Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase.

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Validation and Clinical Application of a Locus-Specific Polymerase Chain Reaction- and Minisequencing-Based Assay for Congenital Adrenal Hyperplasia (21-Hydroxylase Deficiency) Dianne Keen-Kim, Joy B. Redman, Reno U. Alanes, Michele M. Eachus, Robert C. Wilson, Maria I. New, Jon M. Nakamoto, Raymond G. Fenwick The Journal of Molecular Diagnostics Volume 7, Issue 2, Pages 236-246 (May 2005) DOI: 10.1016/S1525-1578(10)60550-8 Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Amplification scheme for CYP21 genes and relative position of mutations within CYP21. A: Primers ME0008 and ME0066 amplify the functional CYP21A2 gene (amplicon 1), whereas primers ME0059 and ME0067 amplify the CYP21A pseudogene (amplicon 2). Relative positions of the EcoRI sites used for demonstration of locus-specific amplification are designated by RI. B: Primers ME0059 and ME0066 amplify the CYP21A/A2 fusion gene created when a 30-kb deletion occurs (amplicon 3). C: Primers ME0008 and ME0067 amplify the CYP21A2/A rearrangement product (amplicon 4). D: Relative positions of the exons, 10 common point mutations and a small deletion (G110Δ8nt) detected in the assay. IVS2-13 A/C→G is designated In2G. The Journal of Molecular Diagnostics 2005 7, 236-246DOI: (10.1016/S1525-1578(10)60550-8) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Examples of PCR data. A: Example of the PCR amplicons produced in a typical wild-type sample. M, molecular size standard (bp). Lane 1: amplicon 1, the CYP21A2 amplicon runs at 3.4 kb, while the internal standard runs at ∼1 kb; lane 2: amplicon 2, the CYP21A amplicon runs at 4.0 kb, while the internal standard runs at ∼1 kb; lanes 3 and 4: amplicons 3 and 4, no rearrangement amplicons are present, but the internal standard is present at ∼1 kb. B: Example of the PCR amplicons produced by a sample containing a pseudogene, 30-kb deletion and gene conversion rearrangement loci, but no CYP21A2 fragment. M, molecular size standard (bp). Lane 1: amplicon 1, no CYP21A2 amplicon present, but the internal standard is present at ∼1 kb; lane 2: amplicon 2, the CYP21A amplicon migrates at 4.0 kb, while the internal standard migrates at ∼1 kb; lane 3: the CYP21A/A2 deletion amplicon migrates at 4.0 kb, and the internal standard migrates at ∼1 kb; lane 4: the CYP21A2/A rearrangement amplicon migrates at 3.4 kb, and the internal standard migrates at ∼1 kb. The Journal of Molecular Diagnostics 2005 7, 236-246DOI: (10.1016/S1525-1578(10)60550-8) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Examples of minisequencing data and reporting scheme. A: Minisequencing results for the amplicons obtained in Figure 2A. Peaks obtained for wild-type alleles in the CYP21A2 amplicon are labeled black and mutant alleles are labeled red. Minisequencing results are not shown for amplicons 3 and 4, which were absent in the PCR. B: Reporting scheme for minisequencing data in A. Rows represent the four possible amplicons from the four PCR reactions. Columns report the presence or absence of each PCR amplicon and the genotype of each locus. Squares are colored black for wild-type alleles and red for mutant alleles. IVS2-13 A/C→G is designated In2G. C: Minisequencing results for the amplicons obtained in Figure 2B. D: Reporting scheme for minisequencing results in C. The Journal of Molecular Diagnostics 2005 7, 236-246DOI: (10.1016/S1525-1578(10)60550-8) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Sample reporting scheme for clinical samples. A: Reporting for sample 7215, a WT/Q318X/IVS2-13 A/C→G (designated In2G) normal carrier. Wild-type alleles are illustrated as black squares, mutant alleles as red squares, and heterozygotes as red cross-hatched squares. This patient is known to be normal from biochemical data, as well as the presence of three CYP21A2 alleles, demonstrated by multiple heterozygosity at the IVS2-13 locus. B: Reporting for sample 7257, a V281L/V281L-affected sample. Although amplicon 3 appears to be a 30-kb deletion fragment with an intron 7 breakpoint, this rearrangement fragment was shown to be a duplicated gene fragment (see text). C: Reporting for sample 7243, a V281L/30-kb del-affected patient. Heterozygosity in amplicon 3 reveals the presence of two rearrangement fragments, one with an intron 3 breakpoint and a second with an intron 7 breakpoint. The Journal of Molecular Diagnostics 2005 7, 236-246DOI: (10.1016/S1525-1578(10)60550-8) Copyright © 2005 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions