Various mutations in coding sequences have been documented in iron metabolism disorders. High throughput DNA sequencing data reveal a growing number of variations in non-coding regions. The impact of these variations need to be assessed in order to guide interpretation and classify the mutation. In this study, we focus on splicing variants identified in our cohort of nation-wide recruited patients harbouring iron metabolism disorders.  The splicing process occurs during RNA maturation and is finely regulated by multi-factor complexes that interact with specific nucleotide domains, depending on tissue specificity, environmental or developmental signals. Variations affecting these sequences may induce non-appropriate alternative splicing and thus lead to a loss of function or expression of the protein. These variants are increasingly studied in all pathologies, preferably by analysis of transcripts on blood samples of patients.

Variants selected for this study were predicted to affect splicing process in different genes of iron metabolism (TFR2, HFE, SLCA11A2, TF and CP). Minigene assay and, when available, patients’ blood transcripts analysis (RNA targeted PCR and/or RNA sequencing), were performed to determine the impact of these variants. Experimental results were compared with patients’ clinical data.

This study showed us that even if these assay can converge and allow to classify certain variants, others present a discrepancy of results depending on the test used. Moreover, due to the low blood expression of these genes, minigene functional test in different cell lines could be more helpful to classify splicing variants in iron metabolism disorders.