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Genetic Analysis of Japanese Patients with 21-Hydroxylase Deficiency: Identification of a Patient with a New Mutation of a Homozygous Deletion of Adenine at Codon 246 and Patients without Demonstrable Mutations within the Structural Gene for CYP21

Department of Pediatrics, School of Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-Chome, Bunkyo-ku, Tokyo 113-8519, Japan

Address all correspondence and requests for reprints to: Dr. Satomi Koyama, Department of Pediatrics, School of Medicine, Tokyo Medical and Dental University, 5-45, Yushima 1-Chome, Bunkyo-ku, Tokyo 113-8519, Japan.

Abstract

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is one of the most common inherited metabolic diseases. We studied 52 Japanese 21-hydroxylase deficiency patients corresponding to 49 families (98 chromosomes) to detect the mutations in 21-hydroxylase genes using Southern blotting, PCR-restriction fragment length polymorphism, and a direct sequencing method. Among the 52 patients (49 families), 35 patients (33 families) were diagnosed as the salt-wasting type, 12 (12 families) as the simple virilizing type, and 5 (4 families) as the nonclassical type.

Our findings were as follows. 1) The complete genotype that had homozygous or compound heterozygous mutations was determined in 43 of 49 families (87.8%). Among the remaining patients, no mutation was found in the structural gene of either allele in 3 cases, and a mutation was detected in only 1 allele in 3 cases. This means that at least 9 of 98 alleles have some unusual mutations or recombinations that we cannot detect by our method or gene defects outside of the structural gene. 2) Although the common mutation of Caucasian nonclassical patients is V281L, none of our 4 nonclassical families showed this mutation, and 3 of them had the P30L mutation at least on 1 allele. 3) We identified a putative new mutation, homozygous deletion of adenine at codon 246, in a salt-wasting patient. Although we have not analyzed the functional consequence of this mutation, it causes substitution noncoding for Met²⁵⁶ in exon 7 and premature termination of the mRNA before the heme-binding region of the P450 polypeptide, which would result in a completely nonfunctional enzyme.

CONGENITAL ADRENAL hyperplasia (CAH) is one of the most common inherited metabolic diseases and is characterized by impaired activity of one of the enzymes required for cortisol biosynthesis. Steroid 21-hydroxylase deficiency (21-OHD) is an autosomal recessive disorder that accounts for 90–95% of CAH cases (1, 2, 3, 4).

The 21-hydroxylase gene (CYP21) is located on the short arm of chromosome 6, between the human leukocyte antigen class I and class II gene clusters, in tandem with a highly homologous pseudogene (CYP21P) (5, 6). The CYP21 and CYP21P genes consist of 10 exons spanning about 3 kb of DNA and display 98% sequence homology (7, 8). Most of the mutations reported to date in Caucasians are the results of gene conversion events between the functional CYP21 gene and the pseudogene (CYP21P). Approximately 90% of cases are caused by either deletion/conversion or nine point mutations: P30L, I2g, del 8-bp, I172N, E6 cluster, V281L, 1761 ins T, Q318X, and R356W in the previous reports (9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20).

We studied 52 Japanese 21-OHD patients from 49 families to detect the mutations in 21-hydroxylase genes using PCR-RFLP and a direct sequencing method. We discuss the genetic features of Japanese patients with 21-OHD compared with Caucasian patients. We investigated the frequency of 21-OHD patients who had gene defects outside the structural gene because this was unclear from previous reports. We also reported a putative new mutation, a deletion of adenine at codon 246.

Subjects and Methods

Subjects

We studied 52 Japanese patients with 21-OHD from 49 families, including 2 pairs of siblings and 1 pair of identical twins. Their parents were not consanguineous. Their parents were also studied in 15 families. The patients were divided into 3 groups according to clinical findings: salt wasting (SW), simple virilizing (SV), and nonclassical (NC). Patients with symptoms and signs of circulatory shock, dehydration, and electrolyte disturbances (hyperkalemia; >6 mmol/liter and hyponatremia; <130 mmol/liter) were categorized as SW. However, some patients had been detected before the appearance of electrolyte disturbance by neonatal mass screening for 21-OHD. Females with ambiguous genitalia and males with sexual precocity and accelerated growth rates with no history of salt-wasting crisis were classed as SV, and the patients presenting with high serum levels of ACTH-stimulated 17-hydroxyprogesterone (17-OHP) but without any symptoms suggesting androgen excess at birth were classed as NC (4, 21, 22, 23, 24). Among the 52 patients (49 families), 35 patients (33 families) were diagnosed as the SW type, 12 as the SV type, and 5 (4 families) as the NC type. The patients were born between 1974 and 1998. Thirty-one patients (22 SW, 7 SV, and 2 NC) in this study were found by neonatal screening.

Strategy

Approximately 90% of 21-OHD cases are reported to be caused by deletion/conversion or nine-point mutations. The common point mutations are the results of gene conversion events between the functional CYP21 gene and the pseudogene (CYP21P) as follows: 1) the missense mutation substituting Leu for Pro³⁰ in exon 1 (P30L) (12), 2) the A/C-655 to G substitution in intron 2 causing aberrant splicing of mRNA (I2g) (13), 3) an 8-bp deletion in exon 3 (del 8-bp) (14), 4) the missense mutation substituting Asn for Ile¹⁷² in exon 4 (I172N) (15, 16), 5) the missense mutations substituting Asn, Glu, and Lys for Ile²³⁶, Val²³⁷, and Met²³⁹, respectively, in exon 6 (E6 cluster) (17, 18), 6) the missense mutation substituting Leu for Val²⁸¹ in exon 7 (V281L) (18, 19), 7) the insertion of T at nucleotide1761 in exon 7 (1761 ins T) (4), 8) the nonsense mutation substituting noncoding for Gln³¹⁸ in exon 8 (Q318X) (20), and 9) the missense mutation substituting Trp for Arg³⁵⁶ in exon 8 (R356W) (16). Other rare mutations (G292S, I7 splice, W406S, P453S, R483P, and R484GtoC) have also been reported previously (25).

First, we detected gene deletions and large gene conversions by the Southern blotting method, and we screened the subjects for six common mutations (P30L, I2g, I172N, V281L, Q318X, and R356W) with a PCR-RFLP method. When we found these six mutations, we confirmed them by direct sequencing. We examined subjects without the above six mutations for del 8-bp, E6 cluster, and 1761 ins T by sequencing the respective areas. Finally, for the patients without these nine mutations, the whole sequence of the CYP21 gene between 90 bp 5'-upstream of the transcription start site and the end of exon 10 was analyzed.

Southern blotting method

Gene deletions and large gene conversions were detected using the nonradioactive Southern blotting method. Genomic DNA was digested with the restriction endonucleases TaqI, BglII, and KpnI. The digests were electrophoresed on 1.0% agarose gels, transferred to nylon membranes, and hybridized with the probe specific for the CYP21 and C4 genes. The hybridization probe used was the 3.1-kb EcoRI-BamHI genomic fragment, which was labeled with [³²P]deoxy-CTP by random priming. After autoradiography, the density of DNA bands was determined using an automated densitometer (6, 11, 14, 26).

PCR-RFLP

PCR was carried out on genomic DNA isolated from peripheral blood lymphocytes by standard procedures. We used mismatched primers to detect I2g, I172N, and R356W mutations (27). These primer pairs were designed to selectively amplify the CYP21 gene only. The PCR reaction was carried out in a final volume of 20 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.2 mM of each dNTP, 1 µM of each nucleotide primer, 1 µg genomic DNA, and 2.5 U Taq DNA polymerase. Hot-start PCR was performed by using Taq Start Antibody (CLONTECH Laboratories, Inc., Palo Alto, CA) so as not to amplify the wrong products. Amplifications were carried out by initial denaturation at 94 C for 5 min, then 32 cycles at 94 C for 0.5 min for denaturation, 65 C for 0.5 min for annealing, 72 C for 0.5 min for elongation, and finally 72 C for 7 min for elongation. We then incubated 10 µl of each PCR product and 5 U of the appropriate restriction enzyme to digest. The restriction endonucleases are listed in Table 1. Electrophoresis was performed in 3–4% agarose gel containing 0.5 µg/ml ethidium bromide. We checked the lengths of the fragments as shown in Table 1 to determine whether mutations were present.

Direct sequencing of the amplified CYP21 gene was performed using an ABI PRISM 310 genetic analyzer (Perkin-Elmer Corp., Foster City, CA). The eight primer pairs for sequencing (F1-R1, F2-R2, F3-R3, F4-R4, F5-R5, F6-R6, F7-R7, and F8-R8) are listed in Table 2. The primer pairs of F1-R1, F3-R3, and F5-R5 were CYP21 specific, but F2-R2, F4-R4, F6-R6, F7-R7, and F8-R8 were not CYP21 specific. Therefore, the first PCR was carried out using F1-R3 and F3-R8 primer pairs, and the second PCR was performed using F2-R2, F4-R4, F6-R6, F7-R7, and F8-R8 primer pairs. The PCR conditions were the same as those described above. The PCR products were purified by Microcon 100 (Amicon, Inc., Beverly, MA) and sequenced with the appropriate sequencing primers (F1, R1, F2, R2, F3, R3, F4, R4, F5, R5, F6, R6, F7, R7, F8, and R8) using a BigDye Terminator Cycle Sequencing FS Reaction Kit (Perkin-Elmer Corp.). The samples were purified by Centri-Sep spin columns (Perkin-Elmer Corp.), and analyzed using an ABI PRISM 310 genetic analyzer.

We screened the subjects for deletions and large gene conversions by Southern blotting and six point mutations (P30L, I2g, I172N, V281L, Q318X, and R356W) with the PCR-RFLP method, because these mutations were reported to be common among Caucasian patients. The frequencies of these mutations in previous reports and the present study are summarized in Table 3. The total frequency of deletion, conversion, and these six mutations in the affected alleles was 79.6% in our study. The V281L mutation was not recognized. We were able to determine 36 complete genotypes in 49 families (73.5%) by this technique. Two pairs of siblings and 1 pair of identical twins had the same mutations. One sibling pair, both of whom were SW phenotypes, had I2g/Q318X, and the other pair, both of whom were NC phenotypes, had the P30L/I172N mutation. The twins, both of whom were SW phenotypes, had the I2g/Q318X mutation.

Finally, we performed direct sequencing of the whole CYP21 gene for the patients without the above-mentioned mutations, and we found three patients who had mutation of only one allele (I172N/unknown, Q318X/unknown, and Conversion/unknown) and three patients who had no mutations in either allele.

The direct sequencing also revealed a putative new mutation, a deletion of adenine at codon 246, as shown in Fig. 1. The patient had the homozygous deletion of adenine.

View larger version (92K): [in this window] [in a new window]   Figure 1. Direct sequencing analysis revealed the homozygous deletion of adenine at codon 246 in a SW patient.

Table 4 shows the relationship between genotype and phenotype in our Japanese 21-OHD patients. In this table the patients were divided into 4 groups according to predicted enzymatic activity as described in some reports (25, 26, 28, 29, 30, 31). Group 0 is composed of deletion, conversion, del 8-bp, E6 cluster, 1761 ins T, Q318X, or R356W mutations, which were predicted to have no enzyme activity in either allele. The putative new mutation, homozygous deletion of adenine at codon 246, was classified as group 0. It was expected to disrupt the normal reading frame, resulting in a truncated protein, because it causes substituting noncoding for Met²⁵⁶ in exon 7. Group A is composed of I2g in at least 1 allele predicted to have almost complete enzyme impairment (predicted degree of enzyme impairment was 0–1%) that had mutations belonging to group 0 or A in another allele. Group B is composed of I172N (predicted degree of enzyme impairment was 2%) that had group 0, A, or B mutations in another allele, and group C is composed of P30L or V281L (predicted enzyme impairment was 20–50%) that had group 0, A, B, or C mutations in another. Twelve patients belonged to group 0. Eleven of them were the SW type, but there was 1 female patient with the NC type. She was a homozygote for the E6 cluster mutation. The result of direct sequencing of this patient is shown in Fig. 2. Of the 21 patients in group A, 20 were SW. The genotype of the 1 SV patient in group A was I2g/R356W. Eight patients were found to be members of group B, and 7 of them were SV patients. The genotype of the 1 SW patient in group B was I172N/I2g. Five patients belonged to group C, and 4 were NC patients. The genotype of the SV patient in group C was P30L/E6 cluster.

View larger version (103K): [in this window] [in a new window]   Figure 2. Direct sequencing analysis revealed E6 cluster mutation in a NC patient.

We studied 52 Japanese 21-OHD patients for the mutations in 21-hydroxylase genes and investigated the relationship between genotype and phenotype. The most frequent mutation in the SW form was I2g (53.0%), followed by R356W (21.2%), whereas the most frequent mutation in the SV was I172N (50.0%). The P30L mutation was found in three of eight NC alleles (37.5%). The distribution of mutation frequencies in the Japanese population was slightly different from the reports of Caucasians as shown in Table 3 (25, 28, 30, 31, 32, 33). The frequency of V281L among Japanese patients was far lower than that in Caucasian patients, whereas the frequency of R356W in Japan was a little higher than that in Caucasians (34). The most common mutation of Caucasian NC patients was V281L, as shown in Table 3. However, none of our five NC patients showed the V281L mutation, and four of them had at least one P30L mutation on one allele. The results of our study were in concordance with those of previous studies in Japan, which reported that no NC patients showed the V281L mutation (13, 32, 35, 36).

The relationship between genotype and clinical phenotype was studied. The patients were divided into four groups according to predicted enzymatic activity, as mentioned above. Four cases showed unexpected phenotypes in our study. They were an NC patient with homozygous E6 cluster mutation in group 0, an SV patient with I2g/R356W mutation in group A, an SW patient with I172N/I2g mutation in group B, and an SV patient with P30L/E6 cluster mutation in group C. Theoretically the case with the homozygote for E6 cluster mutation should have no enzyme activity. None of the NC patients showing this genotype had been reported previously. This girl did not show ambiguous external genitalia and was not diagnosed in neonatal mass-screening, but we identified her because her mother was a 21-OHD patient (SV type). She was diagnosed at 2 months of age with a basal serum 17-OHP level of 6.12 nmol/liter, which rose to 210.2 nmol/liter when stimulated with ACTH. The reason why she is asymptomatic remains unresolved, but extraadrenal monooxygenase, which possesses 21-hydroxylase activity, might have compensated for cortisol synthesis in this patient. Only one NC patient in group 0 has previously been reported; the genotype was a compound heterozygote for del 8-bp/R356W (35). The presence of an SV patient in group A and an SW patient in group B might be conceivable because the difference between enzymatic activity in groups A and B is small. The SV patient with P30L/E6 cluster showed ambiguous external genitalia when she was born and was diagnosed in neonatal mass screening. Her 17-OHP level extracted from dried blood spots was 20.3 nmol/liter at 5 d of age and 53.6 nmol/liter at 19 d of age. She was diagnosed at 22 d of age with a basal serum 17-OHP level of 296.2 nmol/liter, serum sodium of 139 nmol/liter, and serum potassium of 5.4 nmol/liter. Her weight gain was good at 22 d of age.

We identified a putative new mutation. It was a homozygous deletion of adenine at codon 246 in an SW patient. Although we have not analyzed the functional consequence of this mutation, it was expected to disrupt the normal reading frame, resulting in a truncated protein, because it causes substituting noncoding for Met²⁵⁶ in exon 7. This is predicted to cause premature termination of the mRNA before the heme binding region of the P450 polypeptide, which results in a completely nonfunctional enzyme (8, 20). It was reasonable that the patient with the homozygote for this mutation presented the SW form.

We could not confirm any mutations in 9 of the 12 affected alleles in the 6 patients between 90 bp 5'-upstream of the transcription start site and the end of exon 10. No mutation was found in the structural gene of either allele in 3 cases, and the mutation was detected in only 1 allele in 3 cases. This indicates that some patients with 21-OHD may have a normal structural gene for 21-hydroxylase. The clinical and biological data of these patients are shown in Table 5. We speculate that these patients have unusual mutations or recombinations that we cannot detect by our method in the structural gene for CYP21 or mutations in the regulator gene for CYP21, in the binding site of the regulator gene, or in the site where the trans-acting factor combines. Three of these 6 patients were of the SW form and the others were of the SV form. This means that there are 2 or more mutations outside of the structure gene. Recently, Nimkarn et al. (37) reported a female 21-hydroxylase-deficient patient without demonstrable genetic mutations. As we found 6 patients without mutations in the structural gene, such patients might present more frequently than expected. We should investigate further the factors relating to appearance of the gene for 21-hydroxylase.

Acknowledgments

Footnotes

Abbreviations: CAH, Congenital adrenal hyperplasia; NC, nonclassical; 21-OHD, 21-hydroxylase deficiency; 17-OHP, 17-hydroxyprogesterone; SV, simple virilizing; SW, salt wasting.

Received July 14, 2001.

Accepted February 5, 2002.

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This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart 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 Google Scholar Google Scholar Articles by Koyama, S. Articles by Yata, J. Search for Related Content PubMed PubMed Citation Articles by Koyama, S. Articles by Yata, J.