Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4

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Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4 Alison Millson, Tracey Lewis, Tina Pesaran, David Salvador, Katrina Gillespie, Chia-Ling Gau, Genevieve Pont-Kingdon, Elaine Lyon, Pinar Bayrak-Toydemir The Journal of Molecular Diagnostics Volume 17, Issue 5, Pages 576-582 (September 2015) DOI: 10.1016/j.jmoldx.2015.05.005 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Multiplex ligation–dependent probe amplification (MLPA) peak ratio results for the processed pseudogene. Two-copy range: 0.66 to 1.34; duplication range: ≥1.35. Ratios for the SMAD4 duplicated probes, exons 2 to 3 and 5 to 12, are ≥1.35. Two-copy probes cluster around the peak ratio of 1. The SD bars result from the analysis of the patient compared with four wild-type controls. PTEN and BMPR1A are additional genes included in the MLPA kit, along with the two-copy control genes. The Journal of Molecular Diagnostics 2015 17, 576-582DOI: (10.1016/j.jmoldx.2015.05.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Yellow highlighted regions are exons 2 to 12 with the exception of exon 4, which is highlighted in green. Probes detecting two copies center on the black line representing a ratio of 1 when compared with the labeled control DNA. Probes detecting duplicated regions appear above the gray line that denotes the upper limit of the two-copy range. Duplication of probes is seen in all transcribed exons (2 to 12) with the exception of exon 4. The Journal of Molecular Diagnostics 2015 17, 576-582DOI: (10.1016/j.jmoldx.2015.05.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 A: Gel shows products amplified using a forward primer in exon 3 and a reverse primer in exon 4. Patient DNA produces two bands: a 517-bp product containing intron 3 sequence in addition to sequence from exons 3 and 4 and an 82-bp product containing only exonic sequence. The wild-type control DNA produces only the larger 517-bp product. B: Sequencing of the 82-bp product produced by the patient DNA confirms the junction of exons 3 and 4 and the resulting absence of intronic sequence. The Journal of Molecular Diagnostics 2015 17, 576-582DOI: (10.1016/j.jmoldx.2015.05.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Next-generation sequencing data reveal errant variant calls (yellow) produced by the capture of the processed pseudogene. Reads produced from the functional SMAD4 are shown in pink and contain intronic sequence. Reads produced from the processed pseudogene are shown in purple and contain only exonic sequence (exon 6 sequence immediately follows sequence from exon 5). Variants were called at positions when the captured processed pseudogene sequence differed from the functional SMAD4. A: There are 824 total reads at position c.667+3G>C with 153 C variants reads from the processed pseudogene. B: There are 785 total reads at position c. 668-1G>A with 77 reads from the processed pseudogene. The Journal of Molecular Diagnostics 2015 17, 576-582DOI: (10.1016/j.jmoldx.2015.05.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Clinical data from Ambry Genetics: clinical histories of patients with SMAD4 mutations/variants likely pathogenic identified in the first 2079 panels performed at Ambry were compared with the individuals who were found to have the SMAD4 pseudogene on panels. Six of eight patients who had SMAD4 mutations and/or variants reported likely pathogenic juvenile polyps on their test requisition. Zero of eight patients who tested positive for the SMAD4 processed pseudogene duplication reported juvenile polyps. The Journal of Molecular Diagnostics 2015 17, 576-582DOI: (10.1016/j.jmoldx.2015.05.005) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions