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Reduced expression of KIF11 (EG5) and splicing abnormalities suggest haploinsufficiency as the disease mechanism in MLC. (A) EG5 (KIF11) protein domain structure indicating the position of the MLC variants p.L347Efs*8 and p.R387*. (B) qRT-PCR analysis of blood using allele-specific primers (mut: patient variant allele) showing strong reduction but not a complete absence of the mutant mRNA in all samples (F2-I.2, F2-II.1, F1-I.2 and F1-II.1), suggesting nonsense-mediated RNA decay. WT KIF11 mRNA levels decreased by up to 50% in patients compared with controls. Data represent average relative expression (n = 3 experiments) ± SD. (C) Western blot analysis of lymphoblastoid cell line lysates indicate approximately 50% reduction in the levels of WT EG5 protein (119 kDa) in the patient samples (F2-I.2, F2-II.1, and F1-II.1) using C-terminal binding EG5 antibody (NB500-181, Novus Biologicals). Average of 2 technical repeats of identical biological materials is shown with β-actin as loading control. (D) Western blot analysis with an N-terminal binding anti-EG5 antibody (CC10014, Cell Applications) showed the presence of a truncated protein of the expected size in patient F1-I.2 (arrow). The position of molecular mass markers (in kDa) is indicated on the left of the gel. (E) Left panel: Sanger sequencing of gDNA from proband F5-II.1. PCR product shows a compound peak in exon 20, which is the synonymous KIF11 c.2922G>A; p.(P974=) variant. Right panel: Sanger sequencing of cDNA from F5-II.1. Black line indicates the boundary between the last base of exon 20 and first base of exon 21. Notice the heteroduplex in exon 20 indicating exon skipping. Analysis of the full trace identified a loss of the last 108 bases in exon 20 (r.2815_2922del), which is predicted to lead to a shorter EG5 protein similar to that of the synonymous variant c.2922G>T as shown by others (46).
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