Rapid detection of 21-hydroxylase deficiency mutations by allele-specific in vitro amplification and capillary zone electrophoresis Paola Carrera, 1 Anna Maria Barbieri, 1 Maurizio Ferrari, 1 Pier Giorgio Righetti, 2 Marilena Perego, 2 and Cecilia Gelfi 3 * A quick diagnosis of the classic form of 21-hydroxylase deficiency (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treat- ment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractory mutation system (ARMS) with capillary zone electrophoresis (CZE) in sieving liquid polymers. In the first PCR, B genes are selectively amplified. In the nested reaction, ARMS-detected wild- type and mutated alleles are separately pooled and resolved by CZE. CZE is performed in coated capillaries in the presence of 30 g/L hydroxyethyl cellulose in the background electrolyte for size separation of the DNA analytes. For high-sensitivity detection the electrophore- sis buffer contains the fluorescent dye SYBR Green I. Laser-induced fluorescence detection is obtained by excitation at 488 nm and signal collection at 520 nm. Specificity and reproducibility of the protocols were established by using samples from 75 Italian families with 21-hydroxylase deficiency already genotyped by allele-specific oligonucleotide hybridization or direct sequencing. Whereas dot-blot is time consuming be- cause of the high number of hybridizations with radio- active probes, this present protocol is more rapid, giving sufficient separation on CZE after PCR reactions with- out preconcentration or desalting of samples. Steroid 21-hydroxylase deficiency is a recessive inherited disease accounting for ;90% of congenital adrenal hyper- plasia (CAH). 4 Lack of 21-hydroxylation results in the accumulation of 17-hydroxyprogesterone, and stimulates excessive androgen production [1]. The severe form of this disease occurs with a frequency of 1:14 000 live births [2]. In this form prenatal virilization in females is ob- served (simple virilizing, SV) and in 70% of cases it also causes salt wasting (SW). In the classical form, prevention of virilization in females is feasible by prenatal diagnosis and treatment with dexamethasone starting at early stages of pregnancy [3]. The milder nonclassical form (late onset, LO), with postnatal virilization, is more common, with variable frequencies in different ethnic groups (1:27– 1:2000). The LO form may also be asymptomatic, called cryptic form [4]. The 21-hydroxylase enzyme is encoded by the P450c21-B gene, located within the HLA complex, on chromosome 6p21.3. The B gene and a 98% homolo- gous pseudogene form a tandem repeat adjacent to C4B and C4A genes, respectively. A certain heterogeneity of mutations in the active gene has been described, such as whole gene deletion, gene conversion [5, 6], and, more frequently, several common point mutations. The major- ity of small rearrangements, which are also present in the pseudogene, are probably the result of small-scale gene conversions [7]. We recently described the distribution of different classes of mutations in the Italian population [8, 9]. In particular, the distribution of gene rearrange- ments has been compared with that found in other ethnic groups and the rearrangements have been correlated with clinical phenotypes, including classic, nonclassic, and cryptic forms of the disease. Molecular diagnosis of the disease can be performed with direct methods for identifying the different classes of 1 I.R.C.C.S., H.S. Raffaele, Via Olgettina 60, I-20132 Milano, Italy. 2 University of Verona, Department of Agricultural and Industrial Biotech- nologies, Strada Le Grazie, Ca` Vignal, 37134 Verona, Italy. 3 ITBA, CNR, Via Ampe`re 56, Milano, Italy. *Author for correspondence. Fax Int 139-2-26423364; e-mail gelfi@ itba.mi.cnr.it. Received February 19, 1997; revision accepted July 10, 1997. 4 Nonstandard abbreviations: CAH, congenital adrenal hyperplasia; SV, simple virilizing; SW, salt wasting; LO, late onset; ARMS, amplification- refractory mutation system; CZE, capillary zone electrophoresis; ASO, allele- specific oligonucleotide; LIF, laser-induced fluorescence; HEC, hydroxyethyl cellulose; and TBE, Tris– borate–EDTA buffer. Clinical Chemistry 43:11 2121–2127 (1997) Molecular Pathology and Genetics 2121 Downloaded from https://academic.oup.com/clinchem/article-abstract/43/11/2121/5640682 by guest on 24 July 2020