Overcoming the PROSP pseudogene challenge: accurate diagnosis of a novel PROS1 variant in a patient with dural venous...

Hereditary protein S deficiency, a risk factor for venous thromboembolism, presents diagnostic challenges due to the PROS1 gene's homologous pseudogene, PROSP. The pseudogene's homology risks co-amplification and misinterpretation of sequencing data, leading to false results and compromising clinical management. We report a case of a 28-year-old male who presented dural venous sinus thrombosis and low free protein S. After a novel PROS1 variant was identified via next-generation sequencing, a "pseudogene-aware" Sanger sequencing assay was developed to validate the finding. This assay utilized primers designed in silico method and high-temperature PCR to exploit nucleotide mismatches between PROS1 and PROSP, ensuring targeted amplification. The assay successfully validated a novel heterozygous missense variant, PROS1:c.1484T>C. This variant co-segregated with low protein S levels in the family and was classified as likely pathogenic. This pseudogene-aware strategy enabled the accurate identification of a novel variant causing severe thrombophilia, highlighting the importance of such assays for reliable molecular diagnosis and patient management.