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Carrier Analysis and Prenatal Diagnosis of Congenital Adrenal Hyperplasia Caused by 21-Hydroxylase Deficiency in Chinese¹

Department of Medical Research (H.-H.L., J.-G.C., C.-H.T.), Division of Molecular Medicine, China Medical College Hospital, Taichung 404; Department of Obstetrics and Gynecology (H.-T.C.), Veterans General Hospital-Taipei, Taipei 112; Department of Pediatrics (Y.-J.L.), Mackay Memorial Hospital, Taipei 104; and Institute of Molecular Biology (B.-c.C., J.-M.K.), Academia Sinica, Nankang, Taipei 115, Taiwan

Address correspondence and requests for reprints to: Hsien-Hsiung Lee, Department of Medical Research, Division of Molecular Medicine, China Medical College Hospital, No. 2, Yuh Der Rd, Taichung 404, Taiwan. E-mail: hhlee{at}hpd.cmch.org.tw

	Abstract

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Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. We screened 1,000 healthy people, using a previously developed differential PCR method combined with single-strand conformation polymorphism and amplification-created restriction site methods for the carrier detection of the CYP21 gene deficiency. Our results indicated that the rate of occurrence of the heterozygous CAH carrier was about 12 in 1,000, with a gene frequency of 0.0060 and an incidence frequency of 1 in 28,000 in the Chinese population. In addition, 9 cases of CAH families were performed with prenatal diagnosis. Among them, 3 cases were diagnosed as the severe form, 4 cases carried the heterozygous mutation, and 2 were normal. This is the first report of carrier frequency analysis and prenatal diagnosis of 21-hydroxylase deficiency in Chinese.

	Introduction

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CONGENITAL adrenal hyperplasia (CAH) is a common autosomal recessive disorder mainly caused by defects in the steroid 21-hydroxylase (CYP21) gene. Deficiency of 21-hydroxylase prevents synthesis of cortisol and aldosterone, major glucocorticoids, and mineralocorticoids. Thereafter, it finally leads to an excess production of androgens. The wide range of CAH phenotypes is associated with multiple mutations known to affect 21-hydroxylase enzyme activity. Two forms of CAH are classified into the classical form with ambiguous genitalia and the milder nonclassical form, which is usually late-onset. The classical form is again divided into the simple virilizing and the salt-wasting types (1, 2).

More than 90% of cases of CAH are caused by mutation of the CYP21 gene (3, 4, 5). Most mutations of the CYP21 gene identified so far are attributable to conversion of DNA sequences from its neighboring duplicated CYP21P gene (6, 7, 8). The most common mutations of CYP21 deficiency in Chinese Taiwanese occur in 3 loci: intron 2 nucleotide (nt) 656 (nt656G), codon 172 (I172N), and codon 356 (R356W) (9, 10). The most frequent mutations in Chinese were nt656G (36.7%; 69 of 188 chromosomes), I172N (19.7%; 37 of 188 chromosomes), and R356W (12.8%; 24 of 188 chromosomes) in our recent study (unpublished analysis). The mutation of codon 318 (Q318X) was 2.1% (37 of 188 chromosomes). The 4 mutations together covered 71.3% of CYP21 deficiency in our population. In addition, 4 novel mutations not attributable to gene conversion and cross-over from the neighboring CYP21P pseudogene were also detected in our population (11). In these cases, the rate of new mutations not related to gene cross-over events was approximately 5% (6 of 130 chromosomes) (11). To estimate the carrier rate frequency in our population, we set up a strategy to screen normal individuals for CYP21 gene deficiency. Blood samples were collected randomly from 1000 healthy men and women at the time of a health examination in the outpatient department of the hospital. Molecular detection of the CYP21 gene was analyzed directly, because this method is superior to the 17-hydroxyprogesterone (17-OHP) assay. It is difficult to distinguish between 17-OHP levels of normal individuals and carriers. The method of differential PCR of the CYP21 gene, followed by amplification-created restriction site (ACRS) and single-strand conformation polymorphism (SSCP) analysis (10, 11), were first applied to screen and estimate the carrier rate in the Chinese population. In addition, prenatal diagnosis was also performed in 9 CAH families using amniotic cells or chorionic villi.

	Materials and Methods

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Subjects

For carrier analysis in our population, 1000 blood samples, mainly used for biochemical testing at the time of a health examination, were obtained from Veterans General Hospital-Taipei. These samples included 528 men and 472 women. The Institute Review Board of Veterans General Hospital-Taipei approved the protocol, and the study strictly followed their guidelines. For prenatal diagnosis, 9 CAH families with at least 1 child affected by CAH asked for prenatal diagnosis during their next pregnancy. One of the samples came from China, with DNA extracted from amniotic cells (see Table 3, family 9). The other 8 CAH families were from Taiwan. Seven were examined with amniocentesis and 1 by chorionic villus sampling (see Table 3). To avoid maternal contamination, these samples were cultured before DNA extraction.

Fresh blood samples and cultured cells were used for DNA preparation. The method of DNA extraction has been described previously (10).

Molecular analysis of the CYP21 gene

Because of the high homology between the CYP21and CYP21P genes, a strategy to amplify the CYP21 gene differentially was used. After the differential PCR amplification, the primary PCR product was used as a template for secondary PCR amplification. Two methods, the ACRS method and SSCP analysis, were applied to analyze the secondary PCR product. The reaction conditions of ACRS and SSCP have been described previously (10, 11). The primers used for differential PCR of the CYP21 gene and secondary PCR of ACRS are listed in Table 1. Primers of S4 and S8 for SSCP analysis have been described previously (11).

	Results

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The major cause of mutations in CAH families requesting prenatal diagnosis was three common mutations of nt656G (Table 3, families 1, 3, 4, 5, 6, 8, and 9), I172N (Table 3, family 2), and R356W (Table 3, 1 and 2). In addition to the common mutations, we found a novel mutation at codon 316 in family 4. This mutation caused amino acid alteration from ArgCGA->StopTGA (11). Furthermore, a chimeric CYP21P/CYP21 gene was also detected in two CAH families (Table 3, families 8 and 9) by our recently developed method (submitted for publication). In both families, there is a large gene conversion changing the 5'-region of the CYP21 gene into CYP21P, resulting in the chimeric CYP21P/CYP21 gene.

	Discussion

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In this report, we analyzed the CYP21 gene from 1,000 healthy individuals to calculate the carrier rate in Chinese. Based on the experience gained from the previous studies (10, 11), we focused our analysis on the mutations at 3 loci from 1,000 samples. From 1995 to 1998, we had analyzed 65 CAH families (11), from which 3 common mutations, nt656G, I172N, and R356W, were identified in our population. As indicated, the most frequent findings (130 chromosomes) were intron 2 nt 656 = 41.5%, I172N = 22.3%, and R356W = 15.3%. The mutation of codon 318 (Q318X) was 0.7% (130 chromosomes). After analyzing current (188 chromosomes) and past data (130 chromosomes), the 4 mutations totally covered between 71.3 and 79.2% CYP21 deficiency in our population. Thus, based on the 9 healthy persons identified as carriers in the screening of 1,000 people, the carrier rate for CAH is between 12.6 and 11.2 per 1,000. In other words, the carrier rate is 1 in 83 (12 in 1,000 persons). The calculated gene frequency is 0.0060, and incidence is estimated to be 1 in 28,000 for CAH in our population. In addition, the allele frequency of both the intron 2 mutation, a salt-wasting form, and I172N for the simple virilizing form was 0.001 and 0.0025, respectively (Table 2), slightly lower than 0.006 for the white population and 0.0041 worldwide (12). Furthermore, we again found the allele frequency for Q318X at 0.001 (Table 2), which is the first data showing the mutation rate in Chinese. We do not know why we did not find the R356W mutation. Probably, more samples need to be screened to get an accurate estimation of the R356W mutation rate. The incidence frequency of 1: 28,000 in the Chinese is slightly lower than the rate of 1 in 15,800 in Japanese and 1 in 14,199 for the world population (13, 14).

Many factors, using traditional methods to detect 21- hydroxylase deficiency, contribute to diagnostic errors. Because there is high variability in the basal level of 17-OHP between normal and heterozygous individuals (15) and because there is a recombination or haplotype shared between parents in the human leukocyte antigen genetic marker and linked microsatellite markers, diagnostic error can occur. To diagnose more accurately, direct molecular detection of the CYP21 gene is necessary and practical. In conclusion, using the method we developed to rapidly detect gene mutation, we were able to determine the carrier rate for CAH in the Chinese population. We also performed prenatal diagnosis for CAH families. This information will be important for the understanding of molecular defects in Chinese.

	Footnotes

 
¹ This work was supported by Grant DOH88-HR-609 from the National Health Research Institute of the Republic of China and Grant 291 from the Veterans General Hospital-Taipei of the Republic of China.

Received September 13, 1999.

Revised October 21, 1999.

Accepted November 1, 1999.

	References

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