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Molecular Basis of Nonclassical Steroid 21-Hydroxylase Deficiency Detected by Neonatal Mass Screening in Japan

Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health (T.Ta., G.B.C.), Bethesda, Maryland 20892; and the Department of Pediatrics, Hokkaido University School of Medicine (T.Ta., K.F., J.N.), Hokkaido; the Department of Pediatrics, Tokyo Medical and Dental University (T.To., K.S.), Tokyo; the Department of Neonatology, Osaka Medical Center (S.K.), Osaka; the Department of Endocrinology and Metabolism, Hyogo Children’s Hospital (K.G.), Hyogo; and the Department of Pediatrics, Kushiro Red Cross Hospital (T.N.), Kushiro, Japan

Address all correspondence and requests for reprints to: Toshihiro Tajima, M.D., Ph.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

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Since 1989, neonatal mass screening for congenital adrenal hyperplasia (CAH) has been performed in Japan, and the frequency of the classical form of 21-hydroxylase deficiency was found to be nearly identical to that in other countries. However, it has not yet been determined whether our mass screening program can detect the nonclassical (NC) form.

From 1991 to 1994, about 4,500,000 infants underwent CAH mass screening in Japan. During this period, we identified by screening 2 siblings and 2 unrelated patients who had mild elevation of serum 17-hydroxyprogesterone levels at 5 days of age, but who revealed no symptoms of CAH. They were diagnosed as having probable NC steroid 21-hydroxylase deficiency. To clarify the molecular basis of NC CAH detectable by neonatal screening in Japan, the steroid 21-hydroxylase (CYP21) genes from these cases were analyzed. The 2 siblings (patients 1 and 2) had I172N and R356W mutations in 1 allele and in the other allele had local gene conversion, including the P30L mutation in exon 1. Patient 3, who was unrelated, had gene conversion encoding the same P30L mutation in 1 allele and in the other allele had an intron 2 mutation (668–12 AG), causing aberrant ribonucleic acid splicing, and the R356W mutation. Patient 4, also a compound heterozygote, had the R356W and 707del8 mutations.

The estimated rate of detection of the NC form by mass screening (1:1,100,000) seemed low compare to the established detection rate for the classical form (1:18,000). As all of our 4 patients were compound heterozygotes with at least 1 allele bearing 1 or more mutations associated with classic CAH, it may be difficult to detect NC cases carrying only NC-associated alleles using our current neonatal mass screening methods.

	Introduction

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CONGENITAL adrenal hyperplasia (CAH) is one of the most common inborn errors of metabolism. Steroid 21-hydroxylase deficiency accounts for 90–95% of CAH cases (1, 2). The symptoms of steroid 21-hydroxylase deficiency are classified into three distinct clinical phenotypes: the salt-wasting (SW), simple virilizing (SV), and nonclassical (NC) forms (1, 2, 3, 4, 5, 6, 7). SW and SV forms of steroid 21-hydroxylase deficiency produce masculinization of the external genitalia in girls and, therefore, can be detected at birth in girls by careful examination of the external genitalia. By contrast, the NC form does not produce genital abnormalities in the newborn female and, thus, is not detectable at birth by clinical examination. The NC form presents later in life as premature pubarche, hirsutism, acne, or menstrual abnormalities. The NC form may also be cryptic, which denotes asymptomatic individuals with the identical biochemical and genetic abnormalities as family members with the symptomatic NC form (2, 3, 5, 6, 7).

CAH is caused by mutations in the steroid 21-hydroxylase gene (CYP21) (1, 2, 5, 6, 7, 8, 9, 10, 11, 12). The degree of enzymatic impairment caused by the different mutations in vitro correlates roughly with the clinical severity of 21-hydroxylase deficiency (9, 10, 11). In the NC or cryptic form, the most common mutations resulted in about 20–50% of wild-type enzymatic activity (P30L, V281L, P453S) (9, 11, 12, 13, 14, 15).

Mass screening programs for CAH have been available since the 1980s, and the benefits of these programs have been recognized (16, 17, 18). Newborn screening has been particularly useful in detecting affected boys, who are phenotypically normal at birth, before the occurrence of adrenal crisis (16, 17, 18). On the other hand, it is difficult to distinguish 17-hydroxyprogesterone (17-OHP) levels in filter paper samples of patients with the NC form from 17-OHP levels in normal newborns (16). However, several cases of the NC form have been detected because of elevated 17-OHP levels at neonatal mass screening (11, 16). In Japan, mass screening for CAH has been performed, and the frequency of the classical form was essentially identical to that in other countries (17, 18). However, it has not yet been determined whether our program can detect the NC form. In this report, we have determined the rate of NC CAH detection in Japan from 1991–1994 and have analyzed the mutations of the CYP21 gene in the four cases detected.

	Materials and Methods

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Neonatal mass screening

In Japan, nationwide neonatal mass screening for CAH was started in 1989 (17, 18). The newborn screening 17-OHP blood sample was collected at an average age of 5 days (17, 18). Serum 17-hydroxyprogesterone (17-OHP) was determined by an enzyme-linked immunosorbent assay with or without initial steroid extraction from the filter paper sample, followed by a second enzyme-linked immunosorbent assay after high pressure liquid chromatography or organic solvent extraction of steroid on samples with increased 17-OHP levels from the first assay (17, 18). The sensitivity and specificity of screening for classic CAH were 99.66% and 99.97%, respectively (18). The current cut-off limit is 500 ng/dL. In the present study, from 1991–1994 approximately 4,500,000 infants underwent neonatal mass screening, among whom 4 cases were identified who exhibited mild persistent elevations of serum 17-OHP without any signs or symptoms of CAH.

Molecular analysis of CYP21 gene

To determine the CYP21 gene defect, DNA from each patient was subjected to blot hybridization and direct sequencing. In each family, DNA from both parents was analyzed.

DNA blot hybridization

Genomic DNA samples were prepared from blood leukocytes by a standard procedure. DNA was digested with the restriction endonuclease TaqI or BglII. The digests were subjected to 1.2% agarose gel electrophoresis and analyzed by DNA blot analysis (10, 19). The hybridization probe used was the 3.1-kilobase EcoRI-BamHI genomic fragment, which was labeled with [³²P]deoxy-CTP by random priming. Filters were hybridized, washed, and autoradiographed as described previously (10, 19). Hybridization signals were quantitated by densitometry using densitol (DM-303 Funakoshi, Tokyo, Japan).

Nucleotide sequence analysis

The nucleotide sequence of the CYP21 gene from genomic DNA was determined directly from PCR products by the chain termination method using appropriate sequencing primers. For the PCR, we used primer pairs A-B and C-D (Fig. 1 and Table 1), specifically amplifying the CYP21 gene, as described previously (19). The sequences of the oligonucleotide primers used for PCR and sequencing are shown in Table 1. Thirty-five cycles of PCR at 94 C for 60 s for denaturation, at 55 C for 60 s for annealing, and at 72 C for 240 s for polymerization were run using the Expand Long Template PCR system (Boehringer Mannheim, Mannheim, Germany). The PCR products were purified on 1.0% agarose gel and sequenced with appropriate internal sequence primers by automated DNA sequencing employing Taq DyeDeoxy sequencing reagents (Applied Biosystems, Foster City, CA; Fig. 1 and Table 1). The ambiguity of sequence was resolved and the presence of mutations was confirmed by direct manual sequencing with internal sequence primers using Sequenase (U.S. Biochemical Corp., Cleveland, OH) (19).

View larger version (8K): [in this window] [in a new window]   Figure 1. Specific amplification of CYP21 and sequencing using oligonucleotide primers. Black boxes represent the exons. Oligonucleotide primers A, B, C, and D for PCR are indicated by arrows. Primers B and C have sequences specific for the CYP21 gene. TaqI indicates the restriction site of TaqI. Oligonucleotide primers a, b, c, d, e, f, g, h, i, j, k, and l indicate the primers for sequencing.

	Results

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The endocrine evaluation and clitoral index (20) of the four patients who had normal genitalia at birth and mild elevation of serum 17-OHP levels at the age of 5 days are shown in Table 2. Patient 2 was the elder brother of patient 1. When he was evaluated at 4.0 yr of age, his bone age was 3.0 yr and his height was 98.7 cm (-1.1 SD for Japanese children); he has been asymptomatic. His growth pattern was normal (Fig. 2A). Patients 1, 3, and 4 also have remained asymptomatic to age 2 yr, and each has grown normally (Fig. 2, B–D).

View larger version (33K): [in this window] [in a new window]   Figure 2. Growth pattern in each patient. A, Patient 2; B, patient 1; C, patient 3; D, patient 4. The upper line indicates +2 SD, and the lower line indicates -2 SD for normal Japanese height. All four patients have grown normally.

Mutations of the CYP21 gene

DNA blot hybridization from these patients using restriction enzymes TaqI and BglII revealed that the ratio of the CYP21P gene and that of the CYP21 gene were approximately equal (data not shown).

Patients 1 and 2

Direct sequence analysis of DNA from this family revealed that one allele transmitted from the mother bore two missense mutations of AAC (Ile) to ATC (Asn), I172N, in exon 4 (Fig. 3A) and CGG (Arg) to TGG (Trp), R356W, in exon 8 (Fig. 3B). The sequence of PCR fragments amplified by primer pair A-B from patients 1 and 2 and their father contained several heterozygous nucleotide changes from -209 of the 5'-region (Fig. 3C). All of these changes correspond to the only differences found between the CYP21P and CYP21 genes in this portion, indicating a local gene conversion event. Sequencing of the same PCR fragments, including the region of gene conversion, revealed that intron 2 had only the CYP21 gene sequence. Thus, the boundary of gene conversion could not be determined exactly; however, the breakpoint was located upstream of intron 2 (Fig. 3D). Among these changes, CCG (Pro) to CTG (Leu) at codon 30 in exon 1 (P30L) produced the only alteration in the amino acid sequence (Fig. 3C). Finally, patients 1 and 2 were found to be compound heterozygotes for the mutant maternal and paternal alleles despite their lack of symptoms of CAH (Fig. 3E).

View larger version (50K): [in this window] [in a new window]   Figure 3. Nucleotide sequence of patients 1 and 2 and their parents. A, Sequences around the I172N mutation site in exon 4. Arrows indicate the positions of the mutation. There are normal A and mutant T bases in patients 1 and 2 and their mother. B, Sequences around the R356W mutation site in exon 8. Patients 1 and 2 and their mother were heterozygous for the exon 8 mutation site (C and T nucleotides). C, Sequences around the P30L mutation site in exon 1. Patients 1 and 2 and their father had both normal C and mutant T nucleotides. D, Gene structure of patients 1 and 2. Dotted and checkered portions, CYP21P gene; black portions, normal sequence of the CYP21 gene. E, Pedigree analysis of the family of patients 1 and 2. Checkered, Gene conversion including exon 1 (P30L); striped, I172N; black, R356W.

Patient 3 was found to carry, on one allele transmitted from the mother, both the R356W mutation in exon 8 and one base substitution causing aberrant ribonucleic acid splicing in intron 2 (668–12 AG; Fig. 4A). In the other allele, transmitted from the father, a local gene conversion involving exon 1, which was similar to that found in the father of patients 1 and 2, was detected (Fig. 4, B and C).

View larger version (26K): [in this window] [in a new window]   Figure 4. Nucleotide sequence analysis of patient 3. A, Patient 3 and her mother had the heterozygous 668–12 AG mutation. B, Gene structure of patient 3. Dotted and checkered portions, CYP21P gene; black, normal sequence of the CYP21 gene. C, Pedigree analysis of the family of patient 3. Checkered, Gene conversion including exon 1 (P30L); striped, 668–12 AG mutation; black, R356W.

In patient 4, an 8-bp deletion in exon 3 (707del8) and the R356W mutation were detected. Family analysis revealed that the father carried the R356W mutation, and the mother carried the 707del8 mutation (as heterozygotes).

	Discussion

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Previously, Pang et al. reported that 17-OHP levels in filter paper samples of patients with the NC form were usually not distinguishable from those in normal newborns (16). However, they described 4 cases with NC CAH revealing elevated 17-OHP by neonatal screening, and Wedell et al. also reported 5 cases with NC showing elevated 17-OHP at neonatal screening (11, 16). In the present study, over 4 yr approximately 4,500,000 infants underwent screening, and 4 subjects showed slightly elevated 17-OHP with absence of symptoms of CAH. Their 17-OHP levels, which were 600-1900 ng/dL at screening, increased to above 4000 ng/dL by 6 weeks of age. By clinical course and endocrine evaluation, they were diagnosed as having NC steroid 21-hydroxylase deficiency. However, their 17-OHP levels at neonatal screening overlaps those of the classical form, as in Wedell’s report (11, 18). From our study, the estimated rate of detection of NC CAH by neonatal screening was about 1 in 1,100,000. In Japan, the precise frequency of NC CAH has yet to be determined, but the frequency is considered to be lower than that in western countries (10, 19). This may explain why our detection rate for NC CAH is lower than that of a previous report (16). Nonetheless, considering the low frequency of detection of NC CAH by our screening program and in view of the large rise in serum 17-OHP in our NC patients during the first 6 weeks of life, a large number of affected individuals may well have been missed.

Regarding the genotype of NC patients detected by neonatal mass screening, Wedell et al. determined the mutation of 18 cases of NC, 5 of whom showed elevated screening 17-OHP levels (11). These 5 subjects had at least 1 V281L, P30L or P453S mutation in 1 allele, but the researchers did not specify which combination of mutations their patients had (11). Therefore, to clarify the molecular basis of the cases with NC detectable by our neonatal screening program, we analyzed the genotypes of our 4 patients. Two siblings (patients 1 and 2) were found to have 1 allele that had 2 mutations (I172N and R356W) and another allele with local gene conversion, including the P30L mutation. Patient 3 was also a compound heterozygote, carrying the P30L mutation in 1 allele and the 688–12 AG and R356W mutations in the other allele. By previous in vitro expression studies, the R356W mutation is known to completely abolish enzymatic activity and, in its homozygous form, is associated with the SW phenotype (8, 9, 10, 11, 12). The I172N mutation has been shown to result in partial activity and is associated with the SV phenotype (8, 9, 10, 11, 12). The splicing junction mutation of patient 3 can give rise to both the SW and SV phenotypes (8, 9, 10, 11, 12). On the other hand, the P30L mutation, which has about 50% of wild-type enzyme activity in vitro, was associated with the NC or cryptic phenotypes (9, 11, 13). As compound heterozygotes typically exhibit phenotypes corresponding to the less severely affected allele, the presence of one allele carrying the P30L mutation in patients 1, 2, and 3 would predict a NC late-onset or cryptic phenotype and thus would explain their absence of symptoms at birth (and through age 4 yr in patient 2). Consistent with their intermediate biochemical abnormalities after ACTH stimulation on the 17-OHP nomogram, they had 1 NC allele associated with a second allele bearing 2 more deleterious mutations associated with the classical phenotype (21).

Unexpectedly, patient 4 was a compound heterozygote for R356W and 707del8. Previous in vitro expression studies have shown these mutations to have no enzymatic activity, and thus, compound heterozygotes with these mutations would be predicted to have the severe SW phenotype (9, 10, 11). Notwithstanding this prediction, patient 4 has not shown any signs or symptoms of CAH from birth up to the age of 2 yr. Further, her basal 17-OHP level was similar to those in patients 1, 2, and 3, but her ACTH-stimulated 17-OHP level fell within the classical range, corresponding to the SV form on the 17-OHP nomogram (4). Thus, the explanation for why her phenotype, biochemical abnormalities, and genotype are discordant remains unclear, although two previous reports have also described less severe phenotypes than were predicted by the genotype (9, 22). Perhaps some differences in phenotype expression are governed by factors remote from the CYP21 locus (22). For example, several reports have shown that 21-hydroxylase activity is present in extraadrenal organs, where it is associated with a different enzyme from the steroid 21-hydroxylase expressed in the adrenal gland (23, 24). Alternatively, there may be genetic differences in the conversion of 17-OHP into active androgens or in tissue responsiveness to androgens. The basis of such differences in phenotype must be studied further.

As mentioned above, our first three cases (patients 1, 2, and 3) had one NC-associated (P30L) mutation on one allele and two classical-associated mutations on the other allele. Patient 4 had two classical-associated mutations. Considering our results, neonatal mass screening as currently performed may miss subjects who carry only NC-associated mutations on both alleles. Further, in our four cases of NC, three subjects had the P30L mutation. In western countries, more than 60% of NC cases had the V281L mutation, whereas in Japan, including a previous study, no case with NC has had the V281L mutation (9, 11). Thus, the P30L mutation appears more likely to be associated with Japanese NC patients than the V281L mutation, although this conclusion will need to be confirmed in a larger number of patients.

In conclusion, our study demonstrates the low detection rate by neonatal mass screening of NC CAH in Japan and suggests that detection by neonatal screening may be particularly difficult for NC cases in which both alleles contain only NC-associated mutations.

Received July 25, 1996.

Revised December 27, 1996.

Revised April 9, 1997.

Accepted April 11, 1997.

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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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Tajima, T. Articles by Cutler, G. B. Search for Related Content PubMed PubMed Citation Articles by Tajima, T. Articles by Cutler, G. B., Jr.