title: 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.
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah. Electronic address: millsoas@aruplab.com.
authors.forename: Alison
authors.initials: A
authors.lastname: Millson
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Tracey
authors.initials: T
authors.lastname: Lewis
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Tina
authors.initials: T
authors.lastname: Pesaran
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: David
authors.initials: D
authors.lastname: Salvador
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Katrina
authors.initials: K
authors.lastname: Gillespie
authors.affiliation: Ambry Genetics, Aliso Viejo, California.
authors.forename: Chia-Ling
authors.initials: CL
authors.lastname: Gau
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah.
authors.forename: Genevieve
authors.initials: G
authors.lastname: Pont-Kingdon
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Pathology Department, University of Utah, Salt Lake City, Utah.
authors.forename: Elaine
authors.initials: E
authors.lastname: Lyon
authors.affiliation: ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah; Pathology Department, University of Utah, Salt Lake City, Utah.
authors.forename: Pinar
authors.initials: P
authors.lastname: Bayrak-Toydemir
mesh_terms: D004252:DNA Mutational Analysis / Q000379:methods; D003937:Diagnosis, Differential; D005189:False Positive Reactions; D017353:Gene Deletion; D020440:Gene Duplication; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D044483:Intestinal Polyposis / Q000151:congenital / Q000175:diagnosis / Q000235:genetics*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D009386:Neoplastic Syndromes, Hereditary / Q000175:diagnosis / Q000235:genetics; D011544:Pseudogenes / Q000235:genetics*; D016415:Sequence Alignment; D051901:Smad4 Protein / Q000235:genetics*; D013683:Telangiectasia, Hereditary Hemorrhagic / Q000175:diagnosis / Q000235:genetics*
chemical_list: C497796:SMAD4 protein, human; D051901:Smad4 Protein