Unveiling Secondary Mutations in Blended Phenotypes: Dual ERCC4 and OTOA Pathogenic Variants Through WES Analysis

Abstract

This study describes two siblings from consanguineous parents who exhibit intellectual disability, microcephaly, photosensitivity, bilateral sensorineural hearing loss, numerous freckles, and other clinical features that suggest a potential disruption of the nucleotide excision repair (NER) pathway. Whole exome sequencing (WES) identified a novel homozygous missense variant in the ERCC4 gene, which was predicted to be pathogenic. However, a subsequent peculiar audiometric finding prompted further investigation, revealing a homozygous deletion in the OTOA gene linked to neurosensorial hearing loss. Both variants were located within a run of homozygosity (ROH) on chromosome 16p13.12-p12.2, implicating a complex genetic basis for the observed phenotype. While this study reports a potentially novel ERCC4 variant, it underscores the importance of comprehensive analysis and deep phenotyping in WES data to improve diagnostic accuracy. Our findings advocate for an expanded approach in WES analysis, ensuring more precise diagnoses and improved genetic counseling, particularly when specialized tests for structural variant analysis are unavailable.

Keywords: ERCC4; OTOA; blended phenotypes; dual diagnoses; run of homozygosity; whole exome sequencing.

Figure 1

Pedigree of the extended family. Black-filled boxes represent affected individuals with a clinical presentation suggestive of a defect in the NER pathway and carry homozygous variants in both ERCC4 and OTOA. The parents (III−1 and III−2) are healthy individuals heterozygous for both the ERCC4 and OTOA variants.

Figure 2

Clinical appearance of the affected brothers. Panels (A–C) depict patient IV−1, while panels (D–F) show patient IV−2. Numerous freckles are visible on the face, shoulders, and hands of both patients.

Figure 3

Multiple sequence alignment of homologous ERCC4 proteins from various species (generated via UniProt), demonstrating the evolutionary conservation of the arginine residue at position 726 in human ERCC4 (highlighted in a red box). The high conservation of this amino acid suggests its functional or structural importance, indicating selective pressure to preserve this residue across diverse species.

Figure 4

Chromosome 16 analysis of CNVs and ROHs using WES data. (A) Arrows indicate a homozygous deletion of approximately 317 kb (chr16: 21,561,888−21,878,568), detected in both siblings using Excavator2 software, which includes the OTOA gene. (B) B-allele frequency (BAF) analysis using H3M2 revealed that both siblings share an approximately 17 Mb ROH on chromosome 16p13.3-p12.1, encompassing both the deletion involving OTOA and the missense variant in ERCC4.

Figure 5

Array-CGH/SNP analysis of patients. The left panel for each patient displays the log2 ratios along chromosome 16, with red arrows indicating the 180 kb homozygous deletion involving OTOA. The shadow boxes on the right panels highlight the homozygous regions on chromosome 16.