Direct Assessment of Hereditary Hemochromatosis in Preimplantation Genetic Testing.

Objective: Hereditary hemochromatosis (HH) is a common genetic disorder characterized by iron overload, which, if undiagnosed, can lead to severe organ damage. There are four types of HH. Type 1 HH, the most common form, is primarily caused by a common variant in Western Europe (p.Cys282Tyr, C282Y, or c.845 G>A). It is generally preventable during in vitro fertilization (IVF) if proper genetic testing is done before implantation. Here, we demonstrate a direct detection and cost-effective approach using PCR-RFLP in PGT settings.

Design: We began by validating the assay with genomic DNA from Coriell cell lines of known HFE C282Y genotypes, followed by testing patients' genomic DNA samples. After establishing the assay on genomic DNA, we extended the assay to whole-genome amplified DNA from embryo biopsies.

Subjects: The subjects include cell line samples and human specimens, and human embryo biopsies.

Exposure: Patients and embryos either carry or do not carry the HFE C282Y variant in their genome. No intervention was applied.

Main Outcome Measures: The readout includes the genotype of samples at the HFE C282Y locus and the accuracy of PCR-RFLP results.

Results: An accuracy of over 99% was achieved across 80 cell line samples, 38 patient samples, and 81 embryo biopsies.

Conclusion: In this study, we demonstrated the feasibility of using the PCR-RFLP approach to preimplantation genetic testing. Specifically, we validated the assay for the HFE C282Y variant, the primary cause of type 1 hemochromatosis. The assay was tested on genomic DNA and DNA resulting from whole genome amplification, achieving over 99% accuracy, sensitivity, precision, and specificity. These results also suggest the possibility for extending the PCR-RFLP approach to cover a broader range of conditions, such as SMA, to benefit more patients currently ineligible for testing at PGT labs.