RNA sequencing combined with whole-exome sequencing revealed familial homocystinemia due to MTHFR deficiency and its complex splicing events.

5,10-Methylenetetrahydrofolate reductase (MTHFR, MIM #607093) is a key enzyme in the folate cycle that catalyzes the conversion of 5,10-methylenetetrahydrofolate (5,10-MTHF) to 5-methyltetrahydrofolate (5-methylTHF), a critical step for the remethylation of homocysteine to methionine. Methylenetetrahydrofolate reductase deficiency is an autosomal recessive disease and the most common congenital defect in folate metabolism. A deficiency in MTHFR results in elevated serum homocysteine levels. In this study, we evaluated a patient diagnosed with epilepsy and elevated homocysteine levels, who carried compound heterozygous variants c.781-6G>A and c.1316T>C in the MTHFR gene. We primarily focused on the unreported non-canonical splicing variants c.781-6G>A in this patient and identified several complex splicing variant patterns. The c.1316T>C variant results in a substitution of leucine at position 439 with proline and this variant has been previously reported and is considered pathogenic. Our study mainly utilized RNA-seq and TA cloning to reveal the complex splicing patterns exhibited by this non-canonical splicing variant. Additionally, this finding was confirmed through in vitro experiments. This provided deeper insights into the underlying reasons for the patient's disease manifestation. Furthermore, despite apparently normal circulating folate and vitamin B12, we found two family members to exhibit mildly elevated homocysteine levels. While these individuals did not present overt clinical symptoms, the potential harm associated with high homocysteine levels should not be overlooked. This study not only provides additional genetic evidence for the clinical diagnosis of the patient but also broadens our understanding of the clinical manifestations of MTHFR deficiency.