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Phenotype-Genotype Correlation in Eight Chinese 17-Hydroxylase/17,20 Lyase-Deficiency Patients with Five Novel Mutations of CYP17A1 Gene

Shanghai Clinical Center for Endocrine and Metabolic Diseases (J.Y., B.C., S.S., Y.B., J.L., Y.Z., J.C., G.N., X.L.), Shanghai Institute of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiaotong University Medical School, Institute of Health Science (B.C.), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and Endocrine and Metabolic Division (G.N., X.L.), E-Institutes of Shanghai Universities, Shanghai 200025, People’s Republic of China; and Bioinformation Center (T.S., S.Z.), Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, People’s Republic of China

Address all correspondence and requests for reprints to: G. Ning, M.D., Ph.D., and X. Li, M.D., Ph.D., Department of Endocrinology and Metabolism, Ruijin Hospital, Shanghai Jiaotong University Medical School, Ruijin 2nd Road, Shanghai 200025, People’s Republic of China. E-mail: lixy{at}sibs.ac.cn.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Context: P450c17 deficiency (17OHD), caused by mutation in CYP17A1 gene, is characterized by severe hypertension-hypokalemia, sexual infantilism in females, and pseudohermaphroditism in males. We investigated eight Chinese 17OHD patients with five novel mutations of CYP17A1 gene and analyzed phenotype-genotype correlation in a patient with regular menses and seven others with classic presentations by in vitro expression and computer modeling.

Objective: The objective of the study was to explore the phenotype-genotype correlation in patients with subtle and classic manifestations.

Subjects and Methods: Eight patients with 17OHD from seven families were diagnosed according to clinical manifestations and basal hormone assays. The CYP17A1 gene was amplified and sequenced. Haplotyping analysis was performed to determine a common ancestor for those subjects with a frequent mutation 1517_1525del. In vitro enzymatic activities assay and computer modeling were used to analyze the phenotype-genotype correlation.

Results: Five novel CYP17A1 mutations, homozygous D487_F489del (1517_1525del) and F453S, combined compound Y329K and 1047del, P434L and V310_W313del, and R416C and D487_F489del were identified. Haplotyping showed that 1517_1525del might be inherited from a common ancestor. Compared with the mutations in patients with classical manifestations, F453S in the patient with regular menses, occasional hypertension, and hypokalemia showed a partially reduced 17-hydroxylase (29% of those of wild type) and a minor protein conformational change.

Conclusion: The clinical manifestations in patients with 17OHD correlate with CYP17A1 mutations and enzymatic activities by in vitro enzyme assay and computer modeling. F453S mutation results in partially reduced enzymatic activities and a subtle phenotype. The prevalent mutation 1517_1525del in Chinese 17OHD patients might be a founder effect.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
AS ONE OF the crucial enzymes in steroid hormone biosynthesis, cytochrome P450c17 performs both hydroxylation and lyase activities, by converting pregnenolone/progesterone to 17-hydroxy pregnenolone/progesterone (17-hydroxylase) and cleaving the latter to yield the precursors of estrogens and androgens (17,20-lyase) (1). The enzyme is expressed in adrenal zona fasciculata and zona reticularis as well as gonadal Leydig and theca cells (2, 3). Patients with 17-hydroxylase deficiency (17OHD) fail to produce gonadal steroids, resulting in sexual infantilism, primary amenorrhea in females (46XX), and pseudohermaphroditism in males (46XY). Moreover, reduced production of glucocorticoids in turn increases ACTH secretion, which leads to enlargement of bilateral adrenal glands and stimulates the overproduction of mineralcorticoids, causing severe hypertension and hypokalemia (4).

As a 508-amino-acid protein with a molecular mass of approximately 57 kDa, P450c17 is encoded by CYP17A1 gene, which locates at chromosome 10q24.3 (5, 6) and consists of eight exons (7). Since the first mutation was identified in a patient with 17OHD in 1988 (8), approximately 54 mutations of the CYP17A1 gene have been reported (9, 10, 11). These mutations have been known to cause either complete or partial, combined, or isolated 17-hydroxylase/17,20-lyase enzyme deficiencies. Recently some 17OHD patients with mild clinical presentations have been reported (12). However, the correlation between phenotype and genotype has not been well studied.

In the present study, CYP17A1 gene mutations and relevant clinical manifestations were analyzed in eight Chinese patients with P450c17 deficiency from seven kindreds. All patients but one, who presented with regular menses, mild hypertension, and hypokalemia, and carried a novel homozygous F453S missense mutation, had classic presentations. The enzymatic activities of P450c17 were evaluated by in vitro expression and computer modeling.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Eight subjects from seven families were investigated (Table 1), including four females with karyotype of 46XX and four males with 46XY. The phenotypic sexes for all the patients were female. Only kindred 4 was a consanguineous family. The diagnosis for all the patients was established by clinical manifestations and basal hormone assays. The clinical data for these subjects were obtained by senior endocrinologists. The study protocols were approved by the Hospital Ethics Committee for Human Research, and informed consent was obtained from every subject participating in the study.

PCR and sequencing of CYP17A1 gene

Genomic DNA was extracted from peripheral blood leukocytes of eight patients and their family members. All eight exons of CYP17A1 gene were amplified by PCR using eight pairs of primers (Table 2), which were designed with Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA). The PCR was performed in a volume of 50 µl containing 1.5 mM MgCl₂, 10 mM dNTP, 0.2 µg genomic DNA, 20 µmol of each primer, and 2.5 U Taq DNA polymerase (Sangon, Shanghai, China). Amplification was performed with preheating at 95 C for 5 min, followed by denaturation at 94 C for 30 sec, annealing at 55–62 C for 30 sec, and extension at 72 C for 45 sec for 30 cycles. The PCR products were purified using a gel extraction kit (QIAGEN, Mississauga, Ontario, Canada) and sequenced in both sense and antisense direction on a ABI 3700 sequencer (Applied Biosystems PerkinElmer, Foster City, CA).

Subclone sequencing

To confirm the monoallelic mutation, subclone sequencing was performed for those exons that were identified with heterozygous mutations by direct PCR sequencing. The purified PCR products were ligated to pGEM-T Easy vectors (Promega, Madison, WI) and transformed into DH5-competent cells. Positive clones were picked and sequenced by ABI PRISM 3700 genetic analyzer.

Haplotyping analysis

The heterozygous and homozygous deletion at position 1517_1525 (1517_1525del) was identified in four subjects from three kindreds (five of 14, 36%), which was the most frequent mutation in this study. To determine the presence of common ancestors for those subjects who carried the same 1517_1525del mutation, four short tandem repeat (STR) markers around CYP17A1 gene labeled as D10S1697, D10S1692, D10S205, and D10S222 spanning 946 kb were chosen for haplotyping. DNA fragments were labeled with fluorescent dye using M13 universal tailed primers. PCR products were analyzed using a CEQ 8800 sequencer (Beckman Coulter, Fullerton, CA). Data collection and analysis were performed with Fragment Analysis Module (Beckman Coulter).

Site-directed mutagenesis

The wild-type pCMV-SPORT6-CYP17A1, provided by Invitrogen (clone ID 5170736; Carlsbad, CA), was used as the template for F453S, P434L, and F114V construction. These CYP17A1 mutants were constructed using QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA). F114V, which was reported with loss of enzymatic activities (13), was used as a positive control for enzymatic activity assay in in vitro expression. The primers were designed as follows: F453S, forward, 5'-cccgccaggagctctccctcatcatggcctgg-3', reverse, 5'-caggccatgatgagggagagctcctggcggg-3'; P434L, forward, 5'-cgtcagt aagctatttgctcttcggagcaggacc-3', reverse, 5'-ggtcctgctccgaagagcaaatagcttactgacg-3'; and F114V, forward, 5'-cgtaagggtatcgccgtcgctgactctggcg-3', reverse, 5'-cgccagagtcagcg acggcgatacccttacg-3'. The PCR amplification was performed with preheating at 95 C for 30 sec, followed by denaturation at 95 C for 30 sec, annealing at 55 C for 1 min, and extension at 68 C for 6 min for 16 cycles. The parent pCMV-SPORT6 were digested by DpnI enzyme and the mutants then transformed into XL-blue supercompetent cells. Positive clones were picked and sequenced to confirm the site-directed mutations.

Transfection of COS-7 cells and assays for enzyme activities

The wild-type and mutant pCMV-SPORT6-CYP17A1 was purified with EndoFree plasmid maxi kit (QIAGEN). COS-7 cells (CRL-1651; American Type Culture Collection, Manassas, VA) were cultured in 24-well plates to 90% confluence and transfected with 0.8 µg DNA per well using Lipofectamine 2000 reagent (Invitrogen). In 40 h after transfection, various concentrations of progesterone (0.2, 0.5, 1.0, 2.0 µmol/liter) were added and incubated for another 8 h. The medium was collected and frozen at –20 C until the assay of 17-hydroxyprogesterone and androstenedione (RIA kits; Diagnostics Systems Laboratories, Webster, TX). All transfection experiments were performed at least twice.

Computer modeling for P450c17 enzyme

To further understand how those mutations of CYP17A1 gene affect P450c17 enzymatic activity, we constructed a three-dimensional computer model using Modeller software (version 8V1; http://salilab.org/modeller). The crystal structure of human microsomal CYP3A4 (PDB accession code 1tqn) with a resolution of 2.05 Å was chosen as the template for the three-dimensional model of P450c17. The small difference between the CYP3A4 structure and P450c17 model was demonstrated as a high score of pair potential 0.94 using a fold recognition algorithm by Genthreader (http://bioinf.cs.ucl.ac.uk/psipred/). The energy-minimized model was finally selected from five optional P450c17 structures constructed by Modeler software (see Fig. 4A). Furthermore, Procheck (version 3.0; http://www.biochem.ucl.ac.uk/roman/procheck/procheck.html) and ProsaII programs (version 3.0; http://www.came.sbg.ac.at) were used to assess the stereochemistry, the fitness of sequence and structure, and the total protein potential for the selected P450c17 model.

View larger version (37K): [in this window] [in a new window]   FIG. 4. Three-dimensional computer model constructed with Modeler for P450c17 protein. R416, P434, and F453 are located close to the heme (A). F453 locates inside a straightforward helix (B). A hydrogen bond forms between serine at 453 and arginine at 449 when the substitution F453S occurs (C).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

Seven patients from six kindreds presented with severe hypertension, hypokalemia, and sexual infantilism. Subjects K2–1, K4–1, K6–1, and K6–2 with karyotype 46XY presented with male pseudohermaphroditism. The patients from kindreds 1, 3, and 5 with 46XX karyotype presented with primary amenorrhea. The laboratory examinations indicated lower plasma cortisol, 17-hydroxy progesterone, estradiol, and testosterone; elevated blood ACTH, FSH, LH, and aldosterone level; and lower basal renin activity. Computerized tomography (CT) scan showed bilateral adrenal enlargement. X-ray showed remarkably delayed bone age (Tables 1 and 3).

Mutation analysis for CYP17A1 gene

Five different CYP17A1 gene mutations were detected in eight patients from seven families, all of which have not been reported previously (Table 1 and Fig. 1). Mutation 1517_1525del (D487_F489del) was identified in four patients from three kindreds (five of 14, 36%), including a homozygous mutation in two patients from kindreds 1 and 2 and a compound heterozygous mutation combined with a R416C substitution in two patients from kindred 6. Another compound heterozygous mutation of an 8-bp deletion (989_996del) at exon 5 and 1361C>T (P434L) substitution at exon 8 was identified in subject K5–1. The compound mutation 1047C>A substitution and 1045del at exon 6 was identified in subjects K3–1 and K4–1, resulting in Tyr329Lys replacement and an open reading frame shift, and producing a stop codon at position 418.

View larger version (38K): [in this window] [in a new window]   FIG. 1. Subclone sequencing of CYP17A1 gene for all probands. A, 1418T>C (F453S) substitution at exon 8 in kindred 7. B, A compound 1047C>A substitution and 1045del (Y329K) at exon 6 in kindreds 3 and 4. C, 1306C>T (R416C) substitution at exon 8 in kindred 6. D, A 9-bp deletion from 1517 to 1525 (D487_F489) in kindreds 1, 2, and 6. E, 1361C>T (P434L) substitution at exon 8 in kindred 5. F, An 8-bp deletion from 989 to 996 (V310_W313del) in kindred 5.

Haplotyping analysis

Subjects K1–1 and K2–1 had two alleles, and K6–1 one allele in common (Fig. 2), which was demonstrated by identical D10S1697, D10S1692, D10S205, and D10S222 STR markers. It indicated that mutation 1517_1525del (D487_F489del) of CYP17A1 gene in these families may come from common ancestors based on the identical haplotypes.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Haplotyping for all the probands from seven kindreds. STR markers are indicated as D10S1697, D10S1699, D10S205, and D10S222. The patients from kindreds 1, 2, and 6 displayed a common haplotype.

The 17-hydroxylase activities of the mutant proteins were estimated by comparison with wild-type enzyme in transiently transfected COS-7 cells. The conversion of various concentrations of progesterone to 17-hydroxyprogesterone and androstenedione was used as a measure for 17-hydroxylase activity (Fig. 3A). The conversion percentages of progesterone (1 µM) were 39.3, 11.4, 2.4, and 3.1% for wild type, F453S, P434L, and F114V, respectively. The relative production of 17-hydroxyprogesterone (hydroxylase activity) for wild-type and mutant proteins is shown in Fig. 3B. The hydroxylase activity for F453S mutated protein was partially reduced (29%), whereas it was dramatically reduced for P434L (6.1%) and F114V (7.9%) mutated proteins. All the products of wild-type and mutant enzymes paralleled with upgraded concentrations of the substrates (Fig. 3A).

View larger version (14K): [in this window] [in a new window]   FIG. 3. A, Enzymatic activity assay by in vitro expression. Production of 17-hydroxyprogesteron and androstenedione in the presence of various concentrations of steroid precursors (0.2, 0.5, 1.0, and 2.0 µM progesterone) is shown as a measure of 17-hydroxylase activity in COS-7 cells transfected with wild-type, F453S, P434L, and F114V mutants. B, The relative production of 17-hydroxyprogesterone and androstenedione is shown as a measure of 17-hydroxylase activity in COS-7 cells transfected with wild-type, F453S, P434L, and F114V mutants. The product percentages were 100, 29.1, 6.1, and 7.9% for 17-hydroxylase activity for wild-type, F453S, P434L, and F114V mutated proteins. Data are shown as the mean ± SD (n = 3 in A and B).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The majority of CYP17A1 gene mutations result in the absence of 17-hydroxylase and 17,20-lyase activities and cause classic clinical presentations for 17OHD patients. In the present study, seven patients from six families, who had D487_F489del, Y329K (1045T>A,1047del), P434L, and R416C mutations presented with primary amenorrhea, sexual infantilism, severe hypertension, and hypokalemia. The absence of enzymatic activities for D487_F489del, Y329K, and R416C mutated proteins has been reported by the previous studies (16, 19, 20). Distinguished from those patients with classic manifestations, patient K7–1 presented with occasional hypertension, hypokalemia, and lack of pubic hair. She had not been diagnosed as 17OHD because she had well-developed breasts, regular menses, and almost normal hormonal levels until we detected a homozygous F453S mutation of the CYP17A1 gene in the patient and one mutant allele in each parent.

We further performed enzymatic activity assay by in vitro expression of wild-type and mutant CYP17A1 in COS-7 cells and measurement of the production of 17-hydroxyprogesterone and androstenedione (for hydroxylase activity). The results revealed that the 17-hydroxylase activity of F453S mutant protein was reduced to 29% of that of wild type, which was relatively higher than that of P434L and F114V (6.1 and 7.9%). The 17-hydroxylase activities paralleled with upgraded concentrations of the substrates for F453S mutant protein. A Japanese 17OHD patient with a homozygous Y201N substitution was recently reported. The patient also had mild clinical manifestations similar to our patient K7–1 with F453S mutation except irregular menses (21). The reduced percentage of enzymatic activity for F453S was close to that of enzyme with Y201N mutation (33%).

To further understand how these missense mutations, F453S, P434L, and R416C, reduced the enzyme activities partially or completely, we constructed a three-dimensional computer model using the recently developed crystal structure of human microsomal CYP3A4 as the principal template and located the mutant residues accordingly. P434 is very important for the formation of ß-turn, which lies below the heme and the active cavity and seems to function by supporting the heme when it interacts with the substrate. Mutation P434L drives the side chain of the substitute L away from forming hydrogen bonds. Meanwhile the substitution L would hinder the formation of the conserved ß-turn, and in this way the spatial structure of the active cavity of the protein would be changed. We noticed that R416 can form a hydrogen bond network with H407, Q411, and P409, and this hydrogen bond network can stabilize the local loop structure. In other words, this structure could possibly be a binding domain with some other proteins. The mutation of R416C undoubtedly would destruct the conformation of this loop.

F453 is located inside a straightforward helix approaching the heme-binding site (Fig. 4B). The hydrophobic benzene ring of phenylalanine is free and does not connect with any other residues. However, when phenylalanine is substituted by serine, a residue with a branched chain, a new hydrogen bond is formed between this polar residue and arginine 449, which tends to bend the straightforward helix away from heme-binding site (Fig. 4C). As a result, this minor conformational change could affect the volume of the active cavity and consequently the enzyme-substrate interaction. An identical conformational structure alteration was demonstrated for the F453S mutation with a commonly used computational model of human P450c17 (PDB code 2c17) (22) except the new hydrogen bond is formed with glutamine 450 instead of arginine 449.

In the present study, we detected five novel mutations of the CYP17A1 gene in patients with classic and subtle manifestations. Mutations D487_F489del, Y329K (1045T>A,1047del), P434L, and R416C caused severe hypertension, hypokalemia, and sexual infantilism, whereas mutation F453S resulted in regular menses and occasional hypertension, which established a correlation between phenotype and genotype by mutation analysis, in vitro expression assay, and computer modeling.

	Acknowledgments

 
We are indebted to all the patients and their family members who participated in this study. We are grateful to Professor Walter L. Miller (University of California, San Francisco, San Francisco, CA) for giving us wild-type CYP17A1 plasmid as a gift. We are also grateful to Dr. Marco Marcelli (Baylor College of Medicine, Houston, TX) for critical reading of the manuscript.

	Footnotes

 
This work was partially supported by Grant E03007 from the E-Institute of Shanghai Universities, Shanghai Municipal Education Commission.

First Published Online June 13, 2006

Abbreviations: CT, Computerized tomography; 17-hydroxylase, 17-hydroxy pregnenolone/progesterone; 17OHD, 17-hydroxylase deficiency; STR, short tandem repeat.

Received October 18, 2005.

Accepted June 7, 2006.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, J. Articles by Li, X. Search for Related Content PubMed PubMed Citation Articles by Yang, J. Articles by Li, X. Related Collections Adrenal and Hypertension Pediatric Endocrinology Cardiovascular Endocrinology Female Endocrinology Male Endocrinology