Identification of a complex genomic rearrangement in TMPRSS3 by massively parallel sequencing in Chinese cases with prelingual hearing loss

Abstract

Background: Genetic variants in TMPRSS3 have been causally linked to autosomal recessive nonsyndromic hearing loss (HL) at the DFNB8 and DFNB10 loci. These variants include both single nucleotide and copy number variations (CNVs). In this study, we aim to identify the genetic cause in three Chinese subjects with prelingual profound sensorineural HL.

Methods: We applied targeted genomic enrichment and massively parallel sequencing to screen 110 genes associated with nonsyndromic HL in the three affected subjects. CNVplex^® analysis and polymerase chain reaction (PCR) were performed for CNV detection.

Results: We identified biallelic variations in TMPRSS3 including a novel complex genomic rearrangement and a novel missense mutation, c.551T>C. We have mapped the breakpoints of the genomic rearrangement and showed that it consisted of two deletions and an inversion encompassing exon 3 to exon 9 of TMPRSS3.

Conclusion: Our study expanded the mutational spectrum of TMPRSS3 to include complex genomic rearrangements. It showcased the importance of an integrative approach to investigate CNVs and their contribution to HL.

Keywords: TMPRSS3; copy number variation; hearing loss; massively parallel sequencing; rearrangement.

Figure 1

(a) Pedigree and Sanger sequencing of Family CQ‐176. In the pedigree, black and white symbols represent people with hearing loss and normal hearing, respectively. The genotypes are labeled below. (b) Conservation analysis: the residue Leu184 is highly conserved among eight different species. (c) 3D structure prediction: the structure of the wild‐type protein and the p.Leu184Ser protein. Red dotted lines indicate the hydrogen bonds between the 184th residue and other residues

Figure 2

Identification of a novel complex genomic rearrangement in TMPRSS3. (a) Pedigree of family GD‐395. (b) The copy number of each exon calculated from the fluorescence peak ratios in CNVplex^® analysis. (c) The copy number of the exons of interest from real‐time PCR. (d) The drop of read‐depth and the split reads in exon 10 of TMPRSS3. (e) Long‐range PCR conducted by TMPRSS3_In2_F and TMPRSS3_Ex12_R. The inverted allele generated a product of 12K, while the normal allele came to a 16K. Only the inverted allele could be amplified in GD‐395. (f) Gap‐PCR and segregation analysis of family GD‐395. (g) The genomic alignment of one of the split reads conducted by UCSC BLAT. (h) Scheme of the normal and inverted alleles. (i) Sanger sequencing of the inverted allele by TMPRSS3_In2_F and TMPRSS3_Ex12_R covering the two breakpoints

Figure 3

Overview of the copy number variants in TMPRSS3 reported to date