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 Merke, D. P. Articles by Cutler, G. B. Search for Related Content PubMed PubMed Citation Articles by Merke, D. P. Articles by Cutler, G. B., Jr.

Novel CYP11B1 Mutations in Congenital Adrenal Hyperplasia due to Steroid 11ß-Hydroxylase Deficiency

Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Deborah P. Merke, M.D., Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Building 10, Room 10N262, 10 Center Drive, MSC 1862, Bethesda, Maryland 20892-1862.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The second most common cause of congenital adrenal hyperplasia is 11ß-hydroxylase deficiency, an autosomal recessive disorder. We performed genetic analysis of CYP11B1, the gene encoding steroid 11ß-hydroxylase, in three patients with classic 11ß-hydroxylase deficiency. Herein we describe the first splice donor site mutation, a new nonsense mutation, and a new missense mutation in this disorder. An African-American patient was found to be a compound heterozygote for a codon 318+1GA substitution at the 5'-splice donor site of intron 5, in combination with Q356X, a nonsense mutation previously reported in an African-American patient. A Caucasian patient was found to be a compound heterozygote with a novel missense mutation, T318R, in combination with a previously reported 28-bp deletion in exon 2. A different mutation at codon 318 (T318M) has been described previously. A Caucasian patient was heterozygous for a novel nonsense mutation (Q19X) in exon 2. The second mutation was not identified in this patient. Multiple apparent polymorphisms were also observed. Two of these polymorphisms in CYP11B1 represent sequences from CYP11B2, suggesting that gene conversion may have occurred. In summary, we have identified three novel mutations and two previously reported mutations in CYP11B1 patients with 11ß-hydroxylase deficiency. Our data suggest the presence of a mutational hot spot at codon 318 of CYP11B1, and the possibility of a founder effect in frequently identified mutations.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
CONGENITAL adrenal hyperplasia (CAH) denotes a family of disorders with defects in cortisol biosynthesis. Deficient 11ß-hydroxylase activity accounts for 5–8% of cases (1). The gene for 11ß-hydroxylase, CYP11B1, is located on chromosome 8q22 approximately 40 kilobases from the gene for aldosterone synthase, CYP11B2 (2). These two functional genes share 95% sequence homology. Previously reported mutations of CYP11B1 are distributed over the entire coding region, but cluster in exons 2, 6, 7, and 8 (3) (Fig. 1). Two different nonsense mutations have been reported in both codons 384 (4, 5) and 448 (3), suggesting the presence of mutational hot spots.

View larger version (13K): [in this window] [in a new window]   Figure 1. CYP11B1 mutations causing classic 11ß-hydroxylase deficiency. Previously reported mutations are displayed above the CYP11B1 schematic and are distributed over the entire coding region with clusters in exons 2, 6, 7, and 8. Mutational hot spots exist at codons 318, 384, and 448. Three novel mutations are displayed below the line. •, Mutations identified in our three patients.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

Three unrelated patients with 11ß-hydroxylase deficiency were studied. Patient 1, an African-American female, presented at 1 yr of age with hypertension (136/78 mm Hg), mildly virilized genitalia (posterior labial fusion and clitoromegaly), and growth acceleration. Serum hormone levels at diagnosis included a 17-hydroxyprogesterone level of 345 ng/dL (normal, 20–70), 11-deoxycortisol of 16,750 ng/dL (normal, 30–200), testosterone of 82 ng/dL (normal, <10), and undetectable PRA. Blood pressure normalized with hydrocortisone treatment. With hydrocortisone therapy, patient 1 has remained prepubertal and has grown along the 95th percentile.

Patient 2, a Caucasian male, was born prematurely at 30 weeks gestation. Persistent hyponatremia in the neonatal intensive care unit, which may have been due to causes other than CAH, led to the diagnosis of 11ß-hydroxylase deficiency. At diagnosis, the serum 11-deoxycortisol concentration was 16,400 ng/dL. With hydrocortisone therapy, patient 2 has had intermittent hypertension (120/90 mm Hg) and remains prepubertal. Patient 3, a Caucasian male, presented with accelerated growth, precocious virilization, and hypertension (138/102 mm Hg) at 5 yr of age. At diagnosis he had a serum 17-hydroxyprogesterone level of 370 ng/dL, 11-deoxycortisol of 12,800 ng/dL, testosterone of 260 ng/dL, undetectable PRA, and a bone age of 12 yr, 6 months. Growth and blood pressure initially normalized with hydrocortisone treatment, but recurrent hypertension several years later led to further antihypertensive therapy.

Institutional review board approval of the research protocol and informed consent from all patients were obtained.

DNA extraction and amplification

Genomic DNA was prepared from peripheral blood leukocytes using standard procedures. The CYP11B1 gene was amplified by PCR. Three pairs of primers were used to amplify exons 1–2, 3–5, and 6–9 of CYP11B1 without amplifying CYP11B2, as previously described (6).

Nested PCR was performed to obtain smaller PCR products spanning individual exons. These smaller PCR products were then screened for mutations. For nested PCR, the first step PCR reactions were carried out using 20 cycles. Subsequently, 1 µL PCR product was used for the second PCR amplification of each exon using new primers (Table 1). The second PCR reactions were performed as previously described, except a 15-s extension time was used.

Screening for mutations was performed by heteroduplex analysis using mutation detection enhancement gels (AT Biochem, Malvern, PA) (7). Double-stranded PCR products were heated to 95 C to produce single-stranded DNA and then slowly cooled to 37 C to reanneal the DNA. The reannealed PCR products were then subjected to PAGE using 0.5 x mutation detection enhancement gels containing 0.6 x TBE and 15% urea. Gels were run at 15 V/cm for 14–18 h and visualized with ethidium bromide. PCR products were sequenced from both strands by the dideoxy method using an automated fluorescent sequencing system (Applied Biosystems model 373A) (8). The entire CYP11B1 gene was sequenced in both forward and reverse directions in all three patients.

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
CYP11B1 gene analysis

Direct sequencing of PCR-amplified genomic DNA for patient 1 revealed a G to A transition at the first base pair of intron 5 within the splice donor site and a previously reported nonsense mutation (Q356X) in exon 6 (Fig. 2). In patient 2, a novel missense mutation at codon 318 (T318R) in exon 5 and a previously reported 28-bp deletion in exon 2 were identified. Patient 3 showed a C to T transition at position 19 in exon 1 (Q19X), which is a nonsense mutation and should prevent synthesis of a functional enzyme from this allele. Sequencing the opposite strand of the PCR products confirmed these findings (data not shown). As expected, patients 1 and 2 were compound heterozygotes, as there was no family history of consanguinity in any of the patients. Moreover, sequencing of parental DNA showed that each parent carried one of the two mutations. In patient 3, we were unable to identify the second mutation in the coding region and exon-intron boundaries of CYP11B1 despite sequencing both DNA strands of each exon three times. The father of patient 3 was a carrier of Q19X, and no mutation was identified in the mother.

View larger version (22K): [in this window] [in a new window]   Figure 2. Nucleotide sequence of CYP11B1 gene in three families with 11ß-hydroxylase deficiency. Automated fluorescent sequencing was performed using PCR products as templates. The mutations were confirmed by sequencing the opposite strand (data not shown). The numbers indicate the position based on the human sequence (9). The arrows indicate the positions of heterozygous mutations. Patients 1 and 2 are compound heterozygotes, and each parent is a carrier for one of the two mutations. Only one mutation in CYP11B1 was identified in patient 3. This mutation was confirmed in his father. The CYP11B1 coding sequence is normal in his mother.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Splicing errors are a well recognized cause of genetic disease. Most known splice site mutations lie within the CAG 3'-splice acceptor site or the GT 5'-splice donor site. To date, a G+1A substitution at the 5'-splice donor site has been identified in at least 26 human diseases (10, 11, 12, 13, 14, 15, 16, 17). In these diseases, G+1A 5'-splice donor site mutations differed in the degree of splice donor site inactivation, but always resulted in abnormal splicing. Functional studies of other G+1A 5'-splice site mutations have shown either recognition of a 5'-cryptic splice site or exon skipping, and our patient suggests that this mutation similarly produces abnormal splicing and a nonfunctional enzyme.

The novel mutation found at codon 318 (T318R) in exon 5 affects the same residue as a mutation (T318M) in Yemenite cousins that completely abolished 11ß-hydroxylase activity (4). T318 represents an absolutely conserved amino acid, across all known P450 enzymes (18), and is involved in proton transfer to the bound oxygen molecule (19). The occurrence of a second mutation (T318R) at codon 318 suggests a mutational hot spot at this functionally critical site. Other mutational hot spots have been observed at codons 384 (4, 5) and 448 (3), which are also in regions of known functional importance (18). Codon 384 is thought to form part of the substrate binding pocket, whereas codon 448 is in a region known to interact with the heme prosthetic group (19).

The exon 6 nonsense mutation (Q356X) has been reported previously in an African-American (4). This patient was homozygous for the mutation, suggesting consanguineous parentage. Our patient is also African-American, but is heterozygous for the mutation, and has no history of parental consanguinity. The presence of the same mutation in ethnically similar patients suggests the possibility of a founder effect. The other previously reported mutation in our patients, the 28-bp deletion in exon 2 (3), also may indicate a founder effect, as both the original case and our patient are Caucasian.

Gene conversion refers to the changing of part of one gene to the sequence of a nearby homologous gene. We found six apparent polymorphisms in CYP11B1. Two of these polymorphisms correspond to the CYP11B2 sequence, which suggests that gene conversion may have occurred. Both of these polymorphisms were described in a previous report, but their identity to CYP11B2 sequence was not noted (20). Although gene conversion of CYP11B1 (from CYP11B2) has to our knowledge not been recognized previously, the reverse process, gene conversion of CYP11B2 (from CYP11B1), has been recognized and to date has not been associated with impaired functional activity (21). This contrasts with the most common form of CAH, 21-hydroxylase deficiency, in which most of the mutations are gene conversions from the nearby CYP21P pseudogene (22), which is transcribed but not translated (23).

In conclusion, the new mutations identified from these patients with 11ß-hydroxylase deficiency identify the first splice site mutation in CYP11B1 and suggest an additional mutational hot spot at codon 318. Expanding the knowledge of the molecular basis for 11ß-hydroxylase deficiency should facilitate new approaches to diagnosis and treatment.

	Acknowledgments

 
The authors thank Katherine Welch, M.D., of the Walter-Reed Army Medical Center (Washington DC) for her referral of patient 1.

	Footnotes

 
¹ Commissioned officers in the USPHS.

² Current address: Eli Lilly Co., Lilly Research Laboratories, Indianapolis, Indiana 46285.

Received May 15, 1997.

Revised September 10, 1997.

Accepted October 6, 1997.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Zachmann MD, Tassinari D, Prader A. 1983 Clinical and biochemical variability of congenital adrenal hyperplasia due to 11ß-hydroxylase deficiency. J Clin Endocrinol Metab. 56:222–229.[Abstract/Free Full Text]
2. Mornet E, Dupont J, Vitek A, White PC. 1989 Characterization of two genes encoding human steroid 11ß-hydroxylase (P-45011B). J Biol Chem. 35:20961–20967.
3. Geley S, Kapelari K, Johrer K, et al. 1996 CYP11B1 mutations causing congenital adrenal hyperplasia due to 11ß-hydroxylase deficiency. J Clin Endocrinol Metab. 81:2896–2901.[Abstract/Free Full Text]
4. Curnow KM, Slutsker L, Vitek J, et al. 1993 Mutations in the CYP11B1 gene causing congenital adrenal hyperplasia and hypertension cluster in exons 6, 7, and 8. Proc Natl Acad Sci USA. 90:4552–4556.[Abstract/Free Full Text]
5. Nakagawa Y, Yamada M, Ogawa H, Igarashi Y. 1995 Missense mutation in CYP11B1 (CGA [Arg-384]GGA [Gly]) causes steroid 11ß-hydroxylase deficiency. Eur J Endocrinol. 132:286–289.[Abstract/Free Full Text]
6. White PC, Dupont J, New MI, Leiberman E, Hochberg Z, Rosler A. 1991 A mutation in CYP11B1 (Arg 448His) associated with steroid 11ß-hydroxylase deficiency in Jews of Moroccan origin. J Clin Invest. 87:1664–1667.
7. White MB, Carvalho M, Derse D, O’Brien SJ, Dean M. 1992 Detecting single base substitutions as heteroduplex polymorphisms. Genomics. 12:301–306.[CrossRef][Medline]
8. Smith LM, Sanders JZ, Kaiser RJ, et al. 1986 Fluorescence detection in automated DNA sequence analysis. Nature. 321:674–679.[CrossRef][Medline]
9. Mornet E, Dupont J, Vitek A, White PC. 1989 Characterization of two genes encoding human steroid 11ß-hydroxylase (P-450_11ß). J Biol Chem. 35:20961–20967.
10. Ozkara HA, Akerman BR, Ciliv G, Topcu M, Renda Y, Gravel RA. 1995 Donor splice site mutation in intron 5 of the HEXA gene in a Turkish infant with Tay-Sachs disease. Hum Mutat. 5:186–187.[CrossRef][Medline]
11. Purandance SM, Lanyon WG, Arngrimsson R, Connor JM. 1995 Characterisation of a novel splice donor mutation affecting position +1 in intron 18 of the NF-1 gene. Hum Mol Genet. 4:767–768.[Free Full Text]
12. Nelis E, Timmerman V, De Jonghe P, Van Broeckhoven C. 1994 Identification of a 5' splice site mutation in the PMP-22 gene in autosomal dominant Charcot-Marie-Tooth disease type 1. Hum Mol Genet. 3:515–516.[Free Full Text]
13. Yong EL, Roy A, Chua KL, Ratman S, Yang M. 1994 Complete androgen insensitivity due to a splice-site mutation in the androgen receptor gene and genetic screening with single-stranded conformation polymorphism. Fertil Steril. 61:856–862.[Medline]
14. Arredondo-Vega FX, Santisteban I, Kelly S, Schlossman CM, Umetsu DT, Hershfield MS. 1994 Correct splicing despite mutation of the invariant first nucleotide of a 5' splice site: a possible basis for disparate clinical phenotypes in siblings with adensoine deaminase deficiency. Am J Hum Genet. 54:820–830.[Medline]
15. Filie JD, Orrison BM, Wang Q, Lewis MB, Marini JC. 1993 A de novo G⁺¹A mutation at eh 2(I) exon 16 splice donor site causes skipping of exon 16 in the cDAN of one allele of an OI type IV proband. Hum Mutat. 2:380–388.[CrossRef][Medline]
16. NakahashiY, Miyazaki H, Kadota Y, et al. 1993 Human erythropoietic protoporphyria: identification of a mutation at the splice donor site of intron 7 causing exon 7 skipping of the ferrochelatase gene. Hum Mol Genet. 2:1069–1070.[Free Full Text]
17. Krawczak M, Reiss J, Cooper DN. 1992 The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet. 90:41–54.[Medline]
18. White PC, Curnow KM, Pascoe L. 1994 Disorders of steroid 11ß-hydroxylase isozymes. Endocr Rev. 15:421–438.[Abstract/Free Full Text]
19. Ravichandran KG, Boddupalli SS, Hasemann CA, Peterson JA, Deisenhofer J. 1993 Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450’s. Science. 262:731–736.
20. Naiki Y, Kawamoto T, Mitsuuchi Y, et al. 1993 A nonsense mutation (TGG[TRP 116]TAG[STOP]) in CYP11B1 causes steroid 11ß-hydroxylase deficiency. J Clin Endocrinol Metab. 77:1677–1682.[Abstract]
21. Fardella CE, Hum DW, Rodriguez H, et al. 1996 Gene conversion in the CYP11B2 gene encoding P450c11AS is associated with, but does not cause, the syndrome of corticosterone methyloxidase II deficiency. J Clin Endocrinol Metab. 81:321–326.[Abstract]
22. Speiser PW, Dupont J, Zhu D, et al. 1992 Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. J Clin Invest. 90:584–595.
23. Bristow J, Gitelman SE, Tee MK, Staels B, Miller WL. 1993 Abundant adrenal-specific transcription of the human P450c21A ‘pseudogene.’ J Biol Chem. 268:1219–1224.

This article has been cited by other articles:

				N. Krone, Y. Grischuk, M. Muller, R. E. Volk, J. Grotzinger, P.-M. Holterhus, W. G. Sippell, and F. G. Riepe Analyzing the Functional and Structural Consequences of Two Point Mutations (P94L and A368D) in the CYP11B1 Gene Causing Congenital Adrenal Hyperplasia Resulting from 11-Hydroxylase Deficiency J. Clin. Endocrinol. Metab., July 1, 2006; 91(7): 2682 - 2688. [Abstract] [Full Text] [PDF]

				A. Grigorescu Sido, M. M. Weber, P. Grigorescu Sido, S. Clausmeyer, U. Heinrich, and E. Schulze 21-Hydroxylase and 11{beta}-Hydroxylase Mutations in Romanian Patients with Classic Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5769 - 5773. [Abstract] [Full Text] [PDF]

				T. Paperna, R. Gershoni-Baruch, K. Badarneh, L. Kasinetz, and Z. Hochberg Mutations in CYP11B1 and Congenital Adrenal Hyperplasia in Moroccan Jews J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5463 - 5465. [Abstract] [Full Text] [PDF]

				N. Krone, F. G. Riepe, D. Gotze, E. Korsch, M. Rister, J. Commentz, C.-J. Partsch, J. Grotzinger, M. Peter, and W. G. Sippell Congenital Adrenal Hyperplasia Due to 11-Hydroxylase Deficiency: Functional Characterization of Two Novel Point Mutations and a Three-Base Pair Deletion in the CYP11B1 Gene J. Clin. Endocrinol. Metab., June 1, 2005; 90(6): 3724 - 3730. [Abstract] [Full Text] [PDF]

				M. Hampf, N. T. N. Dao, N. T. Hoan, and R. Bernhardt Unequal Crossing-Over between Aldosterone Synthase and 11{beta}-Hydroxylase Genes Causes Congenital Adrenal Hyperplasia J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4445 - 4452. [Abstract] [Full Text] [PDF]

				O. Chabre, S. Portrat-Doyen, P. Chaffanjon, J. Vivier, P. Liakos, F. Labat-Moleur, E. Chambaz, Y. Morel, and G. Defaye Bilateral Laparoscopic Adrenalectomy for Congenital Adrenal Hyperplasia with Severe Hypertension, Resulting from Two Novel Mutations in Splice Donor Sites of CYP11B1 J. Clin. Endocrinol. Metab., November 1, 2000; 85(11): 4060 - 4068. [Abstract] [Full Text]

				S. Kitanaka, A. Murayama, T. Sakaki, K. Inouye, Y. Seino, S. Fukumoto, M. Shima, S. Yukizane, M. Takayanagi, H. Niimi, et al. No Enzyme Activity of 25-Hydroxyvitamin D3 1{alpha}-Hydroxylase Gene Product in Pseudovitamin D Deficiency Rickets, Including That with Mild Clinical Manifestation J. Clin. Endocrinol. Metab., November 1, 1999; 84(11): 4111 - 4117. [Abstract] [Full Text]

				B. I. Cerame, R. S. Newfield, L. Pascoe, K. M. Curnow, S. Nimkarn, T. F. Roe, M. I. New, and R. C. Wilson Prenatal Diagnosis and Treatment of 11{beta}-Hydroxylase Deficiency Congenital Adrenal Hyperplasia Resulting in Normal Female Genitalia J. Clin. Endocrinol. Metab., September 1, 1999; 84(9): 3129 - 3134. [Abstract] [Full Text]

				P. P. Feuillan, J. V. Jones, K. Barnes, K. Oerter-Klein, and G. B. Cutler Jr. Reproductive Axis after Discontinuation of Gonadotropin-Releasing Hormone Analog Treatment of Girls with Precocious Puberty: Long Term Follow-Up Comparing Girls with Hypothalamic Hamartoma to Those with Idiopathic Precocious Puberty J. Clin. Endocrinol. Metab., January 1, 1999; 84(1): 44 - 49. [Abstract] [Full Text]

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 Merke, D. P. Articles by Cutler, G. B. Search for Related Content PubMed PubMed Citation Articles by Merke, D. P. Articles by Cutler, G. B., Jr.