The inheritance assignment of homozygous variants appears inconsistent with the reported methodology. For example, PCSK9 c.G1420A and APOB c.A6937G are reported as homozygous in the proband and inherited from both parents. However, the Sanger sequencing validation was performed using forward primers only, which typically prevents accurate phasing and zygosity confirmation for homozygous calls. How was homozygosity definitively determined without bidirectional sequencing or additional linkage analysis? If the inheritance patterns are misassigned, the core trio-based analysis and variant prioritization could be fundamentally flawed. Could you clarify how zygosity was validated for these critical variants?
Dear Reviewer,
Thank you for your careful reading of the manuscript and for raising an important point regarding the validation of homozygous variants and inheritance assignment in our trio-based analysis.
You are correct that the Sanger sequencing validation for the reported PCSK9 c.G1420A and APOB c.A6937G variants was performed using forward primers only. We acknowledge that unidirectional sequencing has inherent limitations for definitive confirmation of zygosity and does not allow robust phasing or exclusion of allele-specific amplification artefacts.
At the time of analysis, homozygosity was initially inferred based on concordant next-generation sequencing (NGS) variant allele frequencies across the trio, supported by consistent signal intensity observed in the forward Sanger traces. However, we agree that this approach alone is not sufficient to conclusively confirm homozygosity or inheritance patterns.
In response to this concern, we are currently performing additional Sanger sequencing using reverse primers for these variants to enable bidirectional validation. The updated results will allow more confident confirmation of zygosity and reassessment of the inheritance model where necessary. Any changes arising from this validation will be clearly reported, and the interpretation of the trio-based analysis and variant prioritisation will be revised accordingly.
We appreciate this constructive feedback, which has helped strengthen the methodological rigour and transparency of our study.
Thank you.
Sincerely,
Zam Zureena Mohd Rani