Fig. 2.

The novel variants with different variants types locating after vitB12-binding domain are related to partial dysfunction of Cubilin. A ClustalW multiple sequence alignment of Cubilin protein in several species. The novel missense variant (p.C1466Y) identified in one proband was located at a highly conserved position in Cubilin protein, as highlighted in black box. The asterisk signs below the sequence alignment indicate evolutionary conserved residue, the colon signs indicate highly conserved residue and the period signs represent less conserved residue. B CUBN minigene splice assay. The pSPL3 reporter minigene construct used in this functional assay and subcloning of the genomic CUBN fragment from wild- type and mutant alleles. RT-PCR analysis of transcripts derived from the indicated reporter assay in HEK293T cells and sequence analysis of the electrophoresis gel recovery product. C Sequencing of the above bands revealed that the splicing variant (c.6821 + 3A > G) resulted in exon 44 skipping and early termination of the amino acids. D Western blot analysis for Cubilin expression of the four variants. E Immunohistochemistry (IHC) staining showed the expression of Cubilin in cubn probands’ kidney biopsies exhibited markedly decline when compared to MCD and HC. M1: CUBN (c.4397G > A), M2: CUBN (c.6796C > T), M3: c.5153_5154delCT and M4: c.6821 + 3A > G