Steroid 21-Hydroxylasedeficiency: Three Additional Mutated Alleles
Total Page：16
File Type：pdf, Size：1020Kb
Proc. Natl. Acad. Sci. USA Vol. 89, pp. 7232-7236, August 1992 Medical Sciences Steroid 21-hydroxylase deficiency: Three additional mutated alleles and establishment of phenotype genotype relationships of common mutations (congenita adrenal hyperplasa/DNA sequencing/PCR/genetlc d s/mu screening) ANNA WEDELL*, E. MARTIN RITZtNt, BARBRO HAGLUND-STENGLER*t, AND HOLGER LUTHMAN*I *Department of Clinical Genetics, Rolf Luft Center for Diabetes Research, and tDepartment of Pediatrics, Karolinska Hospital, S-104 01 Stockholm, Sweden Communicated by RolfLuft, April 30, 1992 ABSTRACT Lesions in the gene encoding steroid 21- on chromosome 6p21.3 (7, 8), forming tandem repeat units. hydroxylase [steroid hydrogen-donor: oxygen oxidoreductase Considerable variation in the number and gross structure of (21-hydroxylating), EC 1.14.99.10] result in defective adrenal C4/210H repeat units has been detected (9, 10). The CYP21 steroid synthesis; the severe forms are known as congenital gene consists of 10 exons spanning 3 kilobases (kb) of DNA adrenal hyperplasia. To filate complete characterization of (11-13). Studies of upstream sequences in mice have identi- mutations in this region of tademly repeated genes, we have fied a number of potential regulatory elements within 300 developed selective PCR ampltion and direct sequencing of base pairs (bp) 5' ofthe gene that influences gene expression full-length nonpseudogene steroid 21-hydroxylase genes. This in vitro (14). Considerable sequence variation has been technique identifies known mutations, c r or ex- described between human CYP21 genes, some altering the cludes unknown mutations, and determines the gene-copy encoded steroid 21-hydroxylase protein sequence. The pro- number. Three additional defective alleles were found. A tein variants include both apparently neutral amino acid Gly-292 -- Ser mutation and a frameshift mutation at Arg-484 substitutions (11-13, 15, 16), as well as substitutions causing (GG -- C) were identified in patients with severe steroid impairment ofsteroid 21-hydroxylase activity (15, 17-22). All 21-hydroxylase deficiency. An allele with three additional described sequence variants that impair enzyme function are sequence variations-C -> T at 4 bases upstam oftra tion also present in the pseudogene and are thought to be trans- initiation, Pro-106 -* Len, and Pro-454 -+ Ser-were identi- ferred by recombination from CYP2JP to CYP21. The pseu- fied in two sings with late-nset deficiency. Pro-454 is dogene-derived mutations represent a large part but do not conserved in four species, indiating its importance for normal represent all of the steroid 21-hydroxylase deficiency muta- enzyme function. Functional consequences ofindi alleles tions in different populations (16, 21, 23). have been determined in vvo by studying individuals with only Attempts to fully characterize the genetic lesions in the one steroid 21-hydroxylase gene. Detailed analyses of clinical various manifestations of steroid 21-hydroxylase deficiency data revealed that genotyping could predict the clinical course have been hampered by the complicated structure of the of the disease. The locations of disease-causing mutations on CYP21 locus. We have developed rapid direct sequencing of different haplotypes of the steroid 21-hydroxylase gene region selectively PCR-amplified full-length CYP21 genes to obtain are described. information ofall nonpseudogenes present in individuals with various disease manifestations. This technique enabled iden- Steroid 21-hydroxylase [steroid hydrogen-donor: oxygen ox- tification of three additional mutated alleles and establish- idoreductase (21-hydroxylating), EC 1.14.99.10; P450c21] is ment of genotype-phenotype correlations for the common an essential enzyme in adrenal steroid synthesis. Inherited mutations. The functional contribution of individual CYP21 defects of this enzyme lead to various degrees of impaired alleles has been determined in vivo by studying subjects cortisol and aldosterone synthesis accompanied with andro- carrying only one copy of the gene (hemizygous). gen excess (1, 2). Steroid 21-hydroxylase deficiency is inher- ited as an autosomal recessive trait. The disorder exists in a wide range of manifestations that traditionally have been MATERIALS AND METHODS divided into three groups: (i) simple virilizing, presenting Patients. The study 2 with virilization ofexternal genitalia at birth in girls and early comprises healthy controls and 34 pseudoprecocious puberty in boys; (ii) salt-wasting, which in patients affected with steroid 21-hydroxylase deficiency of addition shows severe aldosterone deficiency neonatally; and different severity. This group represents 61 unrelated alleles. (iii) late-onset, which is associated with pseudoprecocious The main symptoms and the time of their initial observation puberty, hirsutism, polycystic ovary syndrome, and de- are given in Table 1. Urine from four patients-B32, B33, creased fertility (3-5). However, the three groups are not B103, and B108-carrying three additional mutated alleles, distinct. The severe forms of the disease occur in 1/12,000 were collected over a 24-hr period, and the steroid pattern Swedish newborns. Based on hormonal analyses, mild ste- was analyzed by using gas/liquid chromatography, display- roid 21-hydroxylase deficiency is estimated to occur in ing typical increases of pregnanetriol. There were no metab- 1/500-1/1000 North Europeans (6). olites suggesting any other defect in adrenal steroidogenesis. There are two human steroid 21-hydroxylase genes In addition, patients B32 and B33 showed pathological re- (210H)-one active gene, CYP21, and a highly homologous sponses of serum cortisol and 17-hydroxyprogesterone con- inactive pseudogene, CYP2JP. These genes are located 3' of centrations during an adenocorticotropic hormone (ACTH) each of the two genes encoding the fourth component of test (10). Two individuals hemizygous for the CYP21 gene, complement, C4A and C4B, in the HLA class III gene region Abbreviations: CYP21, alleles of the active steroid 21-hydroxylase gene; CYP21P, alleles of the steroid 21-hydroxylase pseudogene; The publication costs of this article were defrayed in part by page charge 210H, any of the steroid 21-hydroxylase genes; C4, fourth compo- payment. This article must therefore be hereby marked "advertisement" nent of serum complement; ACTH, adrenocorticotropic hormone. in accordance with 18 U.S.C. §1734 solely to indicate this fact. qTo whom reprint requests should be addressed. 7232 Downloaded by guest on September 30, 2021 Medical Sciences: Wedell et al. Proc. Natl. Acad. Sci. USA 89 (1992) 7233 B21 and B31, exhibited normal hormone levels during the a primer with the most-3' nucleotide base-pairing only with ACTH test. The C4/210H haplotypes of the patients have the allele carrying the additional mutation. previously been characterized and are given in Table 1 (10, Direct Sequencing. The biotinylated PCR fragments were 24). Haplotypes 1 and 2 contain one CYP21P and one CYP21 immobilized by binding to streptavidin-covered magnetic gene, haplotype 5 contains only one CYP21, and haplotypes beads (Dynabeads; Dynal, Oslo), and the nonbiotinylated 6, 7, and 8 contain two CYP21P and one CYP21 (Fig. 1). strands were removed by incubating in 50 pl of0.15 M NaOH Linkage phases of haplotypes and mutations were estab- for 5 min at room temperature (25). The remaining DNA was lished through segregation in families. The study was ap- rinsed three times, suspended in 13 1.L of distilled water, and proved by the Ethics Committee at the Karolinska Hospital. sequenced by the dideoxynucleotide chain-termination PCR. Genomic DNA was prepared from peripheral leuko- method with the Auto-Read sequencing kit (Pharmacia). cytes. A specific restriction site for Taq I is present at base Sequencing was done with fluorescently labeled primers by -210 in the CYP21P gene (Fig. 1), base numbering being using the Pharmacia automated laser fluorescent DNA se- identical to that reported by Rodrigues et al. (13). In cases quencer. Thirteen out of 43 alleles were completely se- where the CYP21 alleles were completely sequenced from quenced from nucleotides -330 to 2735. The remaining nucleotides -330 to 2735, DNA was cleaved with Taq I (New alleles were partly sequenced. Additional mutations were England Biolabs), and a first round of PCR was done by using confirmed by sequence determination of both strands. All primers P1 5'-TTCAGGCGATTCAGGAAGGC (-418 to primer sequences for PCR amplification and DNA sequence -399) and P4 5'-TCTCGCACCCCAGTATGACT (2892- determination are available upon request. 2911) at concentrations of 0.5 ILM (Fig. 1). After an initial denaturation at 96WC for 3 min, 30 cycles at 96WC for 1 min, RESULTS 560C for 30 sec, and 720C for 15 min were done in a buffer containing 1.0mM MgCl2, 0.2 mM dNTP, 50 mM KCl, 10 mM Hemizygous Individuals. The CYP21 alleles from two hem- Tris-HCl (pH 8.4 at 70°C), and 0.1% Tween 20. Shorter izygous individuals with normal steroid 21-hydroxylase func- extension time failed to reproducibly generate the desired tion were completely sequenced from base -330 to base fragment. One microliter of the 3.3-kb PCR product was used 2735. Patient B21 differed from patient B31 in four silent for subsequent amplifications to generate four overlapping positions in exons and seven positions in introns (Table 1). In fragments by incubating 30 cycles at 96WC for 1 min, 58°C for addition, this normal allele varied at base 1650, changing 30 sec, and 72°C for 3 min in 100 ,l, using one normal and one Ser-269 to threonine. biotinylated primer at 0.1 ,uM. Taq polymerase (Perkin- Twenty hemizygous patients with steroid 21-hydroxylase Elmer/Cetus) was used at 2 units per 100 ,lI. For partially deficiency were analyzed (Table 1). Patient B103, affected sequenced alleles, the first round of PCR was performed by with salt-wasting disease, carried an allele with a previously using primers P1 together with P48 (5'-CAGAGCAGGGAG- unreported G -* A transition at nucleotide 1718, changing TAGTCTC) and P55 (5'-CCTGTCCTTGGGAGACTACT) Gly-292 -+ Ser in exon 7 (Fig. 2A). The only additional together with P4 (Fig. 1).