Reclassifying a Novel POMT1 Variant by Integrating Functional Analysis and Bioinformatics: Implications for Preimplantation Genetic Testing.

The advancement of next-generation sequencing has spurred the growing adoption of whole-exome sequencing (WES) for genetic screening. Preimplantation genetic testing for monogenic disorders (PGT-M) can effectively prevent the transmission of pathogenic variants. However, interpreting vast data volumes and ensuring precise genetic counseling, especially with variants of uncertain significance (VUS), remains challenging. In this study, we investigated a family with recurrent fetal malformations detected by prenatal ultrasound. WES identified compound heterozygous POMT1 variants, c.1052 + 1G > A and c.1483G > A in the proband; the latter was initially categorized as a VUS. Then our bioinformatics analysis revealed that c.1483G > A variant was located in a highly conserved domain essential for POMT1's enzymatic activity, potentially altering the protein's 3D structure. In vitro studies using HEK293T cells showed that the variant led to aberrant POMT1 mRNA and protein accumulation, impaired cell viability, and abnormal protein localization in the cytoplasm, indicating disruption of normal glycosylation processes. Combining bioinformatics analysis with in vitro experiments, we reclassified the c.1483G > A variant as likely pathogenic. Subsequently, the couple opted for PGT-M, culminating in the birth of a healthy child. Our findings underscore the pivotal role of genetic testing in recurrent fetal malformations and expand the spectrum of POMT1 variants. The successful reclassification of the variant by integrating in vitro experiments with bioinformatics provides substantial evidence for clinicians implementing PGT-M, offering a feasible strategy for counseling with VUS detected by WES.