Performance Evaluation of the TheraTyper-GJB2 Assay for Detection of GJB2 Gene Mutations  Ji-Yong Chun, Soo-Kyung Shin, Kyung Tae Min, Woojae Cho, Jaeil Kim, Soo-Ok Kim, Sun Pyo Hong  The Journal of Molecular Diagnostics  Volume 16, Issue 5, Pages 573-583 (September 2014) DOI: 10.1016/j.jmoldx.2014.04.006 Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Schematic representation of GJB2 gene and connexin 26 protein illustrating the allelic variants studied. A: Three polymorphisms associated with HL in GJB2. Coding exons are shown as black bars and the 5′ and 3′ untranslated regions are shown as white bars. The first base of the translational start site is denoted as nucleotide +1. Locations and allele frequencies of three mutations are indicated. B: Normal connexin 26 protein. Each amino acid is indicated by a circle. C: Three abnormal connexin 26 protein produced by three mutations. The amino acid changes are represented by black circles. c.35delG, c.167delT, and c.235delC mutations are located in intracellular, extracellular, and transmembrane domains, respectively. The deletion of one residue of nucleotide introduces a stop codon and causes a severe deterioration of gap junction activity. EC, extracellular domain; IC, intracellular domain; TM, transmembrane domain. The Journal of Molecular Diagnostics 2014 16, 573-583DOI: (10.1016/j.jmoldx.2014.04.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Schematic representation of the TheraTyper-GJB2 assay strategy. PCR was performed with primers containing a type IIS restriction endonuclease recognition sequence (GGATG: FokI) ahead of each GJB2 target site: 35delG (black bars), 167delT (dark gray bars), and 235delC (white bars). Enzymatic digestion of the PCR products released a pair of 9 to 13 mer fragments representing nucleotide sequences within the target site, and the masses of the resulting oligonucleotide fragments were analyzed by mass spectrometry. Cleavage sites of FokI are indicated by triangles, restriction endonuclease recognition site and primers are indicated by the light gray bars and arrows, and mutation sites were indicated by the gray blocks in the middle of the bars under “Normal type,” respectively. MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The Journal of Molecular Diagnostics 2014 16, 573-583DOI: (10.1016/j.jmoldx.2014.04.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Representative results of the TheraTyper-GJB2 assay and direct sequencing assays. A: Reference standards were analyzed by the TheraTyper-GJB2 assay and direct sequencing. Molecular masses of 3910.2/4142.2, 3654.9/3743.0, and 3100.1/3173.4 represent the homogenous wild-type c.35delG, c.167delT, and c.235delC, respectively. Each variant region is indicated by boxes in the sequencing chromatogram. B: Molecular masses of 3621.1/3814.0, 3350.9/3429.1, and 2810.3/2844.2 represent the homogenous mutant type of c.35delG, c.167delT, and c.235delC, respectively. AI, absolute intensity; del, deletion; wt, wild type. The Journal of Molecular Diagnostics 2014 16, 573-583DOI: (10.1016/j.jmoldx.2014.04.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Comparison of the TheraTyper-GJB2 assay and direct sequencing assays for the detection of mixed genotypes. Direct sequencing analysis without deletion/frameshift mutations of c.35delG (A), c.167delT (B), and c.235delC (C). Each variant region is indicated by boxes in the sequencing chromatogram. D: Direct sequencing analysis with deletion/frameshift mutation of heterozygote at c.235delC. The variant region is indicated by a box in the sequencing chromatogram. c.35delG and c.167delT were not detected in our study. E: Mass peaks show wild type at c.35delG (3909.8 and 4141.5) and c.167delT (3655.2 and 3742.0). However, mass peaks of c.235delC show c.235delC without a deletion mutation (3099.7 and 3173.5) and c.235delC with a deletion (2810.8 and 2843.9). del, deletion; wt, wild type. The Journal of Molecular Diagnostics 2014 16, 573-583DOI: (10.1016/j.jmoldx.2014.04.006) Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions