Exome sequencing identifies a novel CEACAM16 mutation associated with autosomal dominant nonsyndromic hearing loss DFNA4B in a Chinese family

Abstract

Autosomal dominant nonsyndromic hearing loss (ADNSHL/DFNA) is a highly genetically heterogeneous disorder. Hitherto only about 30 ADNSHL-causing genes have been identified and many unknown genes remain to be discovered. In this research, genome-wide linkage analysis mapped the disease locus to a 4.3 Mb region on chromosome 19q13 in SY-026, a five-generation nonconsanguineous Chinese family affected by late-onset and progressive ADNSHL. This linkage region showed partial overlap with the previously reported DFNA4. Simultaneously, probands were analyzed using exome capture followed by next-generation sequencing. Encouragingly, a heterozygous missense mutation, c.505G>A (p.G169R) in exon 3 of the CEACAM16 gene (carcinoembryonic antigen-related cell adhesion molecule 16), was identified via this combined strategy. Sanger sequencing verified that the mutation co-segregated with hearing loss in the family and that it was not present in 200 unrelated control subjects with matched ancestry. This is the second report in the literature of a family with ADNSHL caused by CEACAM16 mutation. Immunofluorescence staining and western blots also prove CEACAM16 to be a secreted protein. Furthermore, our studies in transfected HEK293T cells show that the secretion efficacy of the mutant CEACAM16 is much lower than that of the wild type, suggesting a deleterious effect of the sequence variant.

Figure 1

Pedigree of a large Chinese family (SY-026) with late-onset ADNSHL carrying the missense G169R mutation in CEACAM16 and the audiograms of four affected subjects from the family. (a) Pedigree of the family shows an autosomal-dominant inheritance pattern. The circular and square symbols represent female and male, and the black and white ones indicate affected and unaffected individuals, respectively. The subjects younger than the onset age whose hearing status is ambiguous are marked by question mark and the deceased are differentiated by a slash. Arrow shows the proband (IV:22) The bars below each symbol indicate individuals involved in this study. Twenty-two family members included in the linkage analysis are designated by asterisk on each right shoulder of the symbol. The genotypes at the c.505G>A mutation site of CEACAM16 are also presented for each enrolled individual. (Note: ^#The affected member of III:12 was excluded from linkage analysis because the hearing-impairment was caused by environmental factors.) (b) Audiograms of 4 affected individuals from the family. By convention, the air conduction results are displayed on audiogram using blue “×” for left ear and red “○” for right ear. And likewise, bone conduction results are displayed on audiogram using blue “greater than” angle brackets “>” for left ear and red “less than” angle brackets “<” for right ear.

Figure 2

Combinational strategy of linkage analysis and exome sequencing identifies a novel CEACAM16 mutation as causing ADNSHL. (a) SNP linkage analysis maps the disorder locus to chromosome 19 (two-point lod scores of more than 3). The critical interval is flanked by genetic markers rs11671074 and rs1236093 and spans approximately 4.3 Mb. (b) Genome bioinformatics analysis maps physical position to chr19: 44,804,119-49,104,308 and physical map to chr19q13.31-13.33 according to UCSC. (c) Schematic structure of CEACAM16 shows both mutations previously reported in American 1070 family and in the Chinese SY-026 family in this report occurred in the IgC-like domain of subtype A. (d) Identification of a novel heterozygous missense G169R mutation in the CEACAM16 gene. Arrow shows the position of the mutation.

Figure 3

Effects of G169R mutation on CEACAM16 trafficking and secretion. (a, b) Confocal images of COS7 cells expressing Flag-tagged wild type (WT, a) and G169R mutant (b) CEACAM16 (green), counterstained for actin (red). Arrows in a and b show secreted proteins. (c) Western blot of protein extracts of HEK293T cells expressing wild type (WT) and G169R mutant Flag-CEACAM16 and of culture media secreted protein precipitates (upper panel). Total protein expression of WT and G169R CEACAM16 is very similar (Cells); total β-actin loads from both preparations are comparable (lower panel). However, the secreted (Medium) amount of mutant CEACAM16 is much lower than the WT CEACAM16. Scale bar: 10 μm.