Why would an SMAD4 (SMAD family member 4) gene mutation be detected in one genetic test but not others?

Why SMAD4 Mutations May Be Detected in One Genetic Test But Not Others

SMAD4 mutations can be missed by genetic tests that perform only sequencing without deletion/duplication analysis, as whole or partial gene deletions have been reported in SMAD4 and will not be detected by sequencing-only approaches. 1

Technical Limitations of Different Testing Methodologies

Sequencing-Only Tests Miss Structural Variants

• Tests performing only DNA sequencing will fail to detect large deletions or duplications affecting one or more exons of SMAD4, which represent a significant proportion of pathogenic variants 1
• Single-strand conformation polymorphism analysis followed by sequencing (an older methodology) may miss certain mutation types that newer comprehensive approaches would detect 2
• Clinical laboratories must offer full gene sequencing including copy number calling to maximize detection sensitivity 1

Gene Panel Composition Varies Between Laboratories

• SMAD4 is not included in all hereditary cancer panels—it appears only in comprehensive panels but is absent from more restricted testing options 3
• The GeneDx Breast/Ovarian Cancer Panel does not include SMAD4, while their Comprehensive Cancer Panel does include it 3
• Similarly, the Baylor College of Medicine Hereditary Breast/Ovarian Cancer Panel excludes SMAD4, but their Comprehensive Hereditary Cancer Panel includes it 3
• A patient tested with a restricted panel may receive a "negative" result simply because SMAD4 was never analyzed 3

Evolution of Testing Technology Over Time

Historical Testing Limitations

• Genetic testing performed years ago may have used older methodologies with lower sensitivity for detecting all mutation types 3
• The medical provider must recognize newer testing options that were not carried out as part of initial testing, particularly for high-risk mutation-negative patients 3
• Simply reporting a "negative panel test" is only partially informative without documenting which specific genes were tested and when 3

Importance of Test Date Documentation

• At minimum, test results should always be reported with the date of testing if the number of genes tested are too numerous to be included in a clinical summary 3
• A woman who tested negative for mutations in 1998 could still potentially be found to be a mutation carrier if complete rearrangement testing was carried out with modern techniques 3

Critical Clinical Implications

Never Rely on Negative Testing Alone

• The diagnosis should not rely on genetic testing alone—clinical criteria for HHT and juvenile polyposis remain the diagnostic foundation 1
• 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 1
• The presence of clinical features consistent with juvenile polyposis syndrome or HHT-juvenile polyposis overlap should prompt comprehensive re-testing even if prior results were negative 3

Recommended Comprehensive Testing Approach

• The American College of Medical Genetics recommends using clinical laboratories that perform simultaneous sequencing AND deletion/duplication analysis of the entire SMAD4 gene 1
• Pathogenic variants are scattered throughout the SMAD4 gene, requiring complete gene analysis rather than hotspot testing 1
• When there is a confirmed diagnosis of SMAD4-related disease, germline testing is strongly recommended regardless of somatic testing results 1

Enhancing Confidence in Results

Complementary Diagnostic Approaches

• Confidence in variant pathogenicity can be enhanced by immunohistochemistry showing loss of SMAD4 protein expression in tumor tissue 1, 4
• In normal and reactive states, SMAD4 protein localizes to both cytoplasm and nucleus, while the protein is lost in high-grade lesions and cancer 4
• Identifying a second pathogenic variant in tumor tissue (loss of heterozygosity) provides additional evidence for pathogenicity 1

Mutation Location Matters

• Most SMAD4 mutations cluster in the C-terminal MH2 domain, with 80% located in the mutational hotspot "mutation cluster region" 5
• However, mutations outside this region still occur and must be detected—comprehensive gene sequencing is essential 1, 5
• SMAD4 mutations affect protein expression to different extents depending on their location within the gene 5

Common Clinical Pitfall to Avoid

The most critical error is accepting a "negative" genetic test result without understanding the specific methodology used, which genes were included in the panel, and when the testing was performed. 3, 1 This is particularly important for SMAD4 given its association with life-threatening complications including gastrointestinal cancers, arteriovenous malformations causing stroke and cerebral abscess, and aortopathy requiring specialized surveillance. 6, 7