Diagnosis Of Steroid 21-hydroxylase Deficiency Research Articles

Molecular Diagnosis of Steroid 21-Hydroxylase Deficiency: A Practical Approach.

Adrenal insufficiency in paediatric patients is mostly due to congenital adrenal hyperplasia (CAH), a severe monogenic disease caused by steroid 21-hydroxylase deficiency (21-OHD, encoded by the CYP21A2 gene) in 95% of cases. CYP21A2 genotyping requires careful analyses that guaranty gene-specific PCR, accurate definition of pseudogene-gene chimeras, gene duplications and allele dropout avoidance. A small panel of well-established disease-causing alterations enables a high diagnostic yield in confirming/discarding the disorder not only in symptomatic patients but also in those asymptomatic with borderline/positive results of 17-hydroxyprogesterone. Unfortunately, the complexity of this locus makes it today reluctant to high throughput techniques of massive sequencing. The strong relationship existing between the molecular alterations and the degree of enzymatic deficiency has allowed genetic studies to demonstrate its usefulness in predicting/classifying the clinical form of the disease. Other aspects of interest regarding molecular studies include its independence of physiological variations and analytical interferences, its usefulness in the diagnosis of simple virilizing forms in males and its inherent contribution to the genetic counseling, an aspect of great importance taking into account the high carrier frequency of CAH in the general population. Genetic testing of CYP21A2 constitutes an irreplaceable tool to detect severe alleles not just in family members of classical forms but also in mild late-onset forms of the disease and couples. It is also helpful in areas such as assisted reproduction and preimplantation diagnosis. Molecular diagnosis of 21-OHD under expert knowledge definitely contributes to a better management of the disease in every step of the clinical course.

Duplication of the CYP21A2 gene complicates mutation analysis of steroid 21-hydroxylase deficiency: characteristics of three unusual haplotypes.

Steroid 21-hydroxylase deficiency, the primary cause of congenital adrenal hyperplasia, is caused by defects of the CYP21A2 gene. As a complement to hormonal measurements, mutation analysis of CYP21A2 is an important tool in the diagnosis of steroid 21-hydroxylase deficiency. Contemporary mutation-detection protocols based on the polymerase chain reaction often depend on the assumption that no more than one CYP21A2 gene is present on each chromosome 6. We describe three haplotypes with two CYP21A2 genes on the same chromosome, with defects typical of salt-losing steroid 21-hydroxylase deficiency in one of those genes, but not necessarily in the other. The frequency of these haplotypes in the general population is 6/365 (1.6%), so they are no less common than other haplotypes that indeed carry steroid 21-hydroxylase deficiency. Chromosomes that carry two CYP21A2 genes therefore represent a significant pitfall in the molecular diagnosis of steroid 21-hydroxylase deficiency. We recommend that, whenever CYP21A2 mutation analysis of an individual who is not a known carrier of steroid 21-hydroxylase deficiency is performed, the overall structure of the CYP21/ C4 region (the RCCX area) is determined by haplotyping to avoid erroneous assignment of carrier status.

Prenatal Diagnosis of Steroid 21-Hydroxylase Deficiency by Allele-Specific Amplification

Objective: Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency (21-OHD) is the most common cause of ambiguous genitalia in females at birth. Here, we report the first prenatal diagnosis of 21-OHD by DNA analysis in Hungary. Methods: Allele-specific amplification (ASA) of the DNA obtained by chorionic villus sampling was performed. Results: The fetus had a homozygous nonsense mutation (Gln318Stop), suggesting a salt-wasting phenotype. Dexamethasone treatment of the mother was started on the 8th gestational week and, as the fetus was an affected female, it was continued until term. The newborn had normal external genitalia at birth, and severe salt-wasting crisis and postnatal virilization was prevented by mineralo- and glucocorticoid replacement therapy. Conclusion: 21-OHD was genotyped by ASA, and virilization of the fetus was prevented by antenatal dexamethasone therapy.

Prenatal diagnosis of steroid 21-hydroxylase deficiency by analysis of polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profiles.

The polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) profile analysis could be applied to the prenatal diagnosis of steroid 21-hydroxylase deficiency. We designed PCR primers to amplify most of the 21-hydroxylase gene, including all the mutations previously reported. PCR-SSCP analysis in eight patients showed at least one polymorphic site in each case. We confirmed that the mobility shifts in SSCP in an affected kindred were transmitted as a Mendelian trait. As these results indicated that PCR-SSCP profiles could be used for DNA-based diagnosis, we attempted to use this technique for prenatal diagnosis. DNA was obtained by chorionic villus sampling of a fetus and PCR-SSCP profiles were analysed in the PCR-amplified fragments in which the mobility shifts had been observed in the SSCP of the proband. We concluded that the fetus was a carrier. Direct nucleotide sequencing and allele-specific oligonucleotide hybridization confirmed that the fetus was heterozygous. At birth, the infant showed no signs of virilization or of abnormal endocrine findings on laboratory study. The results suggest that this new application of PCR-SSCP has advantages over conventional RFLP analysis and is useful in making a prenatal diagnosis of steroid 21-hydroxylase deficiency both rapidly and accurately.

Conventional molecular diagnosis of steroid 21-hydroxylase deficiency using mismatched primers and polymerase chain reaction

We tested a conventional method based on polymerase chain reaction (PCR) and specific primers with one mismatched base at the 3′ end to introduce restriction enzyme sites in order to detect mutations of the CYP21 gene without radioisotope. Using this method, the intron 2 mutation causing aberrant splicing of mRNA (In2G) and the exon 4 mutation (IIe->Asn, Ex4) in the CYP21 gene were analyzed. The nonsense mutation in exon 8 (Ex8NON) of the CYP21 gene was also investigated by PCR and subsequent restriction enzyme digestion.The mismatched primers successfully amplified the CYP21 gene containing the In2G and the Ex4 mutation sites, and the presence of these two mutations could be determined by restriction enzyme digestion after PCR. We used this new method to study 33 patients. Twenty-five of these patients were found to have at least one mutation (In2G and / or Ex4 mutation). By enzyme digestion after PCR, the Ex8NON mutation was also identified (7 out of 33 patients).In conclusion, we have developed a new method to detect point mutations in the CYP21 gene. This method was proved to be sensitive and rapid for the detection of the mutations studied. Therefore, this method is suitable for clinical genetic diagnosis.

A steroid 21-hydroxylase allele concomitantly carrying four disease-causing mutations is not uncommon in the swedish population.

We describe a steroid 21-hydroxylase allele carrying four disease-causing mutations, viz. I173N, V282L, I237N + V238E + M240K, and the insertion of T at 308 L. The first two are established causes of partial enzyme deficiency, whereas the last two are known to result in the most severe, salt-wasting form of the disease. All four mutations are normally found in the pseudogene. This abnormal allele was found in the general Swedish population (6 out of 354 individuals), but has so far not been identified among 21-hydroxylase deficiency patients. The existence of alleles with multiple mutations illustrates the importance of segregating mutations for the correct genetic diagnosis of steroid 21-hydroxylase deficiency; an allele-specific polymerase chain reaction can be successfully employed for this purpose when families are unavailable.

Problems in diagnosis and management of congenital adrenal hyperplasia due to 21-hydroxylase deficiency

A number of biochemical tests have been utilized to assist the diagnosis of steroid 21-hydroxylase deficiency. The specificity and accuracy of plasma 17-hydroxyprogesterone assays are important. A profile of steroids in urine by gas chromatography and mass spectrometry is the definitive test. Molecular biology is not practical for the diagnosis of a new case. The ACTH stimulation test for detection of heterozygotes is a poor discriminant. Fertility in patients with congenital adrenal hyperplasia may be due to excess of progesterone as well as of androgens. Gene amplification offers the best approach in molecular biology for the prenatal diagnosis of 21-hydroxylase deficiency.

Prenatal diagnosis of congenital adrenal hyperplasia by direct detection of mutations in the steroid 21‐hydroxylase gene

Our aim was to develop a rapid and accurate method for the prenatal diagnosis of congenital adrenal hyperplasia using the polymerase chain reaction to detect mutations in the steroid 21-hydroxylase gene. These procedures will help to minimize exposure to dexamethasone treatment of either affected males or unaffected females. Chorionic villus biopsy samples were obtained between 10 and 11 weeks gestation from three females carrying fetuses at risk of steroid 21-hydroxylase deficiency. Blood samples were taken from parents and the index case in each family. Three common mutations in the 21-hydroxylase B gene were detected following DNA amplification. Prenatal diagnosis of congenital adrenal hyperplasia was successful in all three cases. One affected female was treated with dexamethasone to term. In the other two cases, one affected male and one carrier also male, dexamethasone was withdrawn at an early stage. First trimester prenatal diagnosis of steroid 21-hydroxylase deficiency was achieved in three pregnancies with a strategy based on direct detection of gene mutations.

Steroid 21-hydroxylase deficiency: two additional mutations in salt-wasting disease and rapid screening of disease-causing mutations.

A method for genetic diagnosis of steroid 21-hydroxylase deficiency was developed based on allele-specific PCR. With this approach, genotyping of fourteen mutations and diagnosis of homozygous gene deletions can be performed within hours from tissue sampling. One patient with salt-wasting disease had normal genotype at all positions screened. DNA sequencing revealed two novel mutations, a G to C transversion at the conserved splice donor site of intron 7 and a TGG to TAG nonsense mutation at Trp 406 in exon 9. Allele-specific PCR was established also for these mutations and used to screen for their presence in the pseudogene. However, the two novel mutations were not found in at least 34 pseudogenes.