Intragenic DOK7 deletion detected by whole-genome sequencing in congenital myasthenic syndromes

Abstract

Objective: To identify the genetic cause in a patient affected by ptosis and exercise-induced muscle weakness and diagnosed with congenital myasthenic syndromes (CMS) using whole-genome sequencing (WGS).

Methods: Candidate gene screening and WGS analysis were performed in the case. Allele-specific PCR was subsequently performed to confirm the copy number variation (CNV) that was suspected from the WGS results.

Results: In addition to the previously reported frameshift mutation c.1124_1127dup, an intragenic 6,261 bp deletion spanning from the 5' untranslated region to intron 2 of the DOK7 gene was identified by WGS in the patient with CMS. The heterozygous deletion was suspected based on reduced coverage on WGS and confirmed by allele-specific PCR. The breakpoints had microhomology and an inverted repeat, which may have led to the development of the deletion during DNA replication.

Conclusions: We report a CMS case with identification of the breakpoints of the intragenic DOK7 deletion using WGS analysis. This case illustrates that CNVs undetected by Sanger sequencing may be identified by WGS and highlights their relevance in the molecular diagnosis of a treatable neurologic condition such as CMS.

Figure 1. Whole-genome sequencing analysis and allele-specific PCR

(A) Both index case and his mother show reduced read depth (coverage) from exon 1 to deep intron 2 of the DOK7 gene (red arrow). Controls 1–4 correspond to samples sequenced and analyzed through the same pipeline and without the diagnosis of congenital myasthenic syndromes. (B) Split reads were observed at both presumed breakpoints. Nucleotides matching the reference sequence of DOK7 are highlighted in orange/blue. Single unmatched nucleotides are highlighted in yellow, and further unmatched sequences are not highlighted. The unmatched sequence (indicated with red/green underline) of the split reads of the proximal breakpoint aligns to the reference sequence (indicated in green/red boxes) at the distal breakpoint, and vice versa. (C) The expected products amplified by allele-specific PCR were identified in the index case and the mother. (D) The junction of the breakpoint in the allele with the intragenic deletion was confirmed by Sanger sequencing of the PCR product. Coverage and reads were drawn by the graphical user interface of Sequence Miner 5.21.1 (WuXi NextCODE).

Figure 2. Analysis of the breakpoints of the intragenic 6-kb deletion

(A) University of California Santa Cruz genome browser (genome.ucsc.edu/) view of the deleted region showing the Simple Tandem Repeats track (based on Tandem Repeats Finder, TRF) and the Repeating Elements track (based on RepeatMasker). GT-rich repeat regions (green box) are seen around the distal breakpoint, and a G-rich region (green arrow) is located near the proximal breakpoint. (B) The secondary DNA structure with the lowest delta G value was predicted by the mfold tool (unafold.rna.albany.edu/?q=mfold) for the 800 and 200 bp regions around the proximal breakpoint. An enlarged view of the breakpoint area highlighting the complementary nucleotides is also shown. The proximal breakpoint (indicated by the red arrows) is at the boundary of a loop and a 12-bp inverted repeat that may cause stalling of DNA replication. It is possible that deletion/duplication can occur if stalled replication resumes using an alternate location on the same chromosome. Red/blue/green bars represent hydrogen bonds between G-C/T-A/G-T.