Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4

Abstract

Mutations in SMAD4 have been associated with juvenile polyposis syndrome and combined juvenile polyposis/hereditary hemorrhagic telangiectasia syndrome. SMAD4 is part of the SMAD gene family. To date, there has been no report in the literature of a SMAD4 pseudogene. An unusual SMAD4 duplication pattern was seen in multiple patient samples using two different duplication/deletion platforms: multiplex ligation-dependent probe amplification and chromosomal microarray. Follow-up confirmatory testing included real-time quantitative PCR and sequencing of an exon/exon junction, all results leading to the conclusion of the existence of a processed pseudogene. Examination of clinical results from two laboratories found a frequency of 0.26% (12 in 4672 cases) for this processed pseudogene. This is the first report of the presence of a processed pseudogene for SMAD4. We believe that knowledge of its existence is important for accurate interpretation of clinical diagnostic test results and for new assay designs. This study also indicates how a processed pseudogene may confound quantitative results, dependent on placement of probes and/or primers in a particular assay design, potentially leading to both false-positive and false-negative results. We also found that the SMAD4 processed pseudogene affects next-generation sequencing results by confounding the alignment of the sequences, resulting in erroneous variant calls. We recommend Sanger sequencing confirmation for SMAD4 variants.

Development and Validation of a 34‐Gene Inherited Cancer Predisposition Panel Using Next‐Generation Sequencing

“…e unique feature of processed pseudogenes, lacking promoter and intronic sequences, may cause false-positive exonic duplication calls. e presence of an SMAD4 processed pseudogene was first reported in 2015 in a subset of individuals, with a frequency of 0.26% (12/4,672 clinical cases) [41]. A similar frequency (0.25%, 58/23,032 clinical cases, data not shown) was observed from our clinical results.…”

Identification of prognostic molecular biomarkers in 157 HPV‐positive and HPV‐negative squamous cell carcinomas of the oropharynx

“…A transcription factor gene RUNX1 showed a 3 kb inversion involving exon 3 disrupting the RUNT domain. In one case, NTRK2 showed multiple duplications/deletions suggestive of a processed pseudogene . Among HPV positive cases, JAK3 was affected in one case showing a 50 kb deletion involving exon 6 and the remaining downstream exons encoding the entire kinase domain (amino acids 521–777, 822–1,095) and FERM domain.…”

Navigating highly homologous genes in a molecular diagnostic setting: a resource for clinical next-generation sequencing

“…For example, a processed pseudogene that is homologous to SMAD4 has been found in ~0.26% of the population. 19 Further studies are necessary to continue to elucidate the variability in homologous sequences among individuals. Long-read sequencing technologies, such as those developed by Pacific Biosciences and Oxford Nanopore Technologies, can largely overcome the problem of inaccurate alignment of homologous reads, although these platforms have not yet been implemented in many diagnostic laboratories.…”