Rapid screening method for detecting mutations in the 21-hydroxylase gene Josep Oriola, 1 * Isabel Plensa, 1 Isabel Machuca, 1 Carles Pavı ´a, 2 and Francisca Rivera-Fillat 1 Impaired synthesis of adrenal steroid hormones because of steroid 21-hydroxylase deficiency is one of the most common inborn errors of metabolism. To expedite mo- lecular diagnosis in families with 21-hydroxylase defi- ciency, we have designed a rapid strategy to determine nine of the most common mutations in the 21-hydroxy- lase gene. According to the mutation to be detected, we apply either of two simple strategies: digestion with adequate restriction enzyme or use of the amplification- created restriction site (ACRS) approach and subse- quent restriction analysis. Both procedures are rapid and, being nonradioactive, are safer to perform; more- over determination of zygosity in the analyzed muta- tions requires only one tube per mutation. INDEXING TERMS: congenital adrenal hyperplasia • herita- ble disorders • adrenal steroid hormones • amplification- created restriction site analysis Congenital adrenal hyperplasia (CAH) is an inherited disorder of cortisol biosynthesis [1]. Although five differ- ent enzymes are required to synthesize cortisol in the adrenal cortex-mix, steroid 21-hydroxylase deficiency ac- counts for 90% of CAH. The steroid 21-hydroxylase gene, located in the HLA class III gene region on chromo- some 6, has a complicated structure with a high degree of variability. An active gene, CYP21, and a highly homolo- gous inactive pseudogene, CYP21P, are located 3' from each of the two genes encoding the fourth component of complement C4A and C4B, forming a repeated tandem of the units C4/21OH [2, 3]. Because 21-hydroxylase deficiency is one of the most common inborn errors of metabolism in humans [4, 5], procedures are needed that will detect known mutations in the 21-hydroxylase gene to support a rapid diagnosis. We have designed a coordinated strategy to detect nine common 21-hydroxylase mutations, identifying some of them with the amplification-created restriction site (ACRS) method [6]. ACRS, a PCR-based method, can identify previously known allelic mutations in nucleic acid sequences. Materials and Methods Subjects. Eight CAH families were selected according to mutations that had been identified previously by means of allele-specific oligonucleotide hybridization. Preparation of human DNA. Human DNA was prepared from peripheral white blood cells. 10 mL of heparinized blood was mixed with 40 mL of isotonic saline (9 g/L NaCl) and centrifuged at 1500g. The pellet was resus- pended in 30 mL of erythrocytes lysis buffer (5 mmol/L MgCl 2 and 20 mmol/L Tris-HCl, pH 7.8) and centrifuged at 3000g. Leukocytes were lysed by adding to 10 mL of lysis buffer (0.2 mol/L NaCl, 1 mmol/L EDTA, and 10 mmol/L Tris-HCl, pH 7.8) containing 10 mg of sodium dodecyl sulfate and 0.4 mg of proteinase K per milliliter (Merck, Darmstadt, Germany). After having shaken this mixture for 2 h at room temperature, we extracted it once with an equal volume of phenol and then twice with chloroform/isoamyl alcohol (24/1 by vol). The DNA was precipitated from the combined extracts by adding 2.5 volumes of isopropanol as described [7]. The two-wave- length absorbance ratio (A 260 /A 280 ) of the DNA prepara- tions was 1.7. We checked the integrity of the DNA samples by electrophoresis of 2 g of DNA in a 0.9% agarose gel. Methodology. Primers used were synthesized in our hos- pital in a DNA synthesizer (Model 392; Applied Biosys- tems, Foster City, CA); subsequent purification by HPLC was not required. The oligonucleotide probes used for allele-specific oligonucleotide hybridization were essen- tially those described by Owerbach et al. [5]; for the 1 Servei d’Hormonologia, Hospital Clı ´nic i Provincial de Barcelona, and 2 Seccio ´ d’Endocrinologia, Hospital Universitari de Sant Joan de Deu, Spain. *Address for correspondence: Servei d’Hormonologia, Hospital Clı ´nic, Villarroel 170 08036, Barcelona, Spain. Fax 34-3-227.54.54; e-mail labhor@ medicina.ub.es. Received August 28, 1996; revised October 31, 1996; accepted November 15, 1996. Clinical Chemistry 43:4 557–561 (1997) Molecular Pathology 557 Downloaded from https://academic.oup.com/clinchem/article/43/4/557/5640748 by guest on 03 September 2021