21-Hydroxylase deficiency-induced congenital adrenal hyperplasia in 230 Chinese patients: Genotype–phenotype correlation and identification of nine novel mutations

doi:10.1016/j.steroids.2016.01.007

Steroids

Volume 108, April 2016, Pages 47-55

https://doi.org/10.1016/j.steroids.2016.01.007 Get rights and content

Highlights

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The first large cohort of CYP21A2 gene mutations in 230 Chinese patients.
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Identification of nine novel mutations of CYP21A2 gene and prediction of their effects.
•
Genotype and phenotype correlation to provide more evidence for clinical diagnosis.

Abstract

Steroid 21-hydroxylase deficiency (21-OHD) caused by the CYP21A2 gene mutations accounts for more than 90% of congenital adrenal hyperplasia (CAH) cases. In this study, molecular defects of 230 patients with 21-OHD were investigated. Point mutations of CYP21A2 gene were analyzed by Sanger sequencing, and large gene deletions were detected by multiplex ligation-dependent probe amplification (MLPA). Nine micro-conversions and 18 spontaneous mutations accounted for 74.6% of alleles, while large gene deletions and large gene conversions accounted for 25.4% of alleles. The most frequent micro-conversion was c.292-13A/C>G (I2G) (35%), followed by p.I173N (14.3%), p.R357W (5.9%) and p.Q319^∗ (4.6%). Nine novel mutations were identified in these patients, which were predicted to hamper the 21-hydroxylase protein function in varying degrees. Genotype and phenotype correlated well in 89.6% of our patients, but disparity in phenotypic appearance also appeared in a small portion of the patients. 16.1% of the patients carried homozygous genotypes while 83.9% of patients carried compound heterozygous mutations. We concluded that the frequency of CYP21A2 mutations in our study was slightly different from those reported for other ethnic groups. Micro-conversions were the main category of the mutation spectrum, while large deletions and large gene conversions could also cause 21-OHD. A large portion of different types of the compound heterozygous genotypes may partially contribute to the discordance in genotype–phenotype comparison. This study expanded the CYP21A2 mutation spectrum of Chinese patients and could be helpful in prenatal diagnosis and genetic counseling for 21-OHD patients.

Introduction

Congenital adrenal hyperplasia (CAH; OMIM# 201910) is one of the most frequent autosomal recessive disorders. More than 90% of the CAH cases are caused by steroid 21-hydroxylase deficiency due to the CYP21A2 gene mutations. Enzymatic deficiency of steroid 21-hydroxylase can result in inadequate cortisol and aldosterone synthesis and excessive androgen synthesis that leads to the clinical manifestations of adrenal insufficiency and hyperandrogenism [1]. According to the varying degrees of 21-hydroxylase enzymatic activity, the clinical manifestations can be divided into three categories: classic salt-wasting (SW) form with complete enzyme deficiency, classic simple virilizing (SV) form with partial enzyme defect, and nonclassic (NC) form due to the mildest deficiency [2].

The 21-hydroxylase gene (CYP21A2) is located on the short arm of chromosome 6 (6p21.3), approximately 30 kb apart from its pseudogene CYP21A1P. To date, more than 250 different mutant alleles have been identified and listed in the Human Gene Mutation database (HGMD) (http://www.hgmd.cf.ac.uk/ac/index.php). According to previous studies, more than 90% of these mutations result from recombinations between CYP21A2 and CYP21P [3]. Among these recombination-caused mutations, approximately 20% are gene deletions caused by unequal crossing over during meiosis, while the remaining 70% are caused by the transferring of CYP21P into CYP21A2. The latter form was termed “gene conversion” which happened during mitosis. Sanger sequencing of CYP21A2 gene could identify point mutations while multiplex ligation-dependent probe amplification (MLPA) is proved to be an effective method for detecting large gene deletions [3], [4]. Gene deletions and conversions could be distinguished by these two methods.

The prevalence and mutation spectrum of CYP21A2 gene were reported in different ethnic groups. Among Chinese population, the CYP21A2 gene alterations had already been reported in Taiwan [5] and Hong Kong [6]. However, the prevalence and mutation spectrum of CYP21A2 gene in mainland China still remain to be clarified [7], [8]. The genotype–phenotype correlation in 21-OHD patients had long been studied. A few common mutations were thought to correlate well with specific phenotypes [9]. For example, p.Q319^∗ and p.R357W resulted primarily in SW phenotype, while c.292-13A/C>G (the intron 2 splice site mutation, I2G) and p.I173N could result in both SW and SV phenotypes. However, these mutations above had also been reported in cases with unexpected phenotype [9], suggesting disparities in genotype–phenotype correlation. In this study, we investigated the mutational spectrum of CYP21A2 gene in 230 Chinese CAH patients and analyzed the genotype–phenotype correlation, providing a genetic and clinical profile of CAH in the Chinese population.

Section snippets

Patient data

A total of 230 unrelated Chinese CAH patients (131 males and 99 females) were recruited in our pediatric clinic from 2009 to 2014. The diagnoses of 230 patients with CAH were confirmed by molecular genetic testing. In addition, for other 27 patients, no mutant allele was found in 23 patients, and only one mutant allele was found in 4 patients. These 27 individuals were not included in this study, as they either had interrupted follow-up or lacked clinical data. As a result, the overall

Patients

The clinical information and hormone data of the 230 21-OHD patients were shown in Table 1 (detailed information for each patient was listed in Supplementary Table 2). One hundred and sixty-one patients with presenting the classic forms of CAH (101 SW and 60 SV) were identified when they showed adrenal crisis or ambiguous genitalia during a time span from 20 days to 14 years. In the remaining 69 patients, diagnosis was made by molecular and biochemical studies following the indication of neonatal

Discussion

In this study, the phenotypes of 230 CAH patients were analyzed and subcategorized. Two hundred and two patients were found to have classic forms and no patient presented NC form. SW and SV types of the classic form comprised a major portion of the patients based on literature, and similar results were also observed in our study. However, the fact that we did not observe any NC form in our study group was inconsistent from observations in other ethnic groups. The occurrence rate of NC form was

Conflicts of interests and financial disclosure

Nothing to declare.

Acknowledgments

We are grateful to all the participation of the patients and their family members. We also express our gratitude to all of the pediatricians who helped with the study. This study was supported by grants from the national key technology R&D program (2012BAI09B04), the special basic work of science and technology (2014FY110700) and the “Innovation Fund for Translational Medicine Plan” from the Shanghai Jiao Tong University School of Medicine (No.15ZH3003, to YYG).

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Cited by (47)

Genetic analysis and novel variation identification in Chinese patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency
2022, Journal of Steroid Biochemistry and Molecular Biology
Citation Excerpt :
The frequency of common micro-conversions of CYP21A2 gene varies greatly among different ethnic groups, and the disparity between genotype and phenotype is also reported in different races and regions. Among Chinese population, the CYP21A2 gene alterations had already been reported in Taiwan[10], Hong-Kong[6] and Chinese mainland [6,9,11,12]. However, the mutation spectrum of CYP21A2 gene in Chinese populations still remain to be clarified, due to the small sample size of each single study.
Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency is an autosomal-recessive disorder caused by mutations in the CYP21A2 gene. The aim of the study was to analyze the molecular data of 155 21-OHD patients and retrospectively investigated the common allelic mutations of CYP21A2 in 1442 Chinese 21-OHD patients. Clinical features and mutations of CYP21A2 gene in 155 unrelated 21-OHD patients were examined. Of the 155 patients, 103 cases were salt-wasting (SW) forms, 38 were simple virilizing (SV) forms and 14 were non-classical (NC) forms. In general, two types of mutations including common allelic mutations (281/310, 90.6%) and rare mutations (29/310, 9.4%) were detected, among them four novel variants c.835G>T, c.1081C>T, c.1423C>T and c.651 + 2 T > G were identified. In 1442 Chinese 21-OHD patients, the most frequently mutations were I2G (36.2%), large deletion/conversion (20.7%) and p.I173N (17.8%), while p.V282L has the lowest frequency. In this study, we provided detailed clinical data and mutation spectrum in Chinese 21-OHD patients. Moreover, four novel CYP21A2 variants (c.835G>T, c.1081C>T, c.1423C>T and c.651 +2 T > G) were identified and computational structural modeling indicated that these novel variations probably affect structural stability. Our findings improve the understanding of CYP21A2 mutational spectrum and contribute to the precise diagnosis and prenatal counseling.
Assessing the risk of having a child with classic 21-hydroxylase deficiency: a new paradigm
2021, Trends in Endocrinology and Metabolism
Citation Excerpt :
Based on the level of enzyme dysfunction, 21OH mutations are classified as ‘mild’ or ‘severe’ mutations. Although some overlapping exists, there is a high genotype–phenotype correlation [2,4]. Classic 21OH-def occurs only in patients harboring ‘severe’ mutations on both alleles [3].
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is a complicated condition genetically, clinically, and treatment wise. Genetically, there are numerus mutations with different effect on enzyme activity that make genetic diagnosis a challenge. Clinically, there are a wide range of presentations from asymptomatic patients to the severe life-threatening classic CAH. Both an asymptomatic heterozygote and a mildly affected non-classical patient can carry a ‘severe’ mutation and endow it to their offspring. We present a case of non-classic CAH and discuss the problematic relations between biochemical and genetic diagnosis. By integrating the seemingly contradicting literature, we provide a new simple tool to assess the risk of such patients to give birth to a child with classic CAH.
CYP21A2 mutations in pediatric patients with congenital adrenal hyperplasia in Costa Rica
2021, Molecular Genetics and Metabolism Reports
Steroid 21-hydroxylase deficiency accounts for 95% of congenital adrenal hyperplasia (CAH) cases. Newborn screening has allowed for early detection of the disease, and currently, molecular analysis can identify the genotypes of these patients. Phenotype-genotype correlation has been well described in previous studies. In Costa Rica, there is no data about the genetic background of these patients, nor their phenotypic correlation.
Observational, retrospective, descriptive study based on the review of patient records who had a diagnosis of CAH and were performed molecular analysis using gene sequencing or MLPA during the period from 2006 to 2018 (N = 58).
To describe the clinical and genetic characteristics of CAH patients due to 21-hydroxylase deficiency at the National Children's Hospital “Dr. Carlos Sáenz Herrera”, Caja Costarricense de Seguro Social (CCSS) in Costa Rica.
53% (31/58) of the patients were male and 80% (37/46) were born full term; 72% (42/58) had salt wasting phenotype, 9% (5/58) simple virilizing phenotype and 19% (11/58) non-classic phenotype. The most frequent variants were c.292+5G>A in 26% (15/58) of patients and Del/Del in 21% (12/58) of them.
The most frequent mutation in our study population was the c.292+5G>A, which was found in 15/58 patients. This rare variant has only been reported in three other studies so far but as an infrequent mutation in CAH patients. The genetic characteristics of Costa Rican patients differ from what has been documented worldwide and could respond to a founder effect.
Urinary steroidomic profiles by LC-MS/MS to monitor classic 21-Hydroxylase deficiency
2020, Journal of Steroid Biochemistry and Molecular Biology
21-hydroxylase deficiency, the most common enzyme defect associated with congenital adrenal hyperplasia (CAH) is characterized by an impairment of both aldosterone and cortisol biosynthesis. Close clinical and biological monitoring of Hydrocortisone (HC) and 9α-Fludrocortisone (FDR) replacement therapies is required to achieve an optimal treatment. As frequent and repeated reassessments of plasma steroids, 17-hydroxyprogesterone (17-OHP), androstenedione (Δ4-A) and testosterone (TESTO) is needed in childhood, urine steroid profiling could represent an interesting non-invasive alternative.
We developed and validated a LC-MS/MS method for the measurement of 23-urinary mineralocorticoids, glucocorticoids and adrenal androgens. The usefulness of steroid profiling was investigated on single 08h00 am-collected spot urine for discriminating between 61 CAH patients and their age- and sex-matched controls. CAH patients were split into two groups according to their 08h00 am-plasma concentrations of 17-OHP: below (controlled patients, n = 26) and above 20 ng/mL (uncontrolled patients, n = 35).
The lower limit of quantification and the wide analytical range allows to assay both free and total concentrations of the main urinary adreno-corticoids and their tetra-hydrometabolites. Extraction recoveries higher than 75% and intra-assay precision below 20% were found for most steroids.
Urinary steroids upstream of the 21-hydroxylase defect were higher in uncontrolled CAH patients. Among CAH patients, plasma and urinary 17-OHP were closely correlated. As compared to controls, steroids downstream of the enzyme defect collapsed in CAH patients. This fall was more pronounced in controlled than in uncontrolled patients. Androgens (Δ4-A, TESTO and the sum etiocholanolone + androsterone) accumulated in uncontrolled CAH patients. A strong relationship was observed between plasma and urinary levels of androstenedione. Daily doses and urinary excretion of both FDR and HC were similar in both CAH groups. Urinary FDR was inversely related to the sodium-to-potassium ratio in urine. A partial least squares discriminant analysis model allowed to classify the patient’s classes unaffected, controlled and un-controlled CAH patients based on urinary steroidomic profiles.
Our LC-MS/MS method successfully established steroid profiling in urine and represents a useful and non-invasive tool for discriminating CAH patients according to treatment efficiency.
Analysis of phenotypes and genotypes in 84 patients with 21-Hydroxylase deficiency in southern China
2019, Steroids
Citation Excerpt :
Similar mutational spectrum with our study were also reported in other countries [31–34], suggesting that no significant differences in the frequency of the main micro-conversions in 21-OHD patients were present between southern Chinese and various populations of the world. The frequency of large gene deletions and conversions in our study (13.7%) was lower compared with other ethnic populations (e.g., 30.5% in America, 45% in Britain, 32.2% in Sweden and 27.4% in South Germany) [19,33,35,36], but similar findings were reported for Chinese and other Asian populations (3.6–27%) [6,11–15,32,37]. Ethnic variations might explain these results while the inconsistencies in the detecting methods and insufficient number of samples in our study might also contribute to this difference.
21-hydroxylase deficiency (21-OHD) caused by mutation in CYP21A2 gene is the most common form of Congenital adrenal hyperplasia (CAH). This study aimed to analyze the gene mutation frequency and the phenotype-genotype correlation of 21-OHD patients from southern China.
The clinical features, laboratory tests and gene mutational analysis of 84 patients with 21-OHD were retrospectively investigated. Subsequently, the correlation between phenotypes and genotypes of these patients was analyzed.
59 of 84 cases of 21-OHD (70.2%) were classified as salt-wasting (SW) forms presenting adrenal crisis or other signs of salt loss at the age between neonatal period and 2 months, and other 25 cases were classified as simple virilizing (SV) forms. Mutations of CYP21A2 gene on both alleles were found in all 84 patients (168 alleles). The most common types of mutations included micro-conversions (129/168, 76.8%), large gene conversions and deletions (23/168, 13.7%), and bona fide point mutations (16/168, 9.5%). In increasing order of frequency, the most common micro-conversions were I2G (41.1%), p.I172N (13.1%), p.R356W (7.7%), p.Q318* (7.7%) and E6 Cluster (3.0%). Genotypes and phenotypes correlated in 86.1% of the patients analyzed.
Micro-conversions were the most common types of CYP21A2 gene mutations in our study, and the frequency of the identified mutations was not significantly different compared with most other Chinese areas and different ethnic regions. However, fewer large gene conversions and deletions were found compared to studies in other ethnic populations. Genotype-phenotype correlation was found in patients with the SW and SV forms of 21-OHD. This study expanded the number of mutations affecting CYP21A2 gene in Chinese patients with 21-OHD, providing additional information for a precise clinical diagnosis and genetic counseling.
Novel non-classic CYP21A2 variants, including combined alleles, identified in patients with congenital adrenal hyperplasia
2019, Clinical Biochemistry
Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH.
All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed.
The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40–82%). An additive effect on the reduction of enzymatic activity (1–17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro.
Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH.

View all citing articles on Scopus

¹: These authors contributed equally to this work, and should be considered as co-first author.

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21-Hydroxylase deficiency-induced congenital adrenal hyperplasia in 230 Chinese patients: Genotype–phenotype correlation and identification of nine novel mutations

Highlights

Abstract

Introduction

Section snippets

Patient data

Patients

Discussion

Conflicts of interests and financial disclosure

Acknowledgments

Lancet

Mol. Genet. Metab.

Steroids

Clin. Biochem.

J. Biol. Chem.

Genet. Med.

Congenital adrenal hyperplasia

N. Engl. J. Med.

Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: an endocrine society clinical practice guideline

J. Clin. Endocrinol. Metab.

Multiplex ligation-dependent probe amplification assay for diagnosis of congenital adrenal hyperplasia

Ann. Clin. Lab. Sci.

Molecular characterization of 25 Chinese pedigrees with 21-hydroxylase deficiency

Genet. Test. Mol. Biomarkers

Genotype–phenotype correlation in 1507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency

Proc. Natl. Acad. Sci. U.S.A.

Gu XF

Zhonghua Er Ke Za Zhi

Molecular model of human CYP21 based on mammalian CYP2C5: structural features correlate with clinical severity of mutations causing congenital adrenal hyperplasia

Mol. Endocrinol.

Mutational spectrum of the steroid 21-hydroxylase gene in Sweden: implications for genetic diagnosis and association with disease manifestation

J. Clin. Endocrinol. Metab.

Disease expression and molecular genotype in congenital adrenal hyperplasia due to 21-hydroxylase deficiency

J. Clin. Invest.

Aberrant splicing and missense mutations cause steroid 21-hydroxylase [P-450(C21)] deficiency in humans: possible gene conversion products

Proc. Natl. Acad. Sci. U.S.A.

Mutation in the CYP21B gene (Ile-172––Asn) causes steroid 21-hydroxylase deficiency

Proc. Natl. Acad. Sci. U.S.A.

A mutation (Pro-30 to Leu) in CYP21 represents a potential nonclassic steroid 21-hydroxylase deficiency allele

Mol. Endocrinol.

Molecular analysis of Japanese patients with steroid 21-hydroxylase deficiency

J. Hum. Genet.