Fig 1.
Characterization of the test sample set.
A) Samples from previously reported patients with Alu insertion in MAK exon 9 [21] and control samples were PCR amplified to detect homozygous alleles for Alu insertion and WT alleles. B) Sequence of the inserted element (280 bp Alu, 54 bp poly-A and 13 bp duplication of exon 9 sequence). C) Sanger sequence of the exon 9 Alu insertion breakpoints.
Fig 2.
Alignment of standard BWA-based Illumina reads of a control (top) and a MAK-Alu homozygous (bottom) sample.
MAK-Alu alignment produces a coverage gap in exon 9 but does not clearly identify an insertion.
Table 1.
Specificity and Sensitivity of In Silico Method to Detect the MAK-Alu insertion.
Table 2.
Identification of Homozygous and Heterozygous MAK-Alu Insertions in a Discovery Sample Set.
Fig 3.
PCR validation of Alu insertion identified by in silico analysis in patients from the discovery cohort.
A) PCR amplification using primers spanning exon 9 (top) and nested PCR using Alu-specific primer (bottom). The 1,194 bp amplicon containing the Alu insertion (arrow) is present strongly in the homozygous sample and weakly in the heterozygous samples (top); the Alu insertion-specific amplification (491 bp, bottom) confirms the presence of the Alu insertion. B) Pedigree of patient D379_148, carrying a missense mutation (p.Gly16Arg) and the Alu insertion mutation. C) Evolutionary conservation of glycine 16, mutated in the patient D379_148. D) Protein domains in MAK and location of the p.Gly16Arg change. The mutation annotations are based on the NM_001242957 transcript, where A from the ATG start codon is designated as a +1 position.