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. 1989 Feb;83(2):527–536. doi: 10.1172/JCI113914

Rearrangements and point mutations of P450c21 genes are distinguished by five restriction endonuclease haplotypes identified by a new probing strategy in 57 families with congenital adrenal hyperplasia.

Y Morel 1, J André 1, B Uring-Lambert 1, G Hauptmann 1, H Bétuel 1, M Tossi 1, M G Forest 1, M David 1, J Bertrand 1, W L Miller 1
PMCID: PMC303711  PMID: 2913051

Abstract

Congenital adrenal hyperplasia (CAH) is caused by disorders of the P450c21B gene, which, with the P450c21A pseudogene, lies in the HLA locus on chromosome 6. The near identity of nucleotide sequences and endonuclease cleavage sites in these A and B loci makes genetic analysis of this disease difficult. We used a genomic DNA probe that detects the P450c21 genes (A pseudogene, 3.2 kb; B gene, 3.7 kb in Taq I digests) and the 3' flanking DNA not detected with cDNA probes (A pseudogene, 2.4 kb; B gene, 2.5 kb) to examine Southern blots of genomic DNA from 68 patients and 165 unaffected family members in 57 families with CAH. Of 116 CAH-bearing chromosomes, 114 could be sorted into five easily distinguished haplotypes based on blots of DNA digested with Taq I and Bgl II. Haplotype I (76 of 116, 65.6%) was indistinguishable from normal and therefore bore very small lesions, presumably point mutations. Haplotype II (4 of 116, 3.4%) and haplotype III (8 of 116, 6.9%) had deletions and duplications of the P450c21A pseudogene but had structurally intact P450c21B genes presumably bearing point mutations; point mutation thus was the genetic defect in 88 of 116 chromosomes (75.9%). Haplotypes IV and V lack the 3.7-kb Taq I band normally associated with the P450c21B gene. Haplotype IV (13 of 116, 11.2%) retains all other bands, indicating that the P450c21B gene has undergone a gene conversion event, so that it is now also associated with a 3.2-kb band. Haplotype V (13 of 116, 11.2%) lacks the 2.4-kb Taq I fragment and the 12-kb Bgl II fragments normally associated with the P450c21A pseudogene, as well as lacking the 3.7-kb Taq I fragment, indicating deletion of approximately 30 kb of DNA, resulting in a single hybrid P450c21A/B gene. Most (114 of 116, 98%) CAH alleles thus can easily be classified with this new probing strategy, eliminating many ambiguities resulting from probing with cDNA.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amor M., Parker K. L., Globerman H., New M. I., White P. C. Mutation in the CYP21B gene (Ile-172----Asn) causes steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1600–1604. doi: 10.1073/pnas.85.5.1600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amor M., Tosi M., Duponchel C., Steinmetz M., Meo T. Liver mRNA probes disclose two cytochrome P-450 genes duplicated in tandem with the complement C4 loci of the mouse H-2S region. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4453–4457. doi: 10.1073/pnas.82.13.4453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Belt K. T., Carroll M. C., Porter R. R. The structural basis of the multiple forms of human complement component C4. Cell. 1984 Apr;36(4):907–914. doi: 10.1016/0092-8674(84)90040-0. [DOI] [PubMed] [Google Scholar]
  4. Betuel H., Gebuhrer L., Lambert J., Freidel A. C., Farre A. A possible new HLA-DR allele. Hum Immunol. 1983 Nov;8(3):227–237. doi: 10.1016/0198-8859(83)90040-x. [DOI] [PubMed] [Google Scholar]
  5. Carroll M. C., Campbell R. D., Porter R. R. Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man. Proc Natl Acad Sci U S A. 1985 Jan;82(2):521–525. doi: 10.1073/pnas.82.2.521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carroll M. C., Palsdottir A., Belt K. T., Porter R. R. Deletion of complement C4 and steroid 21-hydroxylase genes in the HLA class III region. EMBO J. 1985 Oct;4(10):2547–2552. doi: 10.1002/j.1460-2075.1985.tb03969.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chung B. C., Matteson K. J., Miller W. L. Structure of a bovine gene for P-450c21 (steroid 21-hydroxylase) defines a novel cytochrome P-450 gene family. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4243–4247. doi: 10.1073/pnas.83.12.4243. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donohoue P. A., Van Dop C., Migeon C. J., McLean R. H., Bias W. B. Coupling of HLA-A3,Cw6,Bw47,DR7 and a normal CA21HB steroid 21-hydroxylase gene in the Old Order Amish. J Clin Endocrinol Metab. 1987 Nov;65(5):980–986. doi: 10.1210/jcem-65-5-980. [DOI] [PubMed] [Google Scholar]
  9. Donohoue P. A., van Dop C., McLean R. H., White P. C., Jospe N., Migeon C. J. Gene conversion in salt-losing congenital adrenal hyperplasia with absent complement C4B protein. J Clin Endocrinol Metab. 1986 May;62(5):995–1002. doi: 10.1210/jcem-62-5-995. [DOI] [PubMed] [Google Scholar]
  10. Dunham I., Sargent C. A., Trowsdale J., Campbell R. D. Molecular mapping of the human major histocompatibility complex by pulsed-field gel electrophoresis. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7237–7241. doi: 10.1073/pnas.84.20.7237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dupont B., Oberfield S. E., Smithwick E. M., Lee T. D., Levine L. S. Close genetic linkage between HLA and congenital adrenal hyperplasia (21-hydroxylase deficiency). Lancet. 1977 Dec 24;2(8052-8053):1309–1312. doi: 10.1016/s0140-6736(77)90362-2. [DOI] [PubMed] [Google Scholar]
  12. Forest M. G., Bétuel H., Couillin P., Boué A. Prenatal diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency by steroid analysis in the amniotic fluid of mid-pregnancy: comparison with HLA typing in 17 pregnancies at risk for CAH. Prenat Diagn. 1981 Jul;1(3):197–207. doi: 10.1002/pd.1970010305. [DOI] [PubMed] [Google Scholar]
  13. Garlepp M. J., Wilton A. N., Dawkins R. L., White P. C. Rearrangement of 21-hydroxylase genes in disease-associated MHC supratypes. Immunogenetics. 1986;23(2):100–105. doi: 10.1007/BF00377968. [DOI] [PubMed] [Google Scholar]
  14. Giles C. M., Uring-Lambert B., Boksch W., Braun M., Goetz J., Neumann R., Mauff G., Hauptmann G. The study of a French family with two duplicated C4A haplotypes. Hum Genet. 1987 Dec;77(4):359–365. doi: 10.1007/BF00291427. [DOI] [PubMed] [Google Scholar]
  15. Harada F., Kimura A., Iwanaga T., Shimozawa K., Yata J., Sasazuki T. Gene conversion-like events cause steroid 21-hydroxylase deficiency in congenital adrenal hyperplasia. Proc Natl Acad Sci U S A. 1987 Nov;84(22):8091–8094. doi: 10.1073/pnas.84.22.8091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higashi Y., Tanae A., Inoue H., Fujii-Kuriyama Y. Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency. Am J Hum Genet. 1988 Jan;42(1):17–25. [PMC free article] [PubMed] [Google Scholar]
  17. Higashi Y., Yoshioka H., Yamane M., Gotoh O., Fujii-Kuriyama Y. Complete nucleotide sequence of two steroid 21-hydroxylase genes tandemly arranged in human chromosome: a pseudogene and a genuine gene. Proc Natl Acad Sci U S A. 1986 May;83(9):2841–2845. doi: 10.1073/pnas.83.9.2841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hood L., Campbell J. H., Elgin S. C. The organization, expression, and evolution of antibody genes and other multigene families. Annu Rev Genet. 1975;9:305–353. doi: 10.1146/annurev.ge.09.120175.001513. [DOI] [PubMed] [Google Scholar]
  19. Höller W., Scholz S., Knorr D., Bidlingmaier F., Keller E., Albert E. D. Genetic differences between the salt-wasting, simple virilizing, and nonclassical types of congenital adrenal hyperplasia. J Clin Endocrinol Metab. 1985 Apr;60(4):757–763. doi: 10.1210/jcem-60-4-757. [DOI] [PubMed] [Google Scholar]
  20. Jospe N., Donohoue P. A., Van Dop C., McLean R. H., Bias W. B., Migeon C. J. Prevalence of polymorphic 21-hydroxylase gene (CA21HB) mutations in salt-losing congenital adrenal hyperplasia. Biochem Biophys Res Commun. 1987 Feb 13;142(3):798–804. doi: 10.1016/0006-291x(87)91484-7. [DOI] [PubMed] [Google Scholar]
  21. Liebhaber S. A., Goossens M., Kan Y. W. Homology and concerted evolution at the alpha 1 and alpha 2 loci of human alpha-globin. Nature. 1981 Mar 5;290(5801):26–29. doi: 10.1038/290026a0. [DOI] [PubMed] [Google Scholar]
  22. Matteson K. J., Phillips J. A., 3rd, Miller W. L., Chung B. C., Orlando P. J., Frisch H., Ferrandez A., Burr I. M. P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5858–5862. doi: 10.1073/pnas.84.16.5858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miller W. L. Gene conversions, deletions, and polymorphisms in congenital adrenal hyperplasia. Am J Hum Genet. 1988 Jan;42(1):4–7. [PMC free article] [PubMed] [Google Scholar]
  24. Miller W. L., Levine L. S. Molecular and clinical advances in congenital adrenal hyperplasia. J Pediatr. 1987 Jul;111(1):1–17. doi: 10.1016/s0022-3476(87)80334-7. [DOI] [PubMed] [Google Scholar]
  25. Miller W. L. Molecular biology of steroid hormone synthesis. Endocr Rev. 1988 Aug;9(3):295–318. doi: 10.1210/edrv-9-3-295. [DOI] [PubMed] [Google Scholar]
  26. Mornet E., Couillin P., Kutten F., Raux M. C., White P. C., Cohen D., Boué A., Dausset J. Associations between restriction fragment length polymorphisms detected with a probe for human 21-hydroxylase (21-OH) and two clinical forms of 21-OH deficiency. Hum Genet. 1986 Dec;74(4):402–408. doi: 10.1007/BF00280494. [DOI] [PubMed] [Google Scholar]
  27. Nebert D. W., Adesnik M., Coon M. J., Estabrook R. W., Gonzalez F. J., Guengerich F. P., Gunsalus I. C., Johnson E. F., Kemper B., Levin W. The P450 gene superfamily: recommended nomenclature. DNA. 1987 Feb;6(1):1–11. doi: 10.1089/dna.1987.6.1. [DOI] [PubMed] [Google Scholar]
  28. Palsdottir A., Arnason A., Fossdal R., Jensson O. Gene organization of haplotypes expressing two different C4A allotypes. Hum Genet. 1987 Jul;76(3):220–224. doi: 10.1007/BF00283611. [DOI] [PubMed] [Google Scholar]
  29. Rumsby G., Carroll M. C., Porter R. R., Grant D. B., Hjelm M. Deletion of the steroid 21-hydroxylase and complement C4 genes in congenital adrenal hyperplasia. J Med Genet. 1986 Jun;23(3):204–209. doi: 10.1136/jmg.23.3.204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schneider P. M., Carroll M. C., Alper C. A., Rittner C., Whitehead A. S., Yunis E. J., Colten H. R. Polymorphism of the human complement C4 and steroid 21-hydroxylase genes. Restriction fragment length polymorphisms revealing structural deletions, homoduplications, and size variants. J Clin Invest. 1986 Sep;78(3):650–657. doi: 10.1172/JCI112623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Skow L. C., Womack J. E., Petresh J. M., Miller W. L. Synteny mapping of the genes for 21 steroid hydroxylase, alpha A crystallin, and class I bovine leukocyte antigen in cattle. DNA. 1988 Apr;7(3):143–149. doi: 10.1089/dna.1988.7.143. [DOI] [PubMed] [Google Scholar]
  32. Speiser P. W., New M. I., White P. C. Molecular genetic analysis of nonclassic steroid 21-hydroxylase deficiency associated with HLA-B14,DR1. N Engl J Med. 1988 Jul 7;319(1):19–23. doi: 10.1056/NEJM198807073190104. [DOI] [PubMed] [Google Scholar]
  33. Werkmeister J. W., New M. I., Dupont B., White P. C. Frequent deletion and duplication of the steroid 21-hydroxylase genes. Am J Hum Genet. 1986 Oct;39(4):461–469. [PMC free article] [PubMed] [Google Scholar]
  34. White P. C., Grossberger D., Onufer B. J., Chaplin D. D., New M. I., Dupont B., Strominger J. L. Two genes encoding steroid 21-hydroxylase are located near the genes encoding the fourth component of complement in man. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1089–1093. doi: 10.1073/pnas.82.4.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. White P. C., New M. I., Dupont B. HLA-linked congenital adrenal hyperplasia results from a defective gene encoding a cytochrome P-450 specific for steroid 21-hydroxylation. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7505–7509. doi: 10.1073/pnas.81.23.7505. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. White P. C., New M. I., Dupont B. Structure of human steroid 21-hydroxylase genes. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5111–5115. doi: 10.1073/pnas.83.14.5111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. White P. C., Vitek A., Dupont B., New M. I. Characterization of frequent deletions causing steroid 21-hydroxylase deficiency. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4436–4440. doi: 10.1073/pnas.85.12.4436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wong C., Dowling C. E., Saiki R. K., Higuchi R. G., Erlich H. A., Kazazian H. H., Jr Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. 1987 Nov 26-Dec 2Nature. 330(6146):384–386. doi: 10.1038/330384a0. [DOI] [PubMed] [Google Scholar]

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