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

Why Genetic Tests Show Discordant SMAD4 Results

Different genetic tests may show discordant SMAD4 mutation results because some laboratories perform only gene sequencing while others include deletion/duplication analysis—tests using sequencing alone will miss whole or partial gene deletions, which have been documented in SMAD4. 1

Technical Testing Differences Explain Discordance

Sequencing vs. Comprehensive Analysis

• The primary reason for discordant results is that not all clinical laboratories use the same testing methodology. 1
• Tests performing sequencing without deletion/duplication analysis will miss whole or partial gene deletions that have been reported in SMAD4. 1
• Pathogenic variants are scattered throughout the SMAD4 gene, requiring full gene sequencing including copy number calling to maximize detection sensitivity. 1

Laboratory Standards Vary

• Clinical laboratories must offer simultaneous sequencing AND deletion/duplication analysis of the entire SMAD4 gene to achieve optimal detection rates. 1
• The American College of Medical Genetics recommends using laboratories that perform both sequencing and deletion/duplication analysis simultaneously. 1

Clinical Implications of Missed SMAD4 Mutations

Critical Screening Consequences

• Failure to detect SMAD4 mutations has immediate and serious clinical implications—these patients are at risk for juvenile polyposis-HHT overlap syndrome with significantly increased gastrointestinal cancer risk. 2, 3
• SMAD4 mutations cause a combined syndrome of juvenile polyposis and hereditary hemorrhagic telangiectasia, with 100% penetrance of the polyposis phenotype. 4
• Three JP-HHT patients with SMAD4 mutations developed early onset colorectal cancer at mean age 28 years. 4

Screening Requirements for SMAD4 Carriers

• SMAD4 mutation carriers require upper GI tract surveillance every 1-3 years starting at age 18 years due to 73% prevalence of gastric polyposis. 5
• All gastric cancers in one cohort occurred exclusively in SMAD4 pathogenic variant carriers. 5
• Patients must undergo screening for pulmonary arteriovenous malformations using contrast echocardiography or chest CT, as these can be treated presymptomatically to prevent stroke and cerebral abscess. 5

Diagnostic Algorithm When Results Conflict

Step 1: Verify Testing Methodology

• Contact both laboratories to determine whether deletion/duplication analysis was performed in addition to sequencing. 1
• If one test used sequencing only while the other included copy number analysis, the comprehensive test is more reliable. 1

Step 2: Apply Clinical Diagnostic Criteria

• Never rely on genetic testing alone—clinical Curaçao criteria remain the diagnostic foundation for HHT. 6
• For HHT diagnosis, require 3 of 4 features: spontaneous/recurrent epistaxis, multiple telangiectasias at characteristic sites, visceral arteriovenous malformations, or affected first-degree relative with HHT. 6
• For juvenile polyposis, assess for three to five cumulative histologically proven juvenile GI polyps, or any number with positive family history. 7

Step 3: Consider Repeat Testing at Comprehensive Laboratory

• Retest at a laboratory performing simultaneous sequencing AND deletion/duplication analysis of the entire SMAD4 gene. 1
• When there is confirmed diagnosis of SMAD4-related disease clinically, germline testing is strongly recommended regardless of previous testing results. 1

Step 4: Enhance Variant Pathogenicity Assessment

• 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. 1
• Collect family segregation data by testing affected and unaffected relatives to help reclassify variants of uncertain significance. 6

Common Pitfalls to Avoid

Do Not Assume Negative Test Excludes Diagnosis

• 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
• In unselected HHT patients negative for ENG and ALK1 mutations, 10% (3/30) harbored SMAD4 mutations. 2

Do Not Delay Screening Based on Negative Result

• If clinical features suggest JP-HHT overlap syndrome, initiate appropriate screening regardless of genetic test results. 6
• For patients with gastrointestinal polyps or family history of juvenile polyposis, initiate GI surveillance even with uncertain genetic results. 6
• Begin colonoscopic surveillance at age 15 years or earlier if symptomatic, with 1-3 year intervals based on polyp burden. 6

Recognize Testing Limitations in Asymptomatic Relatives

• Direct genetic testing significantly improves presymptomatic diagnosis of gene carriers in JP families with SMAD4 mutations. 8
• With genetic testing, non-carriers may no longer require frequent screening endoscopy, whereas gene carriers can be targeted for close endoscopic surveillance and early intervention. 8

Management During Diagnostic Uncertainty

Implement Risk-Based Surveillance

• If one test is positive and clinical features support the diagnosis, proceed with full SMAD4-specific screening protocols regardless of the negative test result. 5
• All patients with SMAD4 mutations must be managed in conjunction with a specialist HHT center with experience in evaluating and managing both HHT and juvenile polyposis complications. 5

Coordinate Multisystem Screening

• Perform Doppler ultrasonography as first-line imaging for liver involvement, but never perform liver biopsy due to catastrophic hemorrhage risk. 5
• Screen for cerebral vascular malformations with brain MRI, as cerebral AVMs occur more commonly in HHT1 and nearly one in five HHT patients develop stroke or cerebral abscess. 5