A functional study for verifying the pathogenicity of a TRPM6 variant of uncertain significance: A novel non-canonical splicing-site variant in primary hypomagnesemia with secondary hypocalcemia

•The variant spectrum of TRPM6 is expanded.•Minigene assay uniting whole exome sequencing is applicable in genetic diseases.•The VUS site was confirmed to be a novel non-canonical splicing-site variant. Primary hypomagnesemia with secondary hypocalcemia (HSH) is a rare autosomal recessive disease caused by biallelic variants in TRPM6 gene. In this study, we reported a Chinese patient diagnosed with HSH in Tianjin Children’s Hospital. Detailed clinical examination and laboratory test were performed and whole exome sequencing (WES) was applied to detect the pathogenic gene in the proband. The suspected compound heterozygous variant in TRPM6 gene was verified by Sanger sequencing and quantitative real-time PCR technology. Minigene assay was performed to verify the function of the variant suspected to affect splicing process. The patient presented with seizure and markedly decreased levels of serum magnesium and calcium. WES combined with functional study diagnosed a pediatric patient with HSH caused by a compound heterozygous variant in TRPM6 gene, containing a novel non-canonical splicing-site variant c.5058-26A > G and a heterozygous deletion in exons 27–33 (chr9q21.13: 77357467–77376734). The compound heterozygous variant in TRPM6 gene is the pathogenic cause of the proband. The combined application of WES and functional study contribute to validating the effect of an uncertain genetic variant on splicing, improving the pathogenicity evidence and identifying the etiology of the disease. It is helpful for early diagnosis and treatment of HSH.