Whole-Exome Sequencing of Discordant Monozygotic Twin Families for Identification of Candidate Genes for Microtia-Atresia

Abstract

Objective: We used data from twins and their families to probe the genetic factors contributing to microtia-atresia, in particular, early post-twinning variations that potentially account for the discordant phenotypes of monozygotic twin pairs. Methods: Six families of monozygotic twins discordant for congenital microtia-atresia were recruited for study. The six patients shared a consistent clinical phenotype of unilateral microtia-atresia. Whole-exome sequencing (WES) was performed for all six twin pairs and their parents. Family segregation and multiple bioinformatics methods were applied to identify suspicious mutations in all families. Recurring mutations commonly detected in at least two families were highlighted. All variants were validated via Sanger sequencing. Gene Ontology (GO) analysis was performed to identify candidate gene sets and related pathways. Copy number variation (CNV), linkage analysis, association analysis and machine learning methods were additionally applied to isolate candidate mutations, and comparative genomics and structural modeling tools used to evaluate their potential roles in onset of microtia-atresia. Results: Our analyses revealed 61 genes with suspected mutations associated with microtia-atresia. Five (HOXA4, MUC6, CHST15, TBX10, and AMER1) contained 7 de novo mutations that appeared in at least two families, which have been previously reported as pathogenic for other diseases. Among these, HOXA4 (c.920A>C, p.H307P) was determined as the most likely pathogenic variant for microtia-atresia. GO analysis revealed four gene sets involving 11 pathways potentially related to underlying pathogenesis of the disease. CNVs in three genes (UGT2B17, OVOS, and KATNAL2) were detected in at least two families. Linkage analysis disclosed 13 extra markers for the disease, of which two (FGFR1 and EYA1) were validated via machine learning analysis as plausible candidate genes for the disease.

The role of genetic factors in microtia: A systematic review

“…Characteristics of the studies of isolated microtia.1,9,17,22,41,46,[48][49][50][65][66][67][68][69][70][71][72]132,133 …”

Search for a genetic cause in children with unilateral isolated microtia and congenital aural atresia

“…These findings may be, at least partially, related to the limitations of genome wide SNP array [25] which may not detect very small deletions/duplications, balanced rearrangements or small/single nucleotide variants. Recent studies using whole exome sequencing (WES) in cases of isolated microtia yielded promising results [24,26]. During WES, the entire exome is analyzed to investigate for variants in the coding part of the human genome (the exomes) that could explain the development of CAA/microtia.…”

“…During WES, the entire exome is analyzed to investigate for variants in the coding part of the human genome (the exomes) that could explain the development of CAA/microtia. Data obtained by WES in discordant monozygotic twins with a consistent clinical phenotype (unilateral type 3 microtia and CAA) revealed HOXA4 as a likely pathogenetic variant for microtia-atresia [26]. Soon, we aim to perform WES in a large group of microtia/CAA patients with a similar phenotype.…”

Clinical and molecular characterization of craniofrontonasal syndrome: new symptoms and novel pathogenic variants in the EFNB1 gene

“…Except for mosaicism in other than blood cells, one may suspect the presence of additional modifiers of the phenotype, including epigenetic factors [ 18 – 20 ]. To check whether the variable severity of CFNS in both twin females resulted from skewed X chromosome inactivation, we performed XCI testing.…”