Why do two genetic tests for SMAD4 (Smad family member 4) mutation yield different results?

Why Genetic Tests Show Discordant SMAD4 Results

Discordant SMAD4 genetic test results most commonly occur due to technical limitations in detecting specific mutation types, particularly the presence of a SMAD4 processed pseudogene that confounds quantitative assays, differences in testing methodologies (sequencing-only versus deletion/duplication analysis), and variable coverage of the gene across different testing panels.

Technical Causes of False-Negative Results

Processed Pseudogene Interference

• A SMAD4 processed pseudogene exists and directly confounds deletion/duplication assays, causing both false-positive and false-negative results depending on probe/primer placement. 1
• This pseudogene affects multiple testing platforms including multiplex ligation-dependent probe amplification (MLPA), chromosomal microarray, and next-generation sequencing by confounding sequence alignment and producing erroneous variant calls. 1
• The frequency of this processed pseudogene is approximately 0.26% (12 in 4,672 cases), making it a rare but clinically significant source of discordance. 1

Incomplete Gene Coverage

• Many genetic tests use hotspot panels that cover only approximately 80% of reported SMAD4 mutations, missing mutations outside the tested regions. 2
• Tests that perform sequencing without deletion/duplication analysis will miss whole or partial gene deletions, which have been reported in SMAD4. 3
• Clinical laboratories must offer full gene sequencing including copy number calling, as pathogenic variants are scattered throughout the gene. 3

Mutation Distribution and Detection Challenges

Common Mutation Locations

• The most common SMAD4 alterations are missense mutations at R361 and P356 in the MH2 domain, but mutations occur throughout the gene including frameshift, nonsense, splice site changes, and deletions of various sizes. 2, 4
• Approximately 95% of SMAD4 mutations result in alteration of protein regions involved in homo-oligomer and hetero-oligomer formation. 5

Variant Classification Issues

• Some variants may be classified as variants of uncertain significance (VUS) by one laboratory but as pathogenic by another, depending on available evidence and interpretation criteria at the time of testing. 6
• VUS classification changes over time as new evidence accumulates from additional families, functional studies, and population databases. 6

Recommended Testing Approach

Comprehensive Testing Strategy

• Use clinical laboratories that perform simultaneous sequencing AND deletion/duplication analysis of the entire SMAD4 gene to maximize detection sensitivity. 3
• Sanger sequencing confirmation is recommended for SMAD4 variants identified by next-generation sequencing due to pseudogene interference. 1
• When there is a confirmed diagnosis of SMAD4-related disease (juvenile polyposis-HHT overlap syndrome with appropriate clinical findings plus loss of SMAD4 expression), germline testing is strongly recommended regardless of somatic testing results. 3

Clinical Correlation is Essential

• The diagnosis should not rely on genetic testing alone—clinical Curaçao criteria for HHT and juvenile polyposis criteria remain the diagnostic foundation. 6
• Confidence in variant pathogenicity can be enhanced by immunohistochemistry showing loss of SMAD4 protein expression in tumor tissue, or by identifying a second pathogenic variant in the tumor. 3

Critical Pitfalls to Avoid

• Never assume a negative SMAD4 test excludes the diagnosis if clinical criteria are met—the test may have missed the mutation due to technical limitations. 6
• Do not use genetic testing results in isolation without considering the testing methodology employed (sequencing-only versus comprehensive analysis). 3
• Recognize that different laboratories may use different testing panels with variable gene coverage, leading to discordant results. 2
• If one test is positive and another negative, the positive result should generally be trusted if confirmed by an orthogonal method (such as Sanger sequencing), and clinical correlation with SMAD4 protein expression should be pursued. 1, 3