High‐throughput custom capture sequencing identifies novel mutations in coloboma‐associated genes: Mutation in DNA‐binding domain of retinoic acid receptor beta affects nuclear localization causing ocular coloboma

Abstract

Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high-throughput screening of 38 known colobomaassociated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand-binding domain, was a missense change in the highly conserved DNA-binding domain predicted to affect the protein's DNA-binding ability.In vitro studies revealed lower steady-state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild-type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA-binding domain could result in human disease. K E Y W O R D Scoloboma, custom capture, high-throughput sequencing, retinoic acid receptor beta

Zfp503/Nlz2 is Required for RPE Differentiation and Optic Fissure Closure

“…The developmental processes associated with optic fissure closure have been progressively elucidated over the past half century, however, the genetics of uveal coloboma is far from well understood. Indeed, two recent studies were only able to molecularly solve approximately 8-15% of anophthalmia, microphthalmia and coloboma cases, consistent with our own laboratory’s experience 8, 46, 47 . Higher yields for molecular testing have been noted in patients with a clearly defined syndrome 48 .…”

“…Mutations in more than 20 different genes have been associated with uveal coloboma. These mutations, however, account for less than 10% of cases in the absence of a clear syndromic diagnosis 7, 8 , implying that additional genes and factors are involved.…”

Review of evidence for environmental causes of uveal coloboma

“…This same group of experiments was able to link these events to RAR-regulated genes in both the ventral retina/optic stalk and the periocular mesenchyme and show that genes in both of these regions are independently important in mediating optic fissure closure 86 . Further supporting these findings are several studies that have reported coloboma in both humans and animals associated with mutations in various RA signaling genes 70,86,66,91,90,131,130 .…”

“…The genetic causes of uveal coloboma have been well reviewed 17,155,1,159 . Despite significant effort, the yield of current molecular genetic testing of coloboma patients is low, estimated to be about 8% 108 , suggesting environmental causes may contribute to the etiology of this condition 70,108 . The purpose of this article is to review epidemiologic and basic research evidence for possible environmental contributors to coloboma.…”

Clinical diagnosis of presumed SOX2 gonadosomatic mosaicism

“…Trio exome sequencing was performed as described and variants were filtered for apparent de novo status ( 9 ). Peripheral blood DNA samples from the proband, her two sisters, and her parents and her maternal aunt were also used for a custom panel capture targeting 193 genes followed by Illumina short-read sequencing, as reported ( 10 ). A custom NGS analysis pipeline was used for variant calling and filtering as described ( 11 ).…”