Fig. 1

Fig. 1

a–f Identification of biallelic RNF170 mutations in four families and functional characterization. a Pedigree of the family in which genome sequencing identified a homozygous splice region mutation in RNF170 segregating with the disease. b Confirmation of the intronic variant c.396+3A>G in genomic DNA. c Gel electrophoresis and d consecutive Sanger sequencing confirmed the sole expression of a shorter transcript lacking exon 5 (wildtype transcript: 395bp; aberrant transcript: 321bp). e Quantitative real-time PCR from blood and fibroblast derived cDNA from individual A.4 demonstrated significantly reduced RNF170 expression in comparison with three control samples (Wilcoxon rank sum test, two-sided); f No residual RNF170 expression could be detected in patient fibroblasts. Note the unspecific band in the RNF170 western blot as well as the specific 25 kDa band corresponding to RNF170, that is abolished upon knockout of RNF170 in SH-SY5Y cells. g Pedigree of family B and h variant confirmation by Sanger sequencing. i Pedigree of family C and segregation in the family. j The deletion was confirmed by visual analysis of split reads in the IGV browser. k, l In addition, primers were designed flanking the breakpoints as well as the deletion. m Subsequent Sanger sequencing of the breakpoint fragment was used to further characterize the variant. n The frameshift variant segregating in family D could be confirmed by o Sanger sequencing