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

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

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: 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

OBJECTIVETo discuss the genetic diagnosis of congenital adrenal hyperplasia (CAH) and investigate the resource of gene mutations in CAH.

METHODEnzymatic methods with restriction endonucleases that specifically recognized the mutation sites were used to detect the gene mutations in patients with CAH and their relatives. Polymerase chain reaction and direct sequencing were used to identify the mutations in 21-hydroxylase gene, and short tandem repeat (STR) typing was used to determine the sources of the mutations.

RESULTSOne CAH patient had two known mutations in 21-hydroxylase gene, namely the I2g and I172N mutations. The former mutation was inherited from the biological mother and the latter was not inherited.

CONCLUSIONThe 9 common mutations of CAH are also the hotspots for new mutations.

Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Gene Deletion ; Genotype ; Humans ; Mutation ; Point Mutation ; Polymerase Chain Reaction ; 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

CYP21A2 gene mutations in a child with congenital adrenal hyperplasia (CAH), and the child's parents, were detected in the study. The clinical features, treatment monitoring and molecular genetic mechanism of CAH are reviewed. In the study, DNA was extracted from peripheral blood samples using the QIAGEN Blood DNA Mini Kit; a highly specific PCR primer for CYP21A2 gene was designed according to the sequence difference between CYP2lA2 gene and its pseudogene; the whole CYP2lA2 gene was amplified with PrimeSTAR DNA polymerase (Takara), and the amplification product was directly sequenced to detect and analyze CYP2lA2 gene mutation. The child was clinically diagnosed with CAH (21-hydroxylase deficiency, 21-OHD) at the age of 36 days, and the case was confirmed by genetic diagnosis at the age of 1.5 years. The proband had a homozygous mutation at c.293-13C in the second intron of CYP21 gene, while the parents had heterozygous mutations. Early diagnosis and standard treatment of CAH (21-OHD) should be performed to prevent salt-wasting crisis and reduce mortality; bone aging should be avoided to increase final adult height (FAH), and reproductive dysfunction due to oligospermia in adulthood should be avoided. These factors are helpful for improving prognosis and increasing FAH. Investigating the molecular genetic mechanism of CAH can improve recognition and optimize diagnosis of this disease. In addition, carrier diagnosis and genetic counseling for the proband family are of great significance.
17-alpha-Hydroxyprogesterone ; blood ; Adrenal Hyperplasia, Congenital ; blood ; genetics ; Humans ; Infant ; Male ; Mutation ; Steroid 21-Hydroxylase ; genetics

OBJECTIVETo investigate the steroid 21-hydroxylase gene (CYP21) mutations in families with 21-hydroxylase deficiency (21-OHD).

METHODSThe CYP21 gene mutations were detected in four patients with 21-hydroxylase deficiency and their relatives. The genomic DNA of the patients was isolated from whole blood.Two pairs of primers were used to amplify the CYP21 gene. The amplified PCR products were purified by agarose gel and then directly sequenced.

RESULTSSix kinds of mutations were found. In the first family, the patient was a compound heterozygote carrying four different mutations (cluster E6, Q318X, A391T, P459H) onCYP21 gene, three mutations (cluster E6, Q318X, A391T) were on her maternal allele, a novel mutation was found:P459H. It located at codon 459 in exon 10 and changing a proline (CCC) to a histidine (CAC), and A391T was a rare mutation. In the second family, two kinds of mutations were found:cluster E6 and R483W. R483W was also a rare mutation. In the third family, the sequencing of the CYP21 gene of two patients revealed a homozygous T to A transition in codon 172 leading to substitution of isoleucine by asparagine (I172N).

CONCLUSIONSix kinds of mutations were found in three families with 21-hydroxylase deficiency. Using DNA sequencing we have identified a novel mutation (P459H) and two rare mutations (A391T, R483W) of the CYP21 gene. Although microconversion events are the main cause of mutations in the CYP21 gene, random mutations can also be the cause of 21-hydroxylase deficiency.

Adrenal Hyperplasia, Congenital ; genetics ; Amino Acid Substitution ; Exons ; genetics ; Female ; Humans ; Multigene Family ; Mutation ; Polymerase Chain Reaction ; 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 develop a method for elucidating genetic basis of 21-hydroxylase deficiency.

METHODSSanger sequencing of entire 21-hydroxylase coding gene CYP21A2 was carried out to detect point mutations, and multiplex ligation-dependent probe amplification (MLPA) and locus-specific PCR/enzyme restriction method were used to detect large deletions and conversion mutations.

RESULTSNine children were analyzed. Point mutations of the CYP21A2 gene have been identified as: IVS2 13A/C>G (9 alleles), p.Arg356Trp (1 allele), Cluster E6 (1 allele), p.Gln318X (1 allele), and Prom conv (1 allele). While the former 4 mutations are pathogenic, the role of Prom conv mutation in the pathogenesis was uncertain. Three cases had entire CYP21A2 gene deletions (3 alleles), three had CYP21A1P/CYP21A2 chimeric mutations (3 alleles). The genotypes of all patients were determined. And all of the mutations were inherited from parents.

CONCLUSIONA rational method for detecting point mutations and large deletions/conversions of CYP21A2 gene has been established.

Adrenal Hyperplasia, Congenital ; diagnosis ; genetics ; Alleles ; Base Sequence ; Child ; Child, Preschool ; Female ; Gene Order ; Genotype ; Humans ; Infant ; Male ; Multiplex Polymerase Chain Reaction ; Steroid 21-Hydroxylase ; genetics