Challenges in Whole Exome Sequencing: An Example from Hereditary Deafness

Whole exome sequencing provides unprecedented opportunities to identify causative DNA variants in rare Mendelian disorders. Finding the responsible mutation via traditional methods in families with hearing loss is difficult due to a high degree of genetic heterogeneity. In this study we combined autozygosity mapping and whole exome sequencing in a family with 3 affected children having nonsyndromic hearing loss born to consanguineous parents. Two novel missense homozygous variants, c.508C>A (p.H170N) in GIPC3 and c.1328C>T (p.T443M) in ZNF57, were identified in the same ∼6 Mb autozygous region on chromosome 19 in affected members of the family. Both variants co-segregated with the phenotype and were absent in 335 ethnicity-matched controls. Biallelic GIPC3 mutations have recently been reported to cause autosomal recessive nonsyndromic sensorineural hearing loss. Thus we conclude that the hearing loss in the family described in this report is caused by a novel missense mutation in GIPC3. Identified variant in GIPC3 had a low read depth, which was initially filtered out during the analysis leaving ZNF57 as the only potential causative gene. This study highlights some of the challenges in the analyses of whole exome data in the bid to establish the true causative variant in Mendelian disease

Screening of 38 Genes Identifies Mutations in 62% of Families with Nonsyndromic Deafness in Turkey

More than 60% of prelingual deafness is genetic in origin, and of these up to 95% are monogenic autosomal recessive traits. Causal mutations have been identified in 1 of 38 different genes in a subset of patients with nonsyndromic autosomal recessive deafness. In this study, we screened 49 unrelated Turkish families with at least three affected children born to consanguineous parents. Probands from all families were negative for mutations in the GJB2 gene, two large deletions in the GJB6 gene, and the 1555A>G substitution in the mitochondrial DNA MTRNR1 gene. Each family was subsequently screened via autozygosity mapping with genomewide single-nucleotide polymorphism arrays. If the phenotype cosegregated with a haplotype flanking one of the 38 genes, mutation analysis of the gene was performed. We identified 22 different autozygous mutations in 11 genes, other than GJB2 , in 26 of 49 families, which overall explains deafness in 62% of families. Relative frequencies of genes following GJB2 were MYO15A (9.9%), TMIE (6.6%), TMC1 (6.6%), OTOF (5.0%), CDH23 (3.3%), MYO7A (3.3%), SLC26A4 (1.7%), PCDH15 (1.7%), LRTOMT (1.7%), SERPINB6 (1.7%), and TMPRSS3 (1.7%). Nineteen of 22 mutations are reported for the first time in this study. Unknown rare genes for deafness appear to be present in the remaining 23 families

A complex TFAP2A allele is associated with branchio-oculo-facial syndrome and inner ear malformation in a deaf child

We present a 4-year-old girl with congenital profound sensorineural deafness associated with inner ear malformation (incomplete partition type II, enlarged vestibule, and enlarged vestibular aqueduct). The proposita also had pseudocleft lips, skin defects, auricle abnormalities, and unilateral multicystic dysplastic kidney, leading to the diagnosis of branchio-oculo-facial (BOF) syndrome. Mutation analysis of the TFAP2A gene showed a de novo deletion of 18 and insertion of 6 nucleotides, resulting in deletion of amino acids LPGARR and insertion of RI between amino acids 276 and 281. Altered amino acids are located within the basic DNA binding and dimerization domains of TFAP2A. Previously reported amino acid substitutions in TFAP2A involved only DNA binding domain in four patients with BOF syndrome who were not reported to have profound sensorineural deafness. Our report implies that the localization of mutations in TFAP2A might be responsible with the phenotypic findings in BOF syndrome

Five autozygous regions detected with Affymetrix 6.0 arrays in the family.

<p>To define autozygous regions from the exome sequences (*) the following filters were applied to reduce the incident of false positives (phred-like consensus score ≥100 and a minimum read depth of 20).</p

Pedigree with haplotypes, audiograms and two identified variants.

<p>(A) The pedigree and the third longest autozygous region on chromosome 19 that co-segregates with the phenotype. (B) Audiograms of affected members in the family. (C) Electropherograms showing the wild type, homozygous and heterozygous forms of the variant p.T443M in <i>ZNF57</i>. (D) Electropherograms showing the wild type, homozygous and heterozygous form of the variant p.H170N in <i>GIPC3</i>.</p

Novel missense, nonsense, splice site, and frameshift variants in top five autozygous regions.

a<p>These variants were detected when filter for read depth was ≥8×.</p

The two-dimensional structure of <i>GIPC3</i> and the localization of identified mutations.

<p>The two-dimensional structure of <i>GIPC3</i> and the localization of identified mutations.</p

Molecular modeling of p.T443M in ZNF57.

<p>The zinc finger domain comprises two β-strands (blue) and one α-helix (red); the turns (green) and the loops (light gray) are shown. Amino-acid residue 443 is pink in the wild type (A) and yellow in the mutant (B).</p

Coverage of autozygous regions with whole exome sequencing.

<p>(A) The exome coverage of the five longest autozygous regions. The plot shows the fraction of on-target coverage (Y-axis) and the read depth (X-axis) for the following specified regions. (B) Average coverage at minimum 8× and GC content of five autozygous regions. (C) Coverage of exon 3 in <i>GIPC3</i>. Red arrow indicates mutation point.</p