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A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.
Gene. 2013 Sep 10; 526(2):239-45.GENE

Abstract

CONTEXT

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.

PATIENTS AND 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.

CONCLUSION

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+Show Affiliations

Department of Internal Medicine, School of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, SP, Brazil.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

23570880

Citation

Coeli-Lacchini, Fernanda Borchers, et al. "A Rational, Non-radioactive Strategy for the Molecular Diagnosis of Congenital Adrenal Hyperplasia Due to 21-hydroxylase Deficiency." Gene, vol. 526, no. 2, 2013, pp. 239-45.
Coeli-Lacchini FB, Turatti W, Elias PC, et al. A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Gene. 2013;526(2):239-45.
Coeli-Lacchini, F. B., Turatti, W., Elias, P. C., Elias, L. L., Martinelli, C. E., Moreira, A. C., Antonini, S. R., & de Castro, M. (2013). A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Gene, 526(2), 239-45. https://doi.org/10.1016/j.gene.2013.03.082
Coeli-Lacchini FB, et al. A Rational, Non-radioactive Strategy for the Molecular Diagnosis of Congenital Adrenal Hyperplasia Due to 21-hydroxylase Deficiency. Gene. 2013 Sep 10;526(2):239-45. PubMed PMID: 23570880.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - A rational, non-radioactive strategy for the molecular diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase deficiency. AU - Coeli-Lacchini,Fernanda Borchers, AU - Turatti,Wendy, AU - Elias,Paula Conde Lamparelli, AU - Elias,Lucila Leico Kagohara, AU - Martinelli,Carlos Eduardo,Jr AU - Moreira,Ayrton Custodio, AU - Antonini,Sonir Roberto, AU - de Castro,Margaret, Y1 - 2013/04/06/ PY - 2012/12/20/received PY - 2013/03/12/revised PY - 2013/03/17/accepted PY - 2013/4/11/entrez PY - 2013/4/11/pubmed PY - 2013/10/19/medline KW - 17-OHP KW - 17-hydroxyprogesterone KW - 21-Hydroxylase deficiency KW - 21-hydroxylase deficiency KW - 21OHD KW - 30-kb deletion KW - 3′ untranslated KW - 3′UTR KW - 5′ untranslated KW - 5′UTR KW - ACTH KW - ASO-PCR KW - Adrenocorticotropic Hormone KW - CAH KW - CYP21A1P KW - CYP21A2 gene KW - Congenital adrenal hyperplasia KW - DHEAS KW - Dehydroepiandrosterone Sulfate KW - MLPA KW - Multiplex Ligation dependent Probe Amplification KW - NC KW - RCCX module KW - RFLP KW - RP, C4, CYP21 and TNX genes KW - SV KW - SW KW - allele specific oligonucleotide polymerase chain reaction KW - congenital adrenal hyperplasia KW - deletion of 30-kb including 3′-end CYP21A1P, C4B, and 5′-end CYP21A2 KW - non-classical KW - restriction fragment length polymorphism KW - salt-wasting KW - simple-virilizing SP - 239 EP - 45 JF - Gene JO - Gene VL - 526 IS - 2 N2 - CONTEXT: 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. PATIENTS AND 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. CONCLUSION: 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. SN - 1879-0038 UR - https://www.unboundmedicine.com/medline/citation/23570880/A_rational_non_radioactive_strategy_for_the_molecular_diagnosis_of_congenital_adrenal_hyperplasia_due_to_21_hydroxylase_deficiency_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0378-1119(13)00351-X DB - PRIME DP - Unbound Medicine ER -