Congenital adrenal hyperplasia (CAH) is one of the most common inherited metabolic disorders, and is associated with substantial morbidity and mortality in affected children and adults (1). Steroid 21-hydroxylase deficiency (21-OHD), the most common variant of CAH, accounts for ~90% of CAH cases (2,3). Patients most commonly present with symptoms associated with different extents of androgen accumulation, a hallmark of CAH, with or without aldosterone deficiency (1,3). Based on the broad spectrum of presentations, CAH has been divided into two categories: Classical, which is represented by salt wasting (SW; <2% 21-OH activity) and simple virilizing (SV) forms (an increase of ~1–2% in 21-OH activity compared with SW); and non-classical (NC; 20–50% 21-OH activity).

Inherited in an autosomal recessive manner, 21-OHD is caused by mutations in the cytochrome P450 family 21 subfamily A member 2 (CYP21A2) gene, which is located on chromosome 6p21.3, ~3.1 kb from the highly homologous CYP21A1 pseudogene (CYP21A1P) (4–7). At present, >200 CYP21A2-inactivating mutations are listed in the Human Gene Mutation Database (HGMD; www.hgmd.org; Table I). The majority of these mutations are missense and nonsense mutations. However, splice site mutations, deletions and insertions, in addition to other types of mutations, have been detected. Due to complex mutation types and the variety of clinical presentations, identification of the genotype it is required to correlate with the phenotype.

In the present study, molecular genetic analysis of the CYP21A2 gene was performed in a patient with 21-OHD and other family members. A novel compound heterozygous mutation, not previously described, was identified and confirmed to be responsible for 21-OHD in this Chinese pedigree. Managed with hormone replacement therapy, the proband and the sister of the proband succeeded in conceiving following 6 months of treatment with dexamethasone. The results of the present study provided novel information clarifying the CAH genotype-phenotype association, offering evidence for patient counseling and improved prenatal diagnosis.

In the present study, it was demonstrated that a compound heterozygous mutation, a missense mutation c.1024C>T (p. Arg342Trp) and heterozygous deletion mutations of exons 1, 3, 4, 6 and 7 in the CYP21A2 gene may serve an important role in a Chinese pedigree of four sisters affected by 21-OHD. To the best of the authors' knowledge, this compound heterozygous mutation has not yet been reported elsewhere. The confirmation of a CAH diagnosis by genetic analysis is important to correlate genotype with phenotype. In addition, the patient presented with SV-CAH, and a detailed molecular analysis may provide useful insights into the functions of the CYP21A2 gene.

The 21-OH locus includes a real gene, functional CYP21A2, which encodes a protein of 495 amino acids, and the non-functional CYP21A1P, which is a pseudogene and hence does not code for a protein. CYP21A2 and CYP21A1 Peach consist of 10 exons and 9 introns, with 98% homology among exons and 96% homology among introns (5). Due to their high homology, CYP21A2 and CYP21A1P are prone to complex and diverse genetic recombinations, which is one of the reasons for the relatively high incidence of mutations. A total of ~95% of CYP21A2 mutations result from intergenic recombination between CYP21A2 and the CYP21A1P pseudogene. The complete deletion of neighboring complement C4B and a net deletion of CYP21A1, or apparent gene conversion events that transfer deleterious mutations normally present in CYP21A1P to CYP21A2, may occur due to unequal crossing over during meiosis (12). These defects or repeated fragments stimulate RCCX unit homologous genes to form different chimeras (13).

The sequence variants identified in the present study are hypothesized to be disease-causing mutations in this Chinese pedigree. The missense mutation c.1024C>T (p. Arg342Trp) detected in the mother and four sisters is expected to exert a substantial impact on the structure and function of the CYP21A2 protein due to the replacement of an arginine with a tryptophan. Arginine residues that exist in the cytochrome P450 enzymes of different mammalians are highly conserved in the family and are important for enzymatic activity (14). Arginine is a positively-charged polar amino acid with a long hydrophobic side chain, whereas tryptophan is a non-polar aromatic amino acid and thus is not conducive to the formation of an α-helix. In the present study, the patient and mother of the patient presented with the same hybrid genetic mutations in CYP21A2, indicating that these mutations are of maternal inheritance. The loss of the larger pieces of the gene adversely influences its functions, altering a number of essential amino acids (15,16). In the present study, heterozygous deletion mutations of exons 1, 3, 4, 6 and 7 of paternal origin were additionally detected, leading to early termination of protein translation and the creation of a truncated protein. The remaining amino acids were unable to form the four important structural domains, thereby deactivating CYP21A2.

Clinical manifestations of 21-OHD are complex and various. 21-OHD is primarily characterized by cortisol deficiency and the increased accumulation of progesterone, 17α-hydroxyprogesterone, and androgen, with or without aldosterone deficiency (3,12). In general, 21-OHD may be divided into SV, SW and NC types, according to the level of enzyme activity. The SV form is characterized by virilization of the external genitalia in newborn females and by hypocortisolism and precocious pseudopuberty due to reactive androgen overproduction in the two sexes. In addition to hyperandrogenism signs, the SW type manifests with an insufficient secretion of aldosterone, hyponatremia and hyperkalemia. The symptoms of the NC form are variable and patients are asymptomatic at birth, manifesting symptoms that include hirsutism, oligomenorrhea and anovulation later in life. In the present study, the proband presented clitoral hypertrophy, and biochemical tests indicated increased 17α-hydroxyprogesterone, ACTH and testosterone. Adrenal CT revealed clear bilateral adrenal hyperplasia. The proband was diagnosed with CAH with normal blood pressure and no symptoms of salt loss. Therefore, the proband was diagnosed with SV CAH.

The molecular diagnosis of CAH is complex due to its unusual features. At present, genetic diagnosis of 21-OHD for patients with CAH is associated with two principal difficulties. CYP21A2 is composed of 10 exons, and each exon is likely to present genetic mutations, loss or amplification of small fragments. Thus, it is recommended that the full-length 21-OHD be sequenced for each patient, which is time-consuming (17). The conventional method used for the detection of a 21-OHD mutation is as follows: A specific primer is designed to amplify the gene segments of CYP21A2, the different amplified fragments are directly sequenced via fragment purification, and the full-length sequence of the CYP21A2 gene as-obtained is used to determine the genetic mutation site (18). The all-gene sequencing method is used to identify the mutation site (19). The second difficulty is the deficiency or amplification of large pieces in the 21-OHD gene of patients with CAH, and such complexity and diversity further complicate the detection. The most commonly used method is Southern imprinting (20), although this is not suitable for a large-scale population or for use in the clinic. In addition, certain conventional methods have been adopted, including long PCR-enzyme digestion analysis, PCR product T-A cloning analysis, and quantitative PCR-based methods for determining CYP21A2 gene copy number (21,22). MLPA maybe used for copy number detection of multiple CYP21A2 gene sites simultaneously (23). This technology combined with gene sequencing is therefore commonly used for CYP21A2 gene diagnosis. Additionally, when a single missing exon is detected by MLPA inspection, it maybe verified by sequencing and by long-segment PCR.

The phenotype of 21-OHD is associated with its genotype, including point mutations, frame shift mutations and deletion mutations of small DNA fragments. SW-, SV- or NC-type 21-OHD maybe predicted by clinical gene sequencing. Concolino et al (24) demonstrated that the consensus between genotype and SW CAH patients was 81.8%. Baumgartner-Parzer et al (25) reported that the consistency between genotype and clinical phenotype was 80%. However, genotype and clinical phenotype are not always completely consistent, and the same 21-OHD gene mutation in patients with CAH may result in different clinical phenotypes (26,27). New et al (28) reported that certain mutations, including the P30L, I2Gand I172N mutations, were prone to yield different CAH phenotypes. In SW and NC CAH, a phenotype maybe attributed to a genotype. However, in SV CAH, wide phenotypic variability may be observed, particularly with the exon 4 I172N mutation (28). Janjanin et al (29) described five patients with 21-OHD belonging to three generations of the same family (grandmother, parents and two children), whose genotype corresponded to NC CAH. Notably, in spite of one minorCYP21 mutation, the proband presented with the SV form of 21-OHD and required glucocorticoid replacement therapy from the age of 4 years. In the present study, the CYP21A2 gene sequencing of the patient, in addition to the clinical symptoms and gene sequencing results, revealed c.1024C>T (p. Arg342Trp) and heterozygous deletion mutations, which supported the clinical diagnosis of SV CAH.

The present study provided strong evidence for the presence of a novel compound heterozygous mutation in the CYP21A2 gene that is predicted to result in the partial loss of enzymatic activity of the resulting protein. This mutation caused SV 21-OHD in a Chinese pedigree. The results of the present study provided information for the prenatal diagnosis of CAH caused by 21-OHD using amniotic cells or chorionic villi. The in vitro expression analysis of residual enzymatic activity may be performed to analyze the functional consequences of this newly identified mutation, thereby proving its relevance to the clinical presentation and analyzing its impact on CYP21A2 function. By investigating the molecular genetic mechanisms of CAH, the identification and diagnosis may be optimized, which has substantial clinical value for diagnosis and genetic counseling of carriers in the family of a patient.