Minigene splicing assay

A minigene splicing assay was performed to verify whether the mutation affected splicing products. To test the effect of the candidate mutation c.3470 G > A in HFM1 on splicing, amplicons generated by standard overlapping PCR and digestion procedures were cloned into the pcDNA3.1 reporter vector (Life Technologies, New York, USA). PCR and Sanger sequencing were used to evaluate whether the wild-type (WT) and mutant-type (MT) expression vectors had been successfully constructed.

WT or MT HFM1 minigenes were transiently transfected into HeLa cells and 293 T cells using Liposomal Transfection Reagent(40802ES03, YEASEN, Shanghai, China) according to the manufacturer’s instructions.

All cellular RNAs were extracted using the RNAiso Plus (Code No.9109, Takara, Otsu, Japan) according to the manufacturer’s recommendation. Total RNA was used to produce cDNA using the PrimeScript RT reagent Kit with gDNA Eraser (Cat#RR047A, Takara, Otsu, Japan). Then, the first-strand cDNA was synthesized and amplified. The PCR products were separated and verified by electrophoresis in an agarose gel and characterized by direct Sanger sequencing.