Junction Site Analysis of Chimeric CYP21A1P/CYP21A2 Genes in 21-Hydroxylase Deficiency

Clinical Chemistry - Tập 58 Số 2 - Trang 421-430 - 2012
Wuyan Chen1, Xu Zhi1, Anne Sullivan2, Gabriela P. Finkielstain3, Carol Van Ryzin2, Deborah P. Merke2,3, Nazli B. McDonnell4,1
1Laboratory of Clinical Investigation, National Institute on Aging, Baltimore, MD
2National Institutes of Health Clinical Center, Bethesda, MD
3National Institutes of Health, Program in Developmental Endocrinology and Genetics (PDEGEN), Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD
4Clinical Research Branch, National Institute on Aging, National Institutes of Health, Baltimore, Md

Tóm tắt

Abstract BACKGROUND Chimeric CYP21A1P/CYP21A2 genes, caused by homologous recombination between CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2) and its highly homologous pseudogene CYP21A1P (cytochrome P450, family 21, subfamily A, polypeptide 1 pseudogene), are common in patients with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD). A comprehensive junction site analysis of chimeric CYP21A1P/CYP21A2 genes is needed for optimizing genetic analysis strategy and determining clinical relevance. METHODS We conducted a comprehensive genetic analysis of chimeric CYP21A1P/CYP21A2 genes in a cohort of 202 unrelated 21-OHD patients. Targeted CYP21A2 mutation analysis was performed, and genotyping of chimeric CYP21A1P/CYP21A2 genes was cross-confirmed with Southern blot, RFLP, and multiplex ligation-dependent probe amplification analyses. Junction sites of chimera genes were determined by sequencing the long-PCR products amplified with primers CYP779f and Tena32F. An updated bioinformatics survey of Chi-like sequences was also performed. RESULTS Of 100 probands with a chimeric allele, 96 had a chimera associated with the severe classic salt-wasting form of CAH, and the remaining 4 carried an uncommon attenuated chimera with junction sites upstream of In2G (c.293–13A/C>G), which is associated with a milder phenotype. In addition to 6 of 7 reported chimeras, we identified a novel classic chimera (CH-8) and a novel attenuated chimera (CH-9). Attenuated chimeras explained prior genotype–phenotype discrepancies in 3 of the patients. Sequencing the CYP779f/Tena32F amplicons accurately differentiated between classic and attenuated chimeras. The bioinformatics survey revealed enrichment of Chi-like sequences within or in the vicinity of intron 2. CONCLUSIONS Junction site analysis can explain some genotype–phenotype discrepancies. Sequencing the well-established CYP779f/Tena32F amplicons is an unequivocal strategy for detecting attenuated chimeric CYP21A1P/CYP21A2 genes, which are clinically relevant.

Từ khóa


Tài liệu tham khảo

Merke, 2005, Congenital adrenal hyperplasia, Lancet, 365, 2125, 10.1016/S0140-6736(05)66736-0

Yang, 1999, Modular variations of the human major histocompatibility complex class III genes for serine/threonine kinase RP, complement component C4, steroid 21-hydroxylase CYP21, and tenascin TNX (the RCCX module). A mechanism for gene deletions and disease associations, J Biol Chem, 274, 12147, 10.1074/jbc.274.17.12147

Werkmeister, 1986, Frequent deletion and duplication of the steroid 21-hydroxylase genes, Am J Hum Genet, 39, 461

Lee, 2001, CYP21 mutations and congenital adrenal hyperplasia, Clin Genet, 59, 293, 10.1034/j.1399-0004.2001.590501.x

White, 1986, Structure of human steroid 21-hydroxylase genes, Proc Natl Acad Sci U S A, 83, 5111, 10.1073/pnas.83.14.5111

Higashi, 1986, 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, 83, 2841, 10.1073/pnas.83.9.2841

Speiser, 2003, Congenital adrenal hyperplasia, N Engl J Med, 349, 776, 10.1056/NEJMra021561

Concolino, 2009, A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form, BMC Med Genet, 10, 72, 10.1186/1471-2350-10-72

Vrzalová, 2011, Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia, Eur J Med Genet, 54, 112, 10.1016/j.ejmg.2010.10.005

Krone, 2000, Predicting phenotype in steroid 21-hydroxylase deficiency? Comprehensive genotyping in 155 unrelated, well defined patients from southern Germany, J Clin Endocrinol Metab, 85, 1059, 10.1210/jcem.85.3.6441

Stikkelbroeck, 2003, CYP21 gene mutation analysis in 198 patients with 21-hydroxylase deficiency in The Netherlands: six novel mutations and a specific cluster of four mutations, J Clin Endocrinol Metab, 88, 3852, 10.1210/jc.2002-021681

Jaaskelainen, 1997, Population-wide evaluation of disease manifestation in relation to molecular genotype in steroid 21-hydroxylase (CYP21) deficiency: good correlation in a well defined population, J Clin Endocrinol Metab, 82, 3293

Speiser, 1992, Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency, J Clin Invest, 90, 584, 10.1172/JCI115897

L'Allemand, 2000, How a patient homozygous for a 30-kb deletion of the C4-CYP 21 genomic region can have a nonclassic form of 21-hydroxylase deficiency, J Clin Endocrinol Metab, 85, 4562, 10.1210/jc.85.12.4562

Killeen, 1991, Molecular and endocrine characterization of a mutation involving a recombination between the steroid 21-hydroxylase functional gene and pseudogene, J Steroid Biochem Mol Biol, 38, 677, 10.1016/0960-0760(91)90078-J

Gomes, 2009, Extraadrenal 21-hydroxylation by CYP2C19 and CYP3A4: effect on 21-hydroxylase deficiency, J Clin Endocrinol Metab, 94, 89, 10.1210/jc.2008-1174

Chu, 1992, Identification of the recombination site within the steroid 21-hydroxylase gene (CYP21) of the HLA-B47,DR7 haplotype, Exp Clin Immunogenet, 9, 80

Helmberg, 1992, Identification of molecular defects causing congenital adrenal hyperplasia by cloning and differential hybridization of polymerase chain reaction-amplified 21-hydroxylase (CYP21) genes, DNA Cell Biol, 11, 359, 10.1089/dna.1992.11.359

Lee, 2004, Use of PCR-based amplification analysis as a substitute for the Southern blot method for CYP21 deletion detection in congenital adrenal hyperplasia, Clin Chem, 50, 1074, 10.1373/clinchem.2003.028597

Lee, 2004, The chimeric CYP21P/CYP21 gene and 21-hydroxylase deficiency, J Hum Genet, 49, 65, 10.1007/s10038-003-0115-2

Lee, 2005, Chimeric CYP21P/CYP21 and TNXA/TNXB genes in the RCCX module, Mol Genet Metab, 84, 4, 10.1016/j.ymgme.2004.09.009

Lee, 2003, Deletion of the C4-CYP21 repeat module leading to the formation of a chimeric CYP21P/CYP21 gene in a 9.3-kb fragment as a cause of steroid 21-hydroxylase deficiency, Clin Chem, 49, 319, 10.1373/49.2.319

White, 1984, 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, 81, 7505, 10.1073/pnas.81.23.7505

White, 1988, Characterization of frequent deletions causing steroid 21-hydroxylase deficiency, Proc Natl Acad Sci U S A, 85, 4436, 10.1073/pnas.85.12.4436

Finkielstain, 2011, Comprehensive genetic analysis of 182 unrelated families with congenital adrenal hyperplasia due to 21-hydroxylase deficiency, J Clin Endocrinol Metab, 96, E161, 10.1210/jc.2010-0319

Krone, 2002, Multiplex minisequencing of the 21-hydroxylase gene as a rapid strategy to confirm congenital adrenal hyperplasia, Clin Chem, 48, 818, 10.1093/clinchem/48.6.818

Chung, 2005, Human complement components C4A and C4B genetic diversities: complex genotypes and phenotypes, Curr Protoc Immunol, 10.1002/0471142735.im1308s68

Lee, 2004, PCR-based detection of the CYP21 deletion and TNXA/TNXB hybrid in the RCCX module, Genomics, 83, 944, 10.1016/j.ygeno.2003.11.006

Canturk, 2011, Sequence analysis of CYP21A1P in a German population to aid in the molecular biological diagnosis of congenital adrenal hyperplasia, Clin Chem, 57, 511, 10.1373/clinchem.2010.156893

Wilson, 1995, Steroid 21-hydroxylase deficiency: Genotype may not predict phenotype, J Clin Endocrinol Metab, 80, 2322

Coeli, 2010, Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency, BMC Med Genet, 11, 104, 10.1186/1471-2350-11-104

Tusie-Luna, 1995, Gene conversions and unequal crossovers between CYP21 (steroid 21-hydroxylase gene) and CYP21P involve different mechanisms, Proc Natl Acad Sci U S A, 92, 10796, 10.1073/pnas.92.23.10796

Concolino, 2009, Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of CYP21A2 gene deletions/duplications in congenital adrenal hyperplasia: first technical report, Clin Chim Acta, 402, 164, 10.1016/j.cca.2009.01.008

Jang, 2011, Multiplex ligation-dependent probe amplification assay for diagnosis of congenital adrenal hyperplasia, Ann Clin Lab Sci, 41, 44