21 hydroxylase deficiency (21-OHD), the most common form of congenital adrenal hyperplasia, is caused by defects in CYP21A2 gene, which encodes the cytochrome P450 oxidase (P450C21) involved in glucocorticoid and mineralocorticoid synthesis. The diagnosis of 21-OHD is based on the comprehensive evaluation of clinical manifestation, biochemical alteration and molecular genetics results. Due to the complex structure of CYP21A2, special techniques are required to perform delicate analysis to avoid the interference of its pseudogene. Recently, the state-of-the-art diagnostic methods were applied to the clinic gradually, including the steroid hormone profiling and third generation sequencing. To standardize the laboratory diagnosis of 21-OHD, this consensus was drafted on the basis of the extensive knowledge, the updated progress and the published consensuses and guidelines worldwide by expert discussion organized by Rare Diseases Group of Pediatric Branch of Chinese Medical Association, Medical Genetics Branch of Chinese Medical Doctor Association, Birth Defect Prevention and Molecular Genetics Branch of China Maternal and Child Health Association. and Molecular Diagnosis Branch of Shanghai Medical Association.
Child ; Humans ; Adrenal Hyperplasia, Congenital/genetics* ; Steroid 21-Hydroxylase/genetics* ; Consensus ; China ; Clinical Laboratory Techniques ; Mutation

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.
3-Oxo-5-alpha-Steroid 4-Dehydrogenase/genetics* ; Child ; China/epidemiology* ; Cryptorchidism/genetics* ; Disorders of Sex Development/genetics* ; Female ; Genital Diseases, Male ; Genotype ; Humans ; Hypospadias/genetics* ; Male ; Membrane Proteins/genetics* ; Penis/abnormalities* ; Phenotype ; Retrospective Studies ; Steroid 21-Hydroxylase/genetics*

OBJECTIVE: To explore the genotype-phenotype correlation among 18 patients with 21-hydroxylase deficiency (21-OHD). METHODS: PCR-Sanger sequencing was used to analyze the 10 exons and flanking regions of the CYP21A2 gene among the 18 patients and 20 healthy controls. RESULTS: Seventeen patients had variants of the CYP21A2 gene. Eight patients (44.4%, 8/18) carried homozygous variants including p.Ile 173Asn (62.5%, 5/8), p.Pro31Leu (25.0%, 2/8), and IVS2-13A/C>G (12.5%, 1/8), respectively. Six patients (33.3%, 6/18) carried compound heterozygous variant, among which IVS2-13 A>G+p.Ile 173Asn were most common (50.0%). 94.4% (34/36) of the variant were pathogenic, with the most common variants being p.Ile173Asn (41.7%), IVS2-13A/C>G (19.4%), and p.Ile173Asn (7.5%). No variant was identified among the 20 healthy controls. CONCLUSION The majority of 21-OHD patients carried CYP21A2 gene variants in homozygous or compound heterozygous forms, among which the p.Ile173Asn was the most common one. There is a strong correlation between the genotypes and clinical phenotypes.
Adrenal Hyperplasia, Congenital ; genetics ; Genotype ; Humans ; Mutation ; Phenotype ; Steroid 21-Hydroxylase ; genetics

OBJECTIVE: Genetic screening and prenatal diagnosis was performed in eighteen families with high risk of 21-hydroxylase deficiency (21-OHD) to provide valuable information for genetic counseling in these affected families. METHODS: First, multiplex ligation-dependent probe amplification (MLPA) combined with nested-PCR based Sanger sequencing was used to detect CYP21A2 gene mutations in probands and their parents of eighteen families, with seven probands had been dead. Second, paternity test was applied to exclude the possibility of maternal genomic DNA contamination, and fetal prenatal diagnosis is based on the mutations found in proband or parents of the family. RESULTS: Ten mutations were identified in these eighteen families, including large fragment deletion, I2G, E3del8bp, I172N, V281L, E6 cluster, L307Ffs, Q318X, R356W and R484Pfs. All probands were caused by homozygous or compound heterozygous mutations of CYP21A2 gene and their parents were carriers. By comparing short tandem repeat sites contamination of maternal genomic DNA was not found in fetal DNA. Prenatal diagnosis showed that five fetus were 21-OHD patients, four fetus were carriers and the other nine fetus were normal. CONCLUSION CYP21A2 gene mutation is the etiology of 21-OHD. Genetic testing of CYP21A2 could assist physicians in 21-OHD diagnosis and provided genetic counseling and prenatal diagnosis for parents who are at risk for having a child with congenital adrenal hyperplasia.
Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Female ; Genetic Testing ; Humans ; Mutation ; Pregnancy ; Prenatal Diagnosis ; Steroid 21-Hydroxylase

OBJECTIVE: To identify pathogenic mutations in 25 Chinese pedigrees affected with congenital adrenal hyperplasia (CAH). METHODS: Mutations of the CYP21A2 gene were detected with locus-specific PCR/restriction endonuclease analysis, multiplex ligation-dependent probe amplification assay, and direct sequencing of the entire CYP21A2 gene. Prenatal diagnosis was offered to fetuses at risk for CAH. RESULTS: All 50 alleles of the CYP21A2 gene carried by the 25 pedigrees were successfully delineated. Large deletions and conversions have accounted for 16 (32%) of the alleles, which included 9 entire CYP21A2 gene deletions, 6 chimeric CYP21A1P/CYP21A2 genes, and 1 partial conversion of the CYP21A2 gene. For the remaining 34 alleles, there were 9 micro-conversions and 4 de novo mutations [including a previously unreported c.62G>A (p.Trp21X) mutation]. Prenatal diagnosis was provided for 28 fetuses with a high risk for CAH, among whom 8 were found to be affected. CONCLUSION The detection of CYP21A2 gene mutations can facilitate appropriate genetic counseling and prenatal diagnosis for the affected pedigrees.
Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Asian Continental Ancestry Group ; China ; Female ; Humans ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; Steroid 21-Hydroxylase ; genetics

OBJECTIVETo investigate gene mutations and the relationship between genotypes and clinical phenotypes in Uygur children with 21-hydroxylase deficiency (21-OHD) in Xinjiang, China.

METHODSA total of 20 Uygur children with 21-OHD who visited the hospital between October 2013 and October 2014 were enrolled. Full-length direct sequencing and multiplex ligation-dependent probe amplification (MLPA) were used to detect the mutations of CYP21A2 gene, which encoded 21-hydroxylase. According to the type of mutation, the patients with 21-OHD were divided into different groups to analyze the consistency between predicted clinical phenotypes and actual clinical phenotypes.

RESULTSA total of 9 mutation types were found in the 20 patients, and 8 of them were identified as pathogenic mutations, i.e., Del, conv, I2g, I172N, Cluster E6, 8-bp del, V281L, and R356W. The other mutation is the new mutation occurring in intron 5 (c.648+37A>G), which had not been reported, and its pathological significance remains unknown. Most clinical phenotypes predicted by mutation types had a higher coincidence rate with actual clinical phenotypes (above 67%), and the clinical phenotypes predicted by P30L and V281L had a lower coincidence rate with actual clinical phenotypes (below 33%).

CONCLUSIONSThe genotype of 21-OHD has a good correlation with phenotype, and the clinical phenotype can be predicted by detecting the patient′s genotype. The new mutation (c.648+37A>G) may be related to the pathogenesis of 21-OHD.

Adolescent ; Adrenal Hyperplasia, Congenital ; enzymology ; ethnology ; genetics ; Child ; China ; ethnology ; Female ; Genotype ; Humans ; Male ; Mutation ; Phenotype ; Steroid 21-Hydroxylase ; genetics

OBJECTIVETo assess the frequencies of CYP21A2 gene mutations among patients from Fujian area with classical 21-hydroxylase deficiency.

METHODSFor 19 probands from different families affected with classical steroid 21-hydroxylase deficiency and 74 family members, mutations of the CYP21A2 gene were analyzed with combined nested polymerase chain reaction, Sanger sequencing and multiplex ligation-dependent probe amplification. Time resolved fluorescence immunoassay was performed to determine the level of 17-hydroxyprogesterone (17-OHP) in all family members. Clinical data and laboratory results of the probands and their family members were analyzed.

RESULTSEleven mutations were identified among the 38 alleles from the 19 probands. 92.1% (35/38) of the mutant CYP21A2 alleles were due to recombination between CYP21A2 and CYP21A1P. Gene conversion and deletions were identified in 84.2% (32/38) and 7.9% (3/38) of the alleles, respectively. IVS2-13A/C>G and chimeras were the most common mutations, which respectively accounted for 34.2% (13/38) and 18.4% (7/38) of all mutant alleles. Among these, IVS2+1G>A and Q318X+356W were first reported in China. 74.3% (55/74) of the family members were carriers of heterozygous mutations. However, no significant difference was found in the 17-OHP levels between carriers and non-carriers (P>0.05).

CONCLUSIONThere seems to be a specific spectrum of CYP21A2 gene mutations in Fujian area, where IVS2-13A/C>G and chimeras are the most common mutations.

Adrenal Hyperplasia, Congenital ; genetics ; Alleles ; Female ; Humans ; Male ; Mutation ; genetics ; Steroid 21-Hydroxylase ; genetics

OBJECTIVETo establish an allele-specific PCR method for detect screening of CYP21A2 gene mutation.

METHODSAllele-specific PCR primers and analogy primers were designed based on the sequence alignment of CYP21A2 and CYP21AP genes. Genomic DNA was extracted from blood specimens of 4 patients with 21-hydroxylase deficiency and 5 healthy controls and respectively amplified with allele-specific PCR primers and analogy primers and sequenced.

RESULTSMutations of CYP21A2 including IVS2-13A/C>G, Arg356Trp and Arg149Pro were found with the established method in all of the 4 patients but not in the healthy controls. When detected with the analogy primers set, IVS2-13A/C>G and Arg356Trp were observed in both patients and healthy controls.

CONCLUSIONThe allele-specific PCR-based method is a simple, effective and reliable method for the detection of CYP21A2 gene mutation.

Adrenal Hyperplasia, Congenital ; enzymology ; genetics ; Alleles ; Base Sequence ; DNA Mutational Analysis ; methods ; DNA Primers ; genetics ; Humans ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; methods ; Steroid 21-Hydroxylase ; genetics

OBJECTIVETo analyze CYP21A2 gene mutation in two families with 21-hydroxylase deficiency (21-OHD) and to explore the correlation between genotype and clinical phenotype.

METHODSTwo patients with 21-OHD and their families were investigated. CYP21A2 gene mutation was analyzed by PCR and direct sequencing.

RESULTSThe probands from family 1 and 2 have been respectively diagnosed with simple virilizing and non-classical 21-OHD. Both showed increased baseline serum 17hydroxyprogesterone, testosterone and adrenocorticotropic hormone (ACTH), but had no evidence of salt loss. Computer tomography revealed bilateral adrenal hyperplasia in both patients. After 1 year treatment, both had conceived successfully. DNA sequencing revealed that the proband of family 1 had compound heterozygous mutations for IVS2 13 A>G and Ile172Asn. Her father was heterozygous for Ile172Asn, whilst her mother and brother were heterozygous for IVS213A/C>G. In family 2, the proband was heterozygous for Arg341Trp and Gln318X. Her father, sister and nephew were heterozygous for Arg341Trp, whilst her mother was heterozygous for Gln318X. her brother and niece were non-affected. Carriers of single heterozygous mutations in both families had no clinical sign.

CONCLUSIONIn both families, the disease has been caused by compound heterozygous mutations, for which there has been a good genotype-phenotype agreement. Screening of CYP21A2 gene can facilitate both diagnosis and genetic counseling.

Adrenal Hyperplasia, Congenital ; blood ; enzymology ; genetics ; Adrenocorticotropic Hormone ; blood ; Adult ; Base Sequence ; Child ; Female ; Genotype ; Humans ; Male ; Molecular Sequence Data ; Mutation, Missense ; Pedigree ; Phenotype ; Steroid 21-Hydroxylase ; genetics ; metabolism ; Testosterone ; blood ; Young Adult

Congenital adrenal insufficiency is caused by specific genetic mutations. Early suspicion and definite diagnosis are crucial because the disease can precipitate a life-threatening hypovolemic shock without prompt treatment. This study was designed to understand the clinical manifestations including growth patterns and to find the usefulness of ACTH stimulation test. Sixteen patients with confirmed genotyping were subdivided into three groups according to the genetic study results: congenital adrenal hyperplasia due to 21-hydroxylase deficiency (CAH, n=11), congenital lipoid adrenal hyperplasia (n=3) and X-linked adrenal hypoplasia congenita (n=2). Bone age advancement was prominent in patients with CAH especially after 60 months of chronologic age (n=6, 67%). They were diagnosed in older ages in group with bone age advancement (P<0.05). Comorbid conditions such as obesity, mental retardation, and central precocious puberty were also prominent in this group. In conclusion, this study showed the importance of understanding the clinical symptoms as well as genetic analysis for early diagnosis and management of congenital adrenal insufficiency. ACTH stimulation test played an important role to support the diagnosis and serum 17-hydroxyprogesterone levels were significantly elevated in all of the CAH patients. The test will be important for monitoring growth and puberty during follow up of patients with congenital adrenal insufficiency.
17-alpha-Hydroxyprogesterone/blood ; 46, XY Disorders of Sex Development/drug therapy/*genetics ; Adolescent ; Adrenal Hyperplasia, Congenital/drug therapy/*genetics ; Adrenal Insufficiency/*congenital/diagnosis/drug therapy/genetics ; Adrenocorticotropic Hormone/*metabolism ; Bone Development/genetics ; Child ; Child, Preschool ; DAX-1 Orphan Nuclear Receptor/genetics ; Female ; Genetic Diseases, X-Linked/drug therapy/*genetics ; Genotype ; Glucocorticoids/therapeutic use ; Humans ; Intellectual Disability/complications ; Male ; Mineralocorticoids/therapeutic use ; Obesity/complications ; Phosphoproteins/genetics ; Puberty, Precocious/complications ; Retrospective Studies ; Steroid 21-Hydroxylase/genetics