylase deficiency gene (congenital adrenal hyperplasia). Transplant Proc 11:1315–1316[Medline]
13. WhitePC, New MI, Dupont B 1984 HLA-linked congenital adrenal hyperplasia results from a defective gene enconding a cytochrome P-450 specific for steroid 21-hydroxylation. Proc Natl Acad Sci USA 81:7505–7509[Abstract/Free Full Text]
14. Laron Z, Pollack MS, Zamir R, Roitman A, Dickerman Z, Levine LS, Lorenzen F, O’Neill GJ, Pang S, New MI, Dupont B 1980 Late onset 21-hydroxylase deficiency and HLA in the Ashkenazi population: a new allele at the 21-hydroxylase locus. Hum Immunol 1:55–60[CrossRef][Medline]
15. Speiser PW, Dupont B, Rubinstein P, Piazza A, Kastelan A, New MI 1985 High frequency of nonclassical steroid 21-hydroxylase deficiency. Am J Hum Genet 37:650–657[Medline]
16. Speiser PW, New MI, White PC 1988 Molecular genetic analysis of nonclassic steroid 21-hydroxylase deficiency associated with HLA-B14, DR1. New Engl J Med 319:19–23[Abstract]
17. Wedell 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 499–504
18. White PC, New MI, Dupont B 1986 Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci USA 83:5111–5115[Abstract/Free Full Text]
19. Martin M, Mann D, Carrington M 1995 Recombination rates across the HLA complex: use of microsatellites as a rapid screen for recombinant chromosomees. Hum Mol Genet 4:423–428[Abstract/Free Full Text]
20. Tusie-Luna MT, Traktman P, White PC 1990 Determination of functional effects of mutations in the steroid 21-hydroxylase gene (CYP21) using recombinant vaccinia virus. J Biol Chem 265:20916–20922[Abstract/Free Full Text]
21. Tusie-Luna MT, Speiser PW, Dumic M, New MI, White PC 1991 A mutation (P30L) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele. Mol Endocrinol 5:685–692[Abstract/Free Full Text]
22. Speiser PW 2001 Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Review. Endocrinol Metab Clin North Am 30:31–59[Medline]
23. Higashi Y, Tanae A, Inoue H, Fujii-Kuriyama Y 1988 Evidence for frequent gene conversions in the steroid 21-hydroxylase (P-450c21) gene: implications for steroid 21-hydroxylase deficiency. Am J Hum Genet 42:17–25[Medline]
24. Chaplin DD, Galbraith LJ, Seidman JG, White PC, Parker KL 1986 Nucleotide sequence analysis of murine 21-hydroxylase genes: mutations affecting gene expression. Proc Natl Acad Sci USA 83:9601–9605[Abstract/Free Full Text]
25. Chung BC, Matteson KJ, Miller WL 1985 Cloning and characterization of the bovine gene for steroid 21-hydroxylase deficiency (P-450c21). DNA 4:211–219[Medline]
26. Zhou MY, Vila MC, Gomez-Sanchez EP, Gomez-Sanchez CE, Cloning of two alternatively spliced 21-hydroxylase cDNAs in the rat adrenal. (GenBank accession no. U56853, NID g1336152)
27. Burghelle MC, Geffrotin C, Vaiman M 1992 Sequences of the swine 21-hydroxylase gene (CYP21) and a portion on the opposite-strand overlapping gene of unknown function previously described in human. Biochim Biophys Acta 1171:153–161[Medline]
28. Mornet E, Gibrat JF 2000 A 3D model of human P450c21: study of the putative effects of steroid 21-hydroxylase gene mutations. Hum Genet 106:330–339[CrossRef][Medline]
29. Carrera P, Bordone L, Azzani T, Brunelli V, Garancini MP, Chiumello G, Ferrari M 1996 Point mutations in Italian patients with classic, non-classic, and cryptic forms of steroid 21-hydroxylase deficiency. Hum Genet 98:662–665[CrossRef][Medline]
30. Dupont B, Oberfield SE, Smithwick EM, Lee TD, Levine LS 1977 Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency). Lancet 2:1309–1312[Medline]
31. Carroll MC, Campbell RD, Porter RR 1984 The mapping of 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man. Proc Natl Acad Sci USA 82:521–525
32. Werkmeister JM, New MI, Dupont B, White PC 1986 Frequent deletion and duplication of steroid 21-hydroxylase genes. Am J Hum Genet 39:461–469[Medline]
33. Levo A, Partanen J 1997 Mutation-haplotype analysis of steroid 21-hydroxylase (CYP21) deficiency in Finland. Implications for the population history of defective alleles. Hum Genet 99:488–497[CrossRef][Medline]
34. Ezquieta B, Jariego C, Varela JM, Oliver A, Gracia R 1997 Microsatellite markers in the indirect analysis of the steroid 21-hydroxylase gene. Prenat Diagn 17:429–434[CrossRef][Medline]
35. Day DJ, Speiser PW, Schulze E, Bettendorf M, Fitness J, Barany F, White PC 1996 Identification of non-amplifying CYP21 genes when using PCR-based diagnosis of 21-hydroxylase deficiency in congenital adrenal hyperplasia (CAH) affected pedigrees. Hum Mol Genet 5:2039–2048[Abstract/Free Full Text]
36. Fitness J, Dixit N, Webster D, Torresani T, Pergolizzi R, Speiser PW, Day D 1999 Genotyping of CYP21, linked chromosome 6p markers, and a sex-specific gene in neonatal screening for congenital adrenal hyperplasia. J Clin Endocrinol Metab 84:960–966[Abstract/Free Full Text]

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