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The Journal of Clinical Endocrinology & Metabolism Vol. 89, No. 1 49-60
Copyright © 2004 by The Endocrine Society

Special Feature

Two Prevalent CYP17 Mutations and Genotype-Phenotype Correlations in 24 Brazilian Patients with 17-Hydroxylase Deficiency

Marivânia Costa-Santos, Claudio E. Kater and Richard J. Auchus AND BRAZILIAN CONGENITAL ADRENAL HYPERPLASIA MULTICENTER STUDY GROUP

Division of Endocrinology and Metabolism, Department of Medicine, Escola Paulista de Medicina, Federal University of Sao Paulo (M.C.-S., C.E.K.), Sao Paulo, Brazil 04039-034; and Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Texas Southwestern Medical Center (R.J.A.), Dallas, Texas 75390-8857

Address all correspondence and requests for reprints to: Richard J. Auchus, M.D., Ph.D., Division of Endocrinology and Metabolism, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-8857. E-mail: richard.auchus{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
We performed molecular genetic analysis of 24 subjects from 19 families with 17-hydroxylase deficiency in Brazil. Of 7 novel CYP17 mutations, 2 (W406R and R362C) account for 50% and 32% of the mutant alleles, respectively. Both mutations were completely inactive when studied in COS-7 cells and yeast microsomes; however, phenotypic features varied among subjects. Some 46,XY individuals with these genotypes had ambiguous genitalia, and other subjects had normal blood pressure and/or serum potassium. We found mutations W406R and R362C principally in families with Spanish and Portuguese ancestry, respectively, suggesting that two independent founder effects contribute to the increased prevalence of 17-hydroxylase deficiency in Brazil. Mutations Y329D and P428L retained a trace of activity, yet the two individuals with these mutations had severe hypertension and hypokalemia. The 46,XX female with mutation Y329D reached Tanner stage 5, whereas the 46,XY subject with mutation P428L remained sexually infantile. The severity of hypertension, hypokalemia, 17-deoxysteroid excess, and sex steroid deficiency varied, even among patients with completely inactive CYP17 protein(s). Spontaneous sexual development occurred only in 46,XX females with partial deficiencies. We conclude that other factors, in addition to CYP17 genotype, contribute to the phenotype of individual patients with 17-hydroxylase deficiency.


   Introduction
Top
 Abstract
 Introduction
Subjects and Methods
 Results
 Discussion
 References
 
MUTATIONS IN THE CYP17 gene cause 17-hydroxylase deficiency (17OHD), a rare form of congenital adrenal hyperplasia (CAH) with an estimated incidence of about 1:50,000 newborns (1). Individuals with 17OHD account for roughly 1% of all cases of CAH, and most reports involve isolated cases from consanguineous families (2). Since cloning of the CYP17 gene encoding cytochrome P450c17 (CYP17, 17{alpha}-hydroxylase/17,20-lyase) (3), nearly 40 different mutations in CYP17 have been described (4, 5, 6, 7, 8, 9), although a few more common mutations reoccur in certain ethnic groups (10, 11, 12).

The typical features of complete 17OHD were described almost 40 yr ago (13), as hypertension, hypokalemia, and sexual infantilism in phenotypic females. Subsequent reports identified 17OHD as a cause not only of incomplete male pseudohermaphroditism (14), but also sexual infantilism in 46,XY subjects (15). The lack of adrenal 17{alpha}-hydroxylase activity drives massive overproduction of the 17-deoxysteroids 11-deoxycorticosterone (DOC) and corticosterone (B), which are the mineralocorticoids that cause hypertension and hypokalemia in 17OHD (4). Concomitant lack of gonadal 17,20-lyase activity precludes sex steroid production and hence the development of the male phenotype in utero or of secondary sexual characteristics at puberty.

Nevertheless, there is considerable variation in the clinical and biochemical features of 17OHD (16), including the variant of isolated 17,20-lyase deficiency (17, 18). The severity of clinical disease tends to be milder with mutations that retain partial catalytic activity in assays using heterologous expression systems (4), but the age of onset of hypertension, the degree of hypokalemia, and the aldosterone production rate appear to vary, even among patients with mutations that completely inactivate the enzyme (2). However, because there have been no studies of multiple individuals bearing the same genotype who have been studied by the same investigators, it is not clear to what extent genotype alone determines phenotype in 17OHD.

Worldwide, the most common form of CAH is 21-hydroxylase deficiency (19), and the second most common form appears to be lipoid CAH in Japan and Korea (20) and 11-hydroxylase deficiency in the Middle East (21); founder effects that yield a single prevalent mutation account for the high prevalence of these two disorders in their respective populations. In contrast, 17OHD appears to be the second most common form of CAH in Brazil (16, 22). Founder effects may also contribute to the high prevalence of 17OHD in Brazil, but the population of Brazil is among the most ethnically heterogeneous in the world (23). The Portuguese settled Brazil beginning in the 1500s, and the indigenous Amerindian people, Africans derived from the extensive slave trade, and waves of immigration from Italy, Spain, Germany, Asia, and The Netherlands contribute to the genetic diversity (23, 24, 25).

The Brazilian Congenital Adrenal Hyperplasia Multicenter Study Group has had the opportunity to evaluate the clinical features of 30 subjects with 17OHD from 24 kindreds, the largest group of 17OHD cases studied by a single group. To provide insight into the phenotypic variations in 17OHD and to define the genetics of 17OHD in Brazil, we analyzed the CYP17 gene in these subjects. We now report the results of molecular genetic and functional analyses of the mutations.


   Subjects and Methods
Top
 Abstract
 Introduction
 Subjects and Methods
Results
 Discussion
 References
 
Subjects, clinical presentation, and hormonal evaluation

Of 30 subjects in whom the diagnosis of 17OHD was established (at Escola Paulista de Medicina by C.E.K.), DNA was analyzed in 24, constituting the cohort for the genetic analysis. These 24 subjects derived from 19 kindreds, and consanguinity was known to occur in 6 of the 19 families. The study protocol was approved by the committee on ethics in human research from Escola Paulista de Medicina (n.1703/98), and all patients provided written informed consent. Blood pressure was measured by aneroid sphygmomanometer in the seated position on at least three occasions. For diagnostic studies, blood samples were obtained before and 60 min after the iv injection of 250 µg cosyntropin [synthetic ACTH-(1–24)], and Table 1GoGo lists the mean and ranges of basal and stimulated hormone values in these subjects. The diagnosis of 17OHD was established by the reduced circulating concentrations of cortisol and gonadal steroids, elevated gonadotropins, and high [>3 SD above normal, with or without ACTH-(1–24) stimulation] concentrations of the diagnostic steroids DOC and/or B, as well as frequently elevated concentrations of 18-hydroxydeoxycorticosterone and 18-hydroxycorticosterone (16). In our subjects, basal hormone concentrations alone established the diagnosis. The clinical features are summarized in Table 2Go.


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  		TABLE 1A. Basal and ACTH-stimulated adrenal steroid values in 24 Brazilian patients with 17OHD

 

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  		TABLE 1B. Baseline gonadotropin and gonadal steroid values in 24 Brazilian patients with 17OHD

 

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  		TABLE 2. Clinical characteristics of 24 Brazilian subjects with 17OHD at diagnosis

 
DNA preparation, PCR, and sequencing

DNA was extracted from peripheral leukocytes (Pure Gene DNA Isolation Kit D-5000, Gentra Systems). The 6.4-kb CYP17 gene was amplified into 1–4 pieces from 0.5–1 µg genomic DNA using TaKaRa Ex Taq DNA polymerase (Takara Shuzo Co., Shiga, Japan) in 100-µl reactions using buffer and deoxy-NTPs provided by the manufacturer and 3% dimethylsulfoxide. The primers are listed in Table 3Go, and the locations of the primers are illustrated in Fig. 1Go. To amplify 3- to 4-kb products, PCR parameters included 40 cycles of 3 min at 94 C, 1 min at 65 C, and 3 min at 70 C. For amplification of the entire gene, the annealing time was increased to 1.5 min, and the extension parameters were 72 C for 5.5 min. The final PCR products were precipitated with ethanol and purified on 1% agarose gels using the QIAEX II kit (Qiagen, Chatsworth, CA). Amplicons were submitted for direct sequencing of the 8 exons and flanking intronic DNA by the dye termination method on a PE Applied Biosystems instrument (McDermott Center Sequencing Facility at University of Texas Southwestern Medical Center, Dallas, TX). The mutations were identified by comparison with the GenBank sequence (accession no. M19489) for CYP17 (3) using MacVector 6.5.3 (Accelrys Corp., San Diego, CA). Identified mutations were confirmed by sequencing the product of a second PCR amplification in the opposite direction.


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  		TABLE 3. Oligonucleotide primers for DNA amplification and sequencing

 


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  		FIG. 1. Schematic representation of the human CYP17 gene, indicating the approximate locations and sizes of exons (numbered boxes), oligonucleotide primers (arrows, numbered as explained in Table 3Go), and identified mutations (labeled asterisks).

 
Heterologous expression and enzyme assay

The cDNAs for missense CYP17 mutations were generated by sequential PCR using overlapping mutagenic oligonucleotides (Table 3Go) with template plasmid pLW01-c17 and Ex Taq polymerase with 1% dimethylsulfoxide as previously described (26). The final PCR product was extracted with phenol-chloroform, precipitated with ethanol, digested with BamHI and EcoRI, gel-purified, ligated into the eukaryotic expression vector pcDNA3 (Invitrogen, Carlsbad, CA), and later subcloned into yeast expression vector V10 (27). Each cDNA insert was sequenced in its entirety to ensure that only the desired mutations were introduced.

The enzymatic activities of the four missense mutations were studied by transient transfection of COS-7 and HEK-293 cells with 1–2 µg of the pcDNA3 expression vectors using FuGENE6 (3 µl) in 100 µl serum-free medium as previously described (26). Incubations with 0.1 µM [3H]progesterone, -pregnenolone, or -17{alpha}-hydroxypregnenolone (90,000 cpm; PerkinElmer Life Sciences, Norwalk, CT) for up to 16 h were repeated three times using COS-7 cells and were confirmed with an additional experiment using HEK-293 cells under similar assay conditions. In some cases, incubations were repeated with 0.01 µM steroids to increase assay sensitivity. Extraction, chromatography, and autoradiography were performed as previously described (28).

The P450 content and enzymatic activities of the mutations were also studied in Saccharomyces cerevisiae strain W303B. Yeast were transformed with 1 µg expression vector V10 (empty, and with wild-type or mutant CYP17 cDNA) with or without pYcDE2-OR to provide cytochrome P450-oxidoreductase (CPR) (29), using the lithium acetate method as previously described (26). CO-reduced P450 difference spectra were performed by resuspending yeast harvested from 80 ml culture in 12 ml 0.1 mM potassium phosphate (pH 7.4) with glucose, adding 3-ml aliquots to two cuvettes, and bubbling CO gas into the sample cuvette for 1 min (26). Using the same suspension of whole yeast used for CO-reduced spectra, substrate-induced difference spectra were recorded with up to 40 µM progesterone as previously described (30).

Microsomes were prepared from 1 liter yeast culture grown to an A600 of 1.0–1.8 in defined medium by sonication of spheroplasts as previously described (26), and protein content was determined by colorometric assay. Microsomes containing CPR and wild-type CYP17 (25 µg protein) or the mutations (250 µg) were incubated at 37 C with 0.1 µM [3H]progesterone, -pregnenolone, or -17{alpha}-hydroxypregnenolone for 60 min in 200 µl 50 mM potassium phosphate, pH 7.4, with 1 mM NADPH. Extraction, chromatography, and autoradiography (26, 29) and immunoblotting were performed as previously described (28).


   Results
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
Discussion
 References
 
Mutation analysis

Seven CYP17 gene mutations were found, none of which has been described previously. We found 5 missense mutations in exons 1, 6 (two), 7, and 8; a nonsense mutation in exon 6; and an AG to CG mutation at g.2306 in the splice acceptor site of intron 2 (Table 4Go and Fig. 1Go). Mutation W406R in exon 7 was the most common, accounting for half of the mutant alleles, including 11 homozygotes. Mutation R362C accounted for almost one third of the mutant alleles with 7 homozygotes, and 2 subjects were compound heterozygotes for W406R plus R362C. Together, mutations W406R and R362C accounted for 23 of 28 (82%) of the alleles identified in 25 (52%) and 16 (33%) of the 48 sequenced CYP17 genes, respectively (Table 4Go and Fig. 2Go). One subject was homozygous for P428L, and another was homozygous for Y329X. One 46,XX female who reached Tanner stage 5 (Table 2Go) was a compound heterozygote for Y329D and the AG to CG substitution in the splice acceptor site of intron 2, and 1 subject was heterozygous for M1T and W406R.


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  		TABLE 4. CYP17 mutations in 24 Brazilian subjects

 


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  		FIG. 2. Brazilian CYP17 mutations. A, Electropherograms corresponding to homozygous and heterozygous patients for the common mutations W406R and R362C. PCR-amplified DNA was purified and submitted for direct sequencing using oligonucleotide I6S1 (for W406R) or I4S1 (for R362C) as described in the text. B, PmlI digest from patient and family members bearing the M1T mutation. Half of the DNA, PCR-amplified using oligonucleotides c17PS3 and I1AS1 (1 kb amplicon), is digested by PmlI, indicating heterozygosity for M1T in all three family members. The sequences of this region for the wild-type and mutant alleles are shown with the PmlI site underlined.

 
Only the M1T mutation altered the restriction map of the surrounding region, introducing a PmlI site (CACGTG). Half of the DNA that was amplified from the affected proband with this mutation, from her mother, and from her half-sister was digested by PmlI at the expected site, indicating that all 3 are heterozygous for this mutation (Fig. 2Go). For the family members of all other kindreds, zygosity was determined by sequencing amplified DNA from the region surrounding the mutation(s). DNA from all 29 available parents and from 36 of 46 available siblings (78%) contained 1 copy of the same mutation found in the affected family member.

After sequencing the exons and flanking intronic DNA from 24 patients, we consistently observed 5 differences from the CYP17 sequence M19489 deposited in GenBank (3): 1) a polymorphism at D283 (GAT to GAC) in exon 5, 2) a third C at the CC in positions -26 to -28 at the 3' end of intron 2, 3) a third C at the CC in positions -3 to -5 at the 3' end of intron 3, 4) an A to T substitution in position -5 at the 3' end of intron 7, and 5) a C in place of the T 34 bp upstream from the ATG start codon. This last difference was incorporated into primer c17geneS1a, which gave better PCR amplifications than primer c17geneS1 (Table 3Go). These differences, except for 2 and 5 above, have been noted previously (31), suggesting that these minor changes correspond to the correct sequence in our population.

Heterologous expression, enzyme assay, and difference spectroscopy

To determine whether the mutant enzymes retained any residual 17{alpha}-hydroxylase activity, cDNAs bearing the four missense mutations (W406R, R362C, P428L, and Y329D) were constructed and subcloned into mammalian and yeast expression vectors pcDNA3 and V10, respectively. COS-7 cells transiently transfected with pcDNA3 containing the wild-type CYP17 cDNA metabolized progesterone to the expected 4:1 mixture of 17{alpha}-hydroxyprogesterone and 16{alpha}-hydroxyprogesterone (Fig. 3Go) (32, 33). In contrast, COS-7 cells expressing the CYP17 mutations W406R and R362C produced only the same background metabolites as mock-transfected cells (Fig. 3Go). Unlike mutations W406R and R362C, mutation Y329D always exhibited a small amount (~5%) of residual activity when expressed in COS-7 cells, and mutation P428L yielded a trace of 17{alpha}-hydroxyprogesterone in most experiments (Fig. 3Go).



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  		FIG. 3. Progesterone metabolism by transiently transfected COS-7 cells expressing the cDNA for CYP17, wild type or mutations W406R, R362C, P428L, and Y329D. A representative autoradiogram of chromatographed steroid products is shown, with the locations of steroid standards indicated. The film was intentionally overexposed to reveal trace 17{alpha}-hydroxylation by mutations P428L and Y329D, so that by-products of progesterone metabolism by endogenous enzymes appear more abundant than is typical. Similar results were obtained in three separate experiments with COS-7 cells and in one identical experiment using HEK-293 cells. Prog, Progesterone; 17OHP and 16OHP, 17{alpha}-hydroxy- and 16{alpha}-hydroxyprogesterone, respectively.

 
The activity and spectral properties of the mutations were further characterized in yeast. Yeast microsomes containing wild-type human CYP17 and human P450-oxidoreductase are an abundant source of enzyme activity (29), as demonstrated by the pattern of [3H]pregnenolone metabolism in Fig. 4Go. As was observed when the mutations were expressed in COS-7 and HEK-293 cells, the 17{alpha}-hydroxylase and 17,20-lyase activities of microsomes containing mutations W406R and R362C did not exceed background levels (Fig. 4AGo). These data confirm that mutations W406R and R362C are not active under these in vitro conditions. In contrast, microsomes containing mutations Y329D and P428L metabolized some progesterone and pregnenolone by 17{alpha}-hydroxylation, and both mutant enzymes demonstrated little 17,20-lyase activity even in the presence of cytochrome b5 (Fig. 4BGo). The 17{alpha}-hydroxylation rates for Y329D and P428L were too low to determine meaningful kinetic constants, but we estimate that mutations Y329D and P428L retain less than 5% of the wild-type activity.



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  		FIG. 4. Activities and spectral properties of mutant CYP17 enzymes in S. cerevisciae. A and B, Autoradiograms of thin layer chromatograms (overexposed as in Fig. 3Go) showing pregnenolone (A) or 17{alpha}-hydroxypregnenolone (B) metabolism by yeast microsomes containing human CPR and wild-type (WT) or mutant CYP17 as indicated. Preg, Pregnenolone; DHEA, dehydroepiandrosterone; 17Preg, 17{alpha}-hydroxypregnenolone. B, Purified cytochrome b5 (5 pmol) was added where indicated. C, CO-reduced difference spectra from yeast expressing wild-type CYP17 (top), P428L (middle), or Y329D mutation (bottom). The cursor is set at 450 nm, and the bar represents 0.05 absorbance units (AU). D, Immunoblot of yeast microsomes containing wild-type (WT) CYP17 and for the four missense mutations (30 µg protein/lane). The arrow denotes bands corresponding to full-length CYP17 proteins, which migrate near the chemically modified 62-kDa protein standard (26 ).

 
Intact yeast that express wild-type human CYP17 afford CO-reduced difference spectra with a peak at 450 nm, derived from the CO adduct with the heme thiolate in CYP17 (34). This difference spectrum, characteristic of all cytochromes P450, indicates the presence of properly folded, functional CYP17 protein, and we obtain 50–100 nmol of spectroscopically active wild-type human CYP17 per liter of culture using vector V10 in strain W303B (26). Likewise, addition of progesterone to a suspension of yeast expressing wild-type human CYP17 yields a type I substrate binding spectrum (30), and these difference spectra can be used to monitor the expression of functional protein in yeast. Because mutations Y329D and P428L retain some activity, some of the protein must fold and incorporate heme properly. If these molecules form a stable CO adduct typical of active cytochromes P450, we would observe a peak at 450 nm in the CO-reduced difference spectrum. However, CO-reduced difference spectra using yeast expressing the four missense mutations did not demonstrate detectable P450 absorbances (Fig. 4CGo). Similarly, yeast expressing mutations Y329D and P428L did not form a type I difference spectrum in the presence of up to 40 µM progesterone (not shown). The lack of discernable spectral changes upon addition of CO or progesterone indicates that only a small fraction of the mutant CYP17 proteins is expressed in a functional form in yeast.

Immunoblots using fresh yeast microsomes containing the four missense mutations all contained immunoreactive protein that comigrated with wild-type human CYP17 at approximately 57 kDa as well as degradation products (Fig. 4DGo). The amount of full-length protein remained relatively constant in microsomes containing wild-type CYP17 after multiple freeze-thaw cycles. In contrast, the quantity of full-length CYP17 protein declined rapidly with freeze-thawing or warming in sodium dodecyl sulfate sample buffer for the four missense mutations, and products of lower mass increased in parallel, presumably through proteolysis (not shown). We conclude from these data that all four missense mutations impair activity primarily by destabilizing the enzyme structures, thus impairing the capacity to incorporate and/or retain heme. For mutations Y329D and P428L, a sufficient portion of the protein molecules remain properly folded to exhibit catalytic activity at least transiently, but this activity is barely detectable using sensitive radiochemical assays at low substrate concentrations. Mutations Y329D and P428L are examples of partial, combined deficiencies in both 17{alpha}-hydroxylase and 17,20-lyase activities.

Correlation of genotype and phenotype: mutations W406R and R362C

We compared the phenotypic characteristics in the 11 homozygotes for W406R and the 7 homozygotes for R362C whose CYP17 enzymes are completely inactive in heterologous assay systems. Although DOC concentrations pre- and post-ACTH-(1–24) administration were higher in W406R homozygotes than in subjects homozygous for R362C (P < 0.01), blood pressure and circulating concentrations of potassium or other hormones were similar in the two groups (Table 5Go). Aldosterone values were low in both groups, and in fact, plasma aldosterone values in untreated subjects were uniformly suppressed regardless of genotype (data not shown). Thus, despite equally inactive CYP17 enzymes, homozygotes for mutations W406R and R362C showed some trends to phenotypic differences, and clinical features varied even among subjects with these two common mutations (Tables 2Go and 5Go). Although variations in blood pressure and potassium values may be influenced by dietary and environmental differences, the range of genital differentiation among these male pseudohermaphrodites remains unexplained.


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  		TABLE 5. Clinical and hormonal characteristics of the W406R and R362C homozygotes

 
The finding of the W406R or R362C allele in an affected or obligate heterozygous subject who recently emigrated from the Iberian peninsula would argue against these mutations arising de novo in Brazil. We studied the family of a compound heterozygote for W406R and R362C (Fig. 5Go) whose deceased father was born in Spain (DNA not available). The father migrated to Brazil and married a Brazilian of Portuguese ancestry, and the mother was heterozygous for allele R362C. Of their children, one with 17OHD bore one copy of each mutation, one was a heterozygote for W406R, one is wild type at both alleles, and one died of an unknown cause. The genetics within this kindred suggest that the W406R mutation arose in Spain from an ancestor common to many Brazilians bearing mutation W406R.



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  		FIG. 5. Genetics of kindred 19. The father was born in Spain (asterisk) and carried mutation W406R (shaded). The mother, of Portuguese descent, was heterozygous for mutation R362C (stippled). One offspring was a compound heterozygote for both mutations and had complete, combined 17OHD.

 

   Discussion
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
References
 
In this study we report the largest series of 17OHD subjects studied in a single country by 1 group of investigators. In 1994, Kater and Biglieri (16) first observed an unusual large number of Brazilian cases of 17OHD, and 17OHD remains the second most common cause of CAH in Brazil (22). The finding in the present study that 82% of the mutant alleles can be explained by 2 mutations suggests founder effects for the CYP17 mutations, an unusual result for an autosomal recessive disease in a country with such an extensive racial admixture (24). Only 3 recurring CYP17 mutations have been described previously: a CATC duplication following Ile479 found in Canadian Mennonites and Dutch Frieslanders (10), the deletion of Phe at codon 53 or 54 in Japan (11); and deletion of residues 487–489 in East Asia (35). The fact that founder effects can be manifested in a country with great ethnic heterogeneity also suggests a high coefficient of inbreeding in local areas, even though known consanguinity occurred in only 6 of our 19 families (32%). Recurring mutations have also been identified in Brazilian series of 3 other autosomal recessive disorders, namely 17ß-hydroxysteroid dehydrogenase 3 deficiency (36), 5{alpha}-reductase deficiency (37), and 21-hydroxylase deficiency (38), and these observations are consistent with a high rate of inbreeding in some populations.

Analysis of 6 intronic polymorphisms found within CYP17 itself (www.pga.swmed.edu) provided further evidence of founder effects. All homozygotes for mutations W406R and R362C were homozygous at all 6 positions, including the rare C and T variants at g.3274 (intron 2) and g.7028 (intron 7), respectively. Among 42 family members of subjects bearing mutation W406R, 4 relatives (heterozygotes or wild type at W406) were also heterozygous for C and A at the adjacent polymorphism in the 3' end of intron 6 (g.6787), but all W406R homozygotes had only the C variant. We discovered another polymorphism in intron 6 at g.4617, C or the T found in the GenBank sequence, adjacent to the R362C mutation in exon 6. All R362C homozygotes had 2 copies of the C variant, whereas 8 of 29 wild-type and heterozygous relatives studied possessed 1 copy each with C and T. The patterns at these polymorphisms associated with both mutations R362 and W406R were identical, consistent with our suggestion that the 2 mutations arose in similar genetic backgrounds. Compound heterozygotes for R362C and W406R were also homozygous at all polymorphisms. Finally, both copies of intron 7 from the subject homozygous for P428L contained 2 additional C’s near the 3' end, and the heterozygous relatives exhibited 1 copy of each variant. Based on the uniform patterns of polymorphisms, we scored 1 allele for each homozygote in Table 4Go (28 total), although the true number may be as high as 32.

Mutations W406R and R362C

Previously, only 2 Brazilian patients with 17OHD had been characterized by molecular genetics, and both were cases of isolated 17,20-lyase deficiency (18). In this study, 7 CYP17 mutations not previously described were identified in 19 families; 2 of these (W406R and R362C) are responsible for 82% of the affected alleles. Patients affected by W406R and R362C mutations are distributed in south/southeast and northeast Brazil, respectively, consistent with separate founder effects in these 2 regions. The majority of subjects with W406R and R362C mutations are of Spanish or Portuguese descent, respectively. In contrast, among the nearly 40 currently reported mutations in the CYP17 gene (4, 5, 6, 7, 8, 9), none is of Spanish or Portuguese origin. This discrepancy with our data may be due to the higher prevalence and greater awareness of other conditions with similar clinical features as 17OHD (16), leading to underdiagnosis of 17OHD worldwide. Mutation W406R is now the most common genetic defect known to cause 17OHD. Given the high prevalence of mutations W406R and R362C, neither of which alters a restriction site, we now screen new Brazilian patients with 17OHD with a single 1.5-kb PCR reaction spanning exons 6 and 7 using primers I5S1 and I7AS1, sequencing one or both exons depending on the family’s ethnic background.

The only phenotypic feature that was significantly different between homozygotes for mutations W406R and R362C was the higher DOC concentration found in subjects with mutation W406R, yet mean blood pressure and serum potassium concentrations did not differ in the two groups. When these two mutations were expressed in COS-7 or HEK-293 cells and in yeast, we consistently found no residual enzymatic activity for either mutation. It is therefore unlikely that any clinical divergence found in these two groups can be explained by differences in the enzymatic properties of the CYP17 mutations alone. Regional or ethnic preferences in dietary sodium content as well as other genetic differences related to ethnic origin may influence the typical features of subjects with these common mutations.

Although most subjects homozygous for mutations W406R and R362C presented with hypertension, hypokalemia, and sexual infantilism (13), phenotypic variations were occasionally observed within each group. Among 3 46,XY homozygotes for mutation R362C, 1 presented at birth with ambiguous genitalia, a second had female external genitalia but normokalemia at diagnosis, and the third demonstrated the classical, complete 17OHD syndrome. Analogously, the appearance of the external genitalia can vary among genetically male siblings with the same mutation in the androgen receptor gene (39). None of the 4 46,XX patients manifested any degree of spontaneous sexual development, and all were hypertensive. All 11 homozygotes for mutation W406R remained sexually infantile, yet 1 subject was consistently normotensive for up to 2 yr of continuous follow-up before the diagnosis of 17OHD despite concurrent, marked hypokalemia. Testosterone concentrations varied within the prepubertal range where this RIA lacks accuracy, limiting the significance of these data. We conclude that environmental and other genetic factors may modulate the phenotypic features of patients with severe 17OHD. These genetic modifier loci might influence steroid production and action by altering the activity of transcription factors (40, 41), the CYP17 cofactor proteins CPR and cytochrome b5 (29), or downstream mediators of mineralocorticoid, androgen, and estrogen action.

Other mutations and partial 17OHD

The homozygote for a TAG nonsense mutation at tyrosine 329 in exon 6 had refractory hypertension in childhood. A TAA stop codon at the same position has been previously reported in a compound heterozygote from Japan, and this patient also had severe clinical manifestations (42). Curiously, our subject who is a compound heterozygote for mutations W406R and M1T, like a previously reported case bearing mutation M1I (43), presented with hypokalemic myopathy and serum potassium values as low as 1.0 mEq/liter. The researchers suggested that CYP17 protein translation from mutation M1I might begin at methionine 49, which would yield an inactive protein (43), but it is not known whether a truncated protein is produced from mutation M1I or M1T, or if this process can contribute to potassium wasting.

Partial 17OHD has been reported in women with normal or abnormal menstrual cycles and breast development as well as in males with incomplete virilization (14, 44, 45). In addition, approximately 10–15% of subjects with the diagnosis of 17OHD are normotensive and/or normokalemic at diagnosis (16), although few of these individuals have been genotyped (4). Our 46,XX compound heterozygote for Y329D and the AG to CG substitution in intron 2 had spontaneous Tanner stage 5 breast development at puberty. The AG to CG mutation should alter RNA splicing and introduce a frameshift that yields a truncated, inactive enzyme (See companion paper, Ref.45A ), yet some correctly spliced transcripts may be produced from this allele as well. In addition, mutation Y329D retains approximately 5% of the catalytic activity when expressed in heterologous systems, but the protein is unstable and readily degraded. Although in vitro experiments do not necessarily reflect true in vivo conditions, as little as 5–8% of 17,20-lyase activity may be sufficient to promote secondary sexual development (2, 11), particularly in genetic females due to the potency of estradiol (46).

In contrast, the phenotype of our 46,XY homozygote for mutation P428L was complete, combined 17OHD, despite some residual activity of the mutant enzyme in heterologous expression experiments. The carboxyl terminus of CYP17 is important for both heme and substrate binding (47), and even small C-terminal alterations can destroy most (48) or all (10, 12, 49) enzyme activity. Mutations closer to the heme at C442 tend to destroy all activity (6, 26), although mutation R415C retains some activity (8). Both our homozygote for P428L and our heterozygote for Y329D developed hypertension and hypokalemia, indicating that the low residual activities of P428L and Y329D are insufficient to prevent mineralocorticoid excess. Consequently, the threshold CYP17 activity level to yield atypical phenotypic features in 17OHD appears to be lowest for pubertal breast development in 46,XX patients, which reflects sufficient C19 steroid production, driven by high precursor concentrations, via a defective CYP17 enzyme. In contrast, 17-deoxysteroid metabolism to cortisol is impaired by the low adrenal 17{alpha}-hydroxylase activity, such that 17-deoxysteroids with mineralocorticoid activity accumulate universally in 17OHD. Nonetheless, differences in target tissue sensitivity to these mineralocorticoids may account for the variability in hypertension and hypokalemia seen among individuals with similar genotypes, but the rare examples of partial virilization in subjects with mutant proteins that have no activity in heterologous systems remain enigmatic.

Structural basis of protein instability and phenotype variability

To understand how the four missense mutations (W406R, R362D, P428L, and Y329D) cause partial or complete loss of enzymatic activity, we located the mutated residues in a computer model of human CYP17 (47). Residue Y329 lies in the middle of the J helix and appears to form a hydrophobic packing interaction with L460 on the C-terminal end of the adjacent L helix (Fig. 6Go, A and B). The substitution of a charged aspartate at position 329 weakens this hydrophobic interaction and destabilizes the enzyme structure, but does not directly perturb the active site. Residues R362, W406, and P428 all lay within a contiguous three-dimensional space in a region of the protein that is critical for heme binding and proper folding. Residue R362 comprises part of the ExxR motif at the C terminus of the K helix, a motif present in all known cytochromes P450 (50). The serpiginous chain of residues that follows and leads to the heme-liganding cysteine (C442) tends to unwind the K helix, but hydrogen bonding between the adjacent E and R residues in this motif stabilizes this structure and helps to form the redox partner binding site (47, 51) (Fig. 6CGo). W406 abuts R362 from its position at the start of the meander region (Fig. 6DGo), another conserved motif that precedes the heme-binding decapeptide. Two CYP17 mutations that change residues in or near the meander domain are F417C (52) and P409R (6), and both of these mutations completely destroy enzyme activity (26). The W406R mutation juxtaposes two positively charged arginine residues, which would weaken hydrogen bonding within the ExxR motif. Thus, it is remarkable that the two common Brazilian mutations derived from different ethnic backgrounds alter two residues that lie adjacent in the protein structure, and these two mutations appear to destabilize the enzyme by a common mechanism. Mutation P428L probably impairs heme incorporation directly, rather than by disrupting hydrogen bonding in the ExxR motif.



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  		FIG. 6. Computer model of human CYP17, highlighting locations of missense mutations Y329D, R362C, W406R, and P428L. A, Ribbon diagram of protein backbone, with heme (red) and all atoms of mutated residues (blue) displayed and labeled. Also shown in yellow are residues E359 and L460, which interact with R362 and Y329, respectively. B, Hydrophobic interaction of Y329 (blue, aromatic ring viewed edge-on) in J helix with L460 (yellow) in L helix. Arrows indicate directions of I, J, and L helices. C, Proximity of mutated residues R362, W406, and P428 (blue). Residue E359, the hydrogen-bonding partner of R362, is shown in yellow, and the directions of the I and K helices are indicated. The front clipping plane has been recessed for clarity. D, Redox partner binding surface of human CYP17 viewed face-on with only backbone atoms of residues 347–447 displayed, plus all atoms of heme (red), E359 (yellow), and mutated residues R362, W406, and P428 (blue). The hydrogen bonding of E359 and R362 maintains the integrity of the K helix, and W406 and P428 lay adjacent to R362 in the serpiginous chain of residues connecting the K helix with the heme-binding decapeptide. Images were generated with MidasPlus software on a Silicon Graphics (Mountain View, CA) Octane workstation using CYP17 structure 2c17 (www.rcsb.org).

 
Our genetic and biochemical studies suggest that the missense mutations described in this study, and perhaps most mutations that cause severe 17OHD, primarily impair enzyme activity by destabilizing enzyme structure. This hypothesis is supported by the lack of spectroscopically demonstrable P450 (CO-reduced or substrate-induced difference spectra) in CYP17 mutations studied in this and previous (26, 53) reports. We postulate that intracellular factors may variably enhance the stability of mutant CYP17 proteins within the adrenals and/or gonads of 17OHD individuals, which may allow for transient maintenance of some enzyme activity in rare cases when enzyme expression is maximally induced despite the lack of demonstrable activity in heterologous expression systems. This paradigm may also explain how high doses of gonadotropins enabled oocyte development leading to in vitro fertilization in one female with complete 17OHD (54). Therefore, the activities of CYP17 mutations in heterologous expression systems aid in defining the molecular basis of 17OHD, but additional undefined factors may still modify the clinical features in individual subjects.


   Acknowledgments
 
The Brazilian CAH Multicenter Study Group includes Ayrton C. Moreira, Berenice B. Mendonça, Bernardo Liberman, D. L. Queiroz, Eduardo P. Dias, Eliana T. Pereira, Ivan R. A. Ferreira, João P. B. Vieira-Filho, José Gastão R. Carvalho, Luiz Lacerda Filho, Manuel H. Aguiar-Oliveira, Maria Tereza M. Baptista, Maria Conceição R. Freitas, Marisa Cesar Coral, Mauro Semer, Ney Cavalcanti, Rosângela R. Rea, Suzana Pacheco, Theresa S. S. Lins, and Thomaz R. P. Cruz. We thank Dr. Ed Biglieri, Dr. Morris Schambelan, and Bobby Chang for performing hormone measurements, and we thank Richard Hall and Kerri Kwist for assistance with experiments.


   Footnotes
 
This work was supported by NIH Grants K08-DK-02387 and R03-DK-56641 (to R.J.A.) and by grants from Fundação de Amparo à Pequisa do Estado de Sâo Paulo (96/7449-6) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (0690/00-7; to C.E.K.).

Results from this work were presented in abstract form at the 83rd Annual Meeting of The Endocrine Society, Denver, CO, June 2001.

Abbreviations: CAH, Congenital adrenal hyperplasia; CPR, cytochrome P450-oxidoreductase; DOC, 11-deoxycorticosterone; 17OHD, 17-hydroxylase deficiency.

Received June 12, 2003.

Accepted September 11, 2003.


   References
Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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