Processed Pseudogene Confounding Deletion/Duplication Assays for SMAD4

Area of Science:

• Genetics
• Molecular Biology
• Clinical Diagnostics

Background:

• Mutations in SMAD4 are linked to juvenile polyposis syndrome and related disorders.
• The SMAD gene family includes SMAD4, but a SMAD4 pseudogene has not been previously reported.

Purpose of the Study:

• To report the discovery of a processed SMAD4 pseudogene.
• To highlight the implications of this pseudogene for genetic testing and diagnostic accuracy.

Main Methods:

• Utilized multiplex ligation-dependent probe amplification and chromosomal microarray for initial detection.
• Employed real-time quantitative PCR and exon/exon junction sequencing for confirmation.
• Analyzed clinical data from two laboratories to determine pseudogene frequency.

Main Results:

• Confirmed the existence of a processed SMAD4 pseudogene.
• Found a frequency of 0.26% (12/4672 cases) for the SMAD4 processed pseudogene.
• Demonstrated that the pseudogene can lead to false-positive/false-negative results in quantitative assays and erroneous variant calls in next-generation sequencing.

Conclusions:

• This is the first report of a processed SMAD4 pseudogene.
• Knowledge of this pseudogene is essential for accurate interpretation of clinical diagnostic tests.
• Recommends Sanger sequencing for SMAD4 variant confirmation to avoid pseudogene-related errors.