title: A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.
abstract: BACKGROUND
  Molecular diagnosis of congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OHD) has not been straightforward.
  
  
  OBJECTIVE
  To conduct a comprehensive genetic analysis by Multiplex Ligation dependent Probe Amplification (MLPA) and evaluate its reliability for the molecular CAH-21OHD diagnosis.
  
  
  METHODS
  We studied 99 patients from 90 families with salt-wasting (SW; n=32), simple-virilizing (SV; n=29), and non-classical (NC; n=29) CAH-21OHD. Molecular analysis was sequentially performed by detecting the most frequent point mutations by allele-specific oligonucleotide polymerase chain reaction (ASO-PCR), large rearrangements by MLPA, and rare mutations by direct sequencing. Parental segregation was evaluated.
  
  
  RESULTS
  ASO-PCR detected microconversions in 164 alleles (91.1%). MLPA identified CYP21A1P large conversions to CYP21A2 in 7 of the remaining 16 (43.7%), 30-kb deletions including the 3'-end of CYP21A1P, C4B, and the 5'-end of CYP21A2 in 3 of the 16 (18.7%), and a complete CYP21A2 deletion in one (6.3%). Five alleles (2.7%) required direct sequencing; three mutations located in the CYP21A2 gene and two derived from CYP21A1P were found. No parental segregation was observed in patients with the c.329_336del and/or the CL6 cluster mutations. These cases were not diagnosed by ASO-PCR, but MLPA detected deletions in the promoter region of the CYP21A2 gene, explaining the genotype/phenotype dissociation.
  
  
  CONCLUSIONS
  Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.
authors.affiliation: Department of Internal Medicine, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.
authors.forename: Fernanda Borchers
authors.initials: FB
authors.lastname: Coeli-Lacchini
authors.forename: Wendy
authors.initials: W
authors.lastname: Turatti
authors.forename: Paula Conde Lamparelli
authors.initials: PC
authors.lastname: Elias
authors.forename: Lucila Leico Kagohara
authors.initials: LL
authors.lastname: Elias
authors.forename: Carlos Eduardo
authors.initials: CE
authors.lastname: Martinelli
authors.forename: Ayrton Custodio
authors.initials: AC
authors.lastname: Moreira
authors.forename: Sonir Roberto
authors.initials: SR
authors.lastname: Antonini
authors.forename: Margaret
authors.initials: M
authors.lastname: de Castro
mesh_terms: D000293:Adolescent; D000312:Adrenal Hyperplasia, Congenital / Q000175:diagnosis* / Q000235:genetics*; D000483:Alleles; D002648:Child; D002675:Child, Preschool; D005091:Exons; D005838:Genotype; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D007438:Introns; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D013255:Steroid 21-Hydroxylase / Q000235:genetics*; D055815:Young Adult
chemical_list: C531510:CYP21A2 protein, human; D013255:Steroid 21-Hydroxylase
keywords: 17-OHP; 17-hydroxyprogesterone; 21-Hydroxylase deficiency; 21-hydroxylase deficiency; 21OHD; 30-kb deletion; 3′ untranslated; 3′UTR; 5′ untranslated; 5′UTR; ACTH; ASO-PCR; Adrenocorticotropic Hormone; CAH; CYP21A1P; CYP21A2 gene; Congenital adrenal hyperplasia; DHEAS; Dehydroepiandrosterone Sulfate; MLPA; Multiplex Ligation dependent Probe Amplification; NC; RCCX module; RFLP; RP, C4, CYP21 and TNX genes; SV; SW; allele specific oligonucleotide polymerase chain reaction; congenital adrenal hyperplasia; deletion of 30-kb including 3′-end CYP21A1P, C4B, and 5′-end CYP21A2; non-classical; restriction fragment length polymorphism; salt-wasting; simple-virilizing