Journal: Journal of the American Society of Nephrology : JASN
Article Title: Noninvasive Immunohistochemical Diagnosis and Novel MUC1 Mutations Causing Autosomal Dominant Tubulointerstitial Kidney Disease
Figure Lengend Snippet: MUC1 VNTR sequencing identifies novel mutations causing ADTKD- MUC1 . (A) Sequence logo showing the most conserved regions of the VNTR repeats. Corresponding amino acid sequences of wild-type MUC1 (wt_AA) and MUC1 fs (mut_AA) are shown below. To find novel frameshift mutations that change the open reading frame, different conserved 10-mers of the wild-type repeat were used as sequence anchors (underlined DNA sequence as an example). For each anchor pair, all sequences delimited by these two anchors that are changing an open reading frame ( i.e. , adding or deleting nucleotides) were selected from the FASTQ file. (B) Sequences of the canonical 60 nucleotide long wild-type VNTR repeat (wt) and candidate frameshift mutations identified in this study. (C) Random mutations are generated in DNA molecules during PCR amplification step. To find true germline mutations, the percentage of reads with a given sequence (putative frameshift mutation) from all reads was calculated for each of the analyzed samples ( y -axis), and this needed to be higher than the average+2 SD of the nine wild-type control samples. Indicated are numbers of controls (wt), patients with individual MUC1 mutations (27dupC, 28dupA, 26_27insG, 1–16dup, 23delinsAT, 51dupC), and individuals with still unknown MUC1 mutation(s) who have urinary cell smears positive for MUC1fs and who tested negative for 27dupC by conventional genotyping assay (unknown). (D) 27dupC, confirmed by a mass spectrometry-based primer extension assay. The 27dupC extension product is observed at 5904 D (red asterisk). (E) 28dupA, confirmed by a mass spectrometry-based assay. The 28dupA extension product is observed at 6571 D (red asterisk). (F) 26_27insG, confirmed by a mass spectrometry-based assay. The 26_27insG extension product is observed at 5944.85 D (red arrow). (G) 1–16dup confirmed by restriction analysis. The mutation creates new restriction site for Eci I enzyme. The electrophoretogram shows amplified VNTR regions of the affected patient (P1), two healthy relatives (H1, H2), and one unrelated control (NC) after (EciI) and before restriction by Eci I (PCR). The patient’s (P1) mutated allele (5000 bp) was cut into two fragments of 3000 and 2000 bp. (H) 23delinsAT, confirmed by restriction analysis. The mutation creates new restriction site for Fok I enzyme. The electrophoretogram is showing amplified VNTR regions of two affected patients (P1, P2) and one unrelated control (NC) after (FokI) and before restriction by Fok I (PCR). The patients’ (P1, P2) mutated alleles (3000 bp) were cut into two fragments of 2000 and 1000 bp.
Article Snippet: These sequences were then read with the Illumina system (see the following references for an explanation of the Illumina system)., The VNTR region was directly amplified from genomic DNA using primers PS2F-T7 (5′-GGATCCTAATACGACTCACTATAGGAACAGACCACCATGGGAGAAAAGGAGACTTCGGCTACCCAG-3′) and PS3 (specified above) and long-range PCR (TaKaRa LA Taq DNA Polymerase with GC Buffers; Takara, Mountain View, CA).
Techniques: Sequencing, Generated, Polymerase Chain Reaction, Amplification, Mutagenesis, Genotyping Assay, Mass Spectrometry, Primer Extension Assay