Study of complex structural variations of X-linked deafness-2 based on single-molecule sequencing

Abstract

X-linked deafness-2 (DFNX2) is cochlear incomplete partition type III (IP-III), one of inner ear malformations characterized by an abnormally wide opening in the bone separating the basal turn of the cochlea from the internal auditory canal, fixation of the stapes and cerebrospinal fluid (CSF) gusher upon stapedectomy or cochleostomy. The causative gene of DFNX2 was POU3F4. To investigate the genetic causes of X-linked deafness-2 (DFNX2) and compare the efficiency of different sequencing methods, twelve unrelated patients were enrolled in this study. Targeted next-generation sequencing (NGS) and long-read sequencing were used to analyze the genetic etiology of DFNX2. Six variants of POU3F4 were identified in this cohort by NGS. Three patients with a negative diagnosis based on NGS were enrolled in further long-read sequencing. Two of them were all found to carry structural variations (SVs) on chromosome X, consisting of an 870-kb deletion (DEL) at upstream of POU3F4 and an 8-Mb inversion (INV). The 870-kb DEL may have been be due to non-homologous end joining, while non-allelic homologous recombination within a single chromatid may have accounted for the 8-Mb INV. Common POU3F4 mutations in DFNX2 included point mutations, small insertions and deletions (INDELs), and exon mutations, which can be detected by Sanger sequencing and NGS. Single-molecule long-read sequencing constitutes an additional and valuable method for accurate detection of pathogenic SVs in IP-III patients with negative NGS results.

Genetic findings of Sanger and nanopore single-molecule sequencing in patients with X-linked hearing loss and incomplete partition type III

“…In this study, we analyzed the clinical characteristics, molecular variants and hearing intervention outcomes in 18 patients with IP-III from 14 unrelated families. To the best of our knowledge, it was so far the largest cohort with IP-III [ 24 ]. Ten different variants were identified in the exon region of POU3F4 , of which six were novel.…”

“…For the remaining 28.6% (4/14) patients, 80–486 kb DELs upstream of POU3F4 were identified using nanopore long-read single-molecule sequencing. In previous studies, DELs identified upstream of POU3F4 varied from 6 kb to 1.75 Mb [ 24 – 27 ]. Entire deletions, insertions and translocations of POU3F4 were also recognized to be pathogenic in some cases [ 25 , 28 – 30 ].…”

Pseudoexon activation by deep intronic variation in GNE myopathy with thrombocytopenia

“…Long‐read single‐molecule sequencing (LRS) was carried out on P1 using PromethION sequencer (Oxford Nanopore Technologies) as described in a previous study 19–21 . The mean aligned coverage reached 14.07–19.74.…”

Considering gene therapy to protect from X‐linked deafness DFNX2 and associated neurodevelopmental disorders

“…To date, over 80 deafness‐causative mutations in the coding sequence of POU3F4 have been identified in some 20 countries, including missense, nonsense, deletion, frameshift, and extension mutations (Figure 1). 6–46 Moreover, deletions of the entire gene as well as deletions, paracentric inversions, and duplications upstream of the gene (containing the putative regulatory elements of POU3F4 transcription) were also reported 12,13,24,47–52 . X‐linked deafness type 2 (DFNX2, locus Xq21.1), caused by POU3F4 mutations, accounts for nearly 50% of all cases of X‐linked hearing loss.…”

“… 5 , 6 To date, over 80 deafness‐causative mutations in the coding sequence of POU3F4 have been identified in some 20 countries, including missense, nonsense, deletion, frameshift, and extension mutations (Figure 1 ). 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 Moreover, deletions of the entire gene as well as deletions, paracentric inversions, and duplications upstream of the gene (containing the putative regulatory elements of POU3F4 transcription) were also reported. 12 , 13 , 24 , 47 , 48 , 49 , 50 ...…”