Deep‐intronic variants in CNGB3 cause achromatopsia by pseudoexon activation

Abstract

Our comprehensive cohort of 1100 unrelated achromatopsia (ACHM) patients comprises a considerable number of cases (~5%) harboring only a single pathogenic variant in the major ACHM gene CNGB3. We sequenced the entire CNGB3 locus in 33 of these patients to find a second variant which eventually explained the patients’ phenotype. Forty‐seven intronic CNGB3 variants were identified in 28 subjects after a filtering step based on frequency and the exclusion of variants found in cis with pathogenic alleles. In a second step, in silico prediction tools were used to filter out those variants with little odds of being deleterious. This left three variants that were analyzed using heterologous splicing assays. Variant c.1663–1205G>A, found in 14 subjects, and variant c.1663‐2137C>T, found in two subjects, were indeed shown to exert a splicing defect by causing pseudoexon insertion into the transcript. Subsequent screening of further unsolved CNGB3 subjects identified four additional cases harboring the c.1663–1205G>A variant which makes it the eighth most frequent CNGB3 variant in our cohort. Compound heterozygosity could be validated in ten cases. Our study demonstrates that whole gene sequencing can be a powerful approach to identify the second pathogenic allele in patients apparently harboring only one disease‐causing variant.

Phenotype Driven Analysis of Whole Genome Sequencing Identifies Deep Intronic Variants that Cause Retinal Dystrophies by Aberrant Exonization

“…The c.1148delC is the most recurrent pathogenic variant reported in CNGB3 . 8 A comprehensive analysis of GS did not reveal any other candidate gene with biallelic coding or canonical splice variants ( Supplementary Fig. S3 ).…”

“…Repeated attempts to amplify the cDNA from control and patient lymphoblast cell line were unsuccessful due to the poor expression of CNGB3 outside the retina, and this challenge has been reported previously. 8 …”

“… 52 Panel-based clinical testing revealed the previously reported most common pathogenic variant (c.1148delC) in CNGB3 ; this variant results in the introduction of a premature stop codon. 8 , 17 , 53 Targeted GS analysis in candidate genes identified a deep intronic second variant in CNGB3 (c.852+4751A>T) that strongly predicted to cause aberrant exonization, and the variants segregated with the disease phenotype. A splicing reporter minigene assay confirmed the occurrence of aberrant exonization consequent to the deep intronic mutation.…”

“…A recent publication identified three additional deep intronic splice variants in CNGB3 in a large cohort of achromatopsia. 8 …”

“… 61 , 65 Hence, using patient-derived cell lines is advantageous in that this system likely better recapitulates the in vivo context but might not be available or suitable in all instances. 8 , 10 , 63 Furthermore, for genes such as NR2E3 , GPR179 , and CNGB3 that are expressed almost exclusively in the retina, it can be challenging to detect transcript levels in nonnative tissue types like fibroblasts or lymphoblasts cell lines. 67 , 68 In these instances, splice reporting minigene or modified minigene constructs are a more feasible approach to validate aberrant splicing.…”