Annotation of uORFs in the OMIM genes allows to reveal pathogenic variants in 5′UTRs

Filatova, Alexandra; Reveguk, Ivan; Piatkova, Mariia; Bessonova, Daria; Kuziakova, Olga; Demakova, Victoria; Romanishin, Alexander; Fishman, Veniamin; Imanmalik, Yerzhan; Chekanov, Nikolay; Skitchenko, Rostislav K.; Barbitoff, Yury A.; Kardymon, Olga L.; Skoblov, Mikhail

doi:10.1093/nar/gkac1247

Cited by 6 publications

(4 citation statements)

References 54 publications

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“…To aid the assessment of 5’UTR variant pathogenicity, in silico tools have recently been developed, with an emphasis put on uORF-perturbing variants [ 20 , 26 ]. A systematic characterization of this class of 5’UTR variants showed that they are subject to strong negative selection, which could even be equivalent to that observed against missense variants [ 11 ].…”

Section: Discussionmentioning

confidence: 99%

“…Finally, functional evidence is necessary to support their pathogenicity and hence confirm the molecular diagnosis. Recently, to close the annotation and interpretation gap, several in silico tools [ 20 – 26 ] and guidelines [ 27 ] have been developed, although their specific applicability to the accurate and comprehensive interpretation of the diverse pathogenic mechanism of 5’UTR variants is yet to be fully established.…”

Section: Introductionmentioning

confidence: 99%

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Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Dueñas Rey,

del Pozo Valero,

Bouckaert

et al. 2024

Genome Med

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Background 5’ untranslated regions (5’UTRs) are essential modulators of protein translation. Predicting the impact of 5’UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5’UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs). Methods We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5’UTRs by different whole exome sequencing (WES) kits. The selected 5’UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5’UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches. Results Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5’UTR compared to that of their canonical isoform. Depending on the probe design, 3–20% of IRD genes have 5’UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5’UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels. Conclusions This study demonstrates the importance of 5’UTR variants implicated in IRDs and provides a systematic approach for 5’UTR annotation and validation that is applicable to other inherited diseases.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Dueñas Rey,

del Pozo Valero,

Bouckaert

et al. 2024

Genome Med

View full text Add to dashboard Cite

show abstract

“…Finally, functional evidence is necessary to support their pathogenicity and hence confirm the molecular diagnosis. Recently, to close the annotation and interpretation gap, several in silico tools [20][21][22][23][24][25][26] and guidelines 27 have been developed, although their specific applicability to the accurate and comprehensive interpretation of the diverse pathogenic mechanism of 5'UTR variants is yet to be fully established.…”

Section: Introductionmentioning

confidence: 99%

Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Rey,

del Pozo Valero,

Bouckaert

et al. 2023

Preprint

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Background| 5' untranslated regions (5'UTRs) are essential modulators of protein translation. Predicting the impact of 5'UTR variants is challenging and typically not performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and subsequent functional assays to evaluate 5'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs).Methods| We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of these 5'UTRs by different whole exome sequencing (WES) capture kits. The selected 5'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2,417 WGS) and an in-house database (n = 1,682 WES), respectively. Identified variants were annotated for 5'UTR-relevant features and classified into 7 distinct categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse experimental approaches.Results| Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5'UTR compared to that of their canonical isoform. Depending on the probe-design 3-20% of IRD genes have 5'UTRs fully captured by WES. After analyzing these regions in both IRD cohorts we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3,MERTK,NDP,NMNAT1,NPHP4,PAX6,PRPF31,PRPF4,RDH12,RD3), of which 8 were novel. Functional analyses further supported the pathogenicity of 2 variants. TheMERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. TheRDH12:c.-123C>T variant was foundin ciswith the reported hypomorphicRDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels.Conclusions| This study demonstrates the importance of 5'UTR variants implicated in IRDs and provides a systematic approach for 5'UTR annotation and validation that is applicable to other inherited diseases.

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“…Although long-read sequencing technology improves the identification of structural variations, the analytical challenge around variant interpretation persists, especially for balanced structural variations with noncoding breakpoints [6][7][8] . Noncoding regions, representing approximately 98% of the human genome, have recently attracted attention in relation to genetically unsolved patients with Mendelian disorders [9][10][11] . To date, a number of noncoding variants have been identified, most of which were recognized according to a physical location in or adjacent to the known disease-causing gene, such as intronic, regulatory, upstream or downstream regions of known genes.…”

mentioning

confidence: 99%

Altered chromatin topologies caused by balanced chromosomal translocation lead to central iris hypoplasia

Sun,

Xiong,

Ouyang

et al. 2024

Nat Commun

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Despite the advent of genomic sequencing, molecular diagnosis remains unsolved in approximately half of patients with Mendelian disorders, largely due to unclarified functions of noncoding regions and the difficulty in identifying complex structural variations. In this study, we map a unique form of central iris hypoplasia in a large family to 6q15-q23.3 and 18p11.31-q12.1 using a genome-wide linkage scan. Long-read sequencing reveals a balanced translocation t(6;18)(q22.31;p11.22) with intergenic breakpoints. By performing Hi-C on induced pluripotent stem cells from a patient, we identify two chromatin topologically associating domains spanning across the breakpoints. These alterations lead the ectopic chromatin interactions between APCDD1 on chromosome 18 and enhancers on chromosome 6, resulting in upregulation of APCDD1. Notably, APCDD1 is specifically localized in the iris of human eyes. Our findings demonstrate that noncoding structural variations can lead to Mendelian diseases by disrupting the 3D genome structure and resulting in altered gene expression.

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Annotation of uORFs in the OMIM genes allows to reveal pathogenic variants in 5′UTRs

Cited by 6 publications

References 54 publications

Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Combining a prioritization strategy and functional studies nominates 5’UTR variants underlying inherited retinal disease

Altered chromatin topologies caused by balanced chromosomal translocation lead to central iris hypoplasia

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