Detection of Complex Structural Variations of X-Linked Deafness-2 (DFNX2) by Single-Molecule Sequencing

Abstract

Purpose: To investigate genetic causes of X-linked deafness-2 (DFNX2) and analyse the efficiencies and advantages/disadvantages of different sequencing methods. Methods: From 2016 to 2018, 12 unrelated patients with characteristic high-resolution computed tomographic images showing incomplete partition type III (IP-III) were enrolled in this study. Targeted next-generation sequencing (NGS) and Nanopore long-read sequencing were used to analyse the genetic etiology. Results: Six variants of POU3F4 were identified in this cohort by NGS. The six variants were all located upstream of the POU-specific domain and were considered pathogenic due to truncated protein. Among six patients with negative NGS results, three patients were volunteered to choose further Nanopore sequencing and were all found to have structural variations (SVs). We detected an 870 kb deletion in J0012, located at ChrX:81,140,313-82,011,100, approximately 750 kb upstream of the POU3F4 gene. Non-homologous end joining may be a deletion mechanism. An 8 Mb inversion was found in J0011 at ChrX:82,146,071-90,188,492. The sequences of the breakpoint analysis suggested non-allelic homologous recombination occurred within a single chromatid. A 6 kb deletion at ChrX:81,096,652-81,102,705 was found in J0007, which is a region with a high density of retrotransposons. Conclusions: The most common mutations in patients with IP-III were point mutations and small INDELs or exons that can be detected by Sanger sequencing and NGS. Single-molecule long-fragment sequencing technology provides a new technique to detect the pathogenic SVs in patients with negative NGS results. Thus, the DNA diagnostic strategy for IP-III and its efficiency have been greatly improved. At the same time, the sequence characteristics around the breakpoint and the possible mechanism of SV formation should be explored further. Funding Statement: This work was supported by National Key Research and Development Program of China (2016YFC1000700, 2016YFC1000704 and 2017YFC1001800), Shanghai Key Laboratory of Translational Medicine on Ear and Nose diseases (14DZ2260300). Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: This investigation was performed with the approval of the ethics committees of the Chinese PLA General Hospital. All of the subjects or their guardians signed the informed consent forms prior to blood sampling.