PMC4306731_01
Deletion of DOCK8 in a patient with HIES and PML. A. Telomeric region of chromosome 9p showing the deleted region and the impacted genes. B. Hypothetical ploidy observed in whole genome sequence. Note apparent single copy coverage near the telomere, then nearly 500 kb with no coverage, and then diploid coverage starting at approximately 586,000 bases. The letters a,b,c and d show the approximate location of PCR primers used to confirm the nature of the deletion. C. Results of PCR using the primers shown in 1B. Gel a: lanes 1,2, and 4 are controls, lane 3 is the HIES subject. Gels b, c and d: lane 1 is the HIES subject, lanes 2 and 3 are control subject, lane 4 is a no template control. Primer sets a, b, and c produce bands from the controls but not from the HIES subject, and primer set d only produces a band from the HIES subject
10875_2014_114_Fig1_HTML.jpg
PMC4306731_01_10875_2014_114_Fig1_HTML.jpg
Fig1
The WGS CNV analysis estimated that the patient was homozygous null for DOCK8 and some of KANK1, but could be hemizygous towards the telomere encompassing CBWD1, FOXD4, FAM138C, WASH1, and DDX11L5 (Fig. 1 panels A,B), and was consistent with the hypothesis that the patient had some DNA telomeric to DOCK8. To resolve the breakpoints for the individual's deletion(s) we designed targeted PCR around SNPs rs12353065, rs7853676 and rs11794423 which the WGS SNP analysis called high confidence SNPs, the WGS CNV analysis estimated to be hemizygous, and PCR primers unique to chromosome 9 could be designed (Fig. 1 panels B,C). Figure 1 panel C shows that the PCR with primers for the 3 SNPs (gels a, b and c) are all negative illustrating that the patient is in fact homozygous null from around the telomere into KANK1. To resolve the exact breakpoints we designed PCR primers close to the telomere and in KANK1 (PCR pair d Fig. 1 panel B,C). We performed a global gapped alignment with the Needleman-Wunsch algorithm of the resulting DNA fragment to chromosome 9 from bases 1-700,000 (NC_000009.11) and it revealed the alignment in Supplementary Figure 1.