Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

Reurink, Janine; Weisschuh, Nicole; Garanto, Alejandro; Dockery, Adrian; Born, L. Ingeborgh van den; Fajardy, I.; Haer‐Wigman, Lonneke; Kohl, Susanne; Wissinger, Bernd; Farrar, G. Jane; Ben-Yosef, Tamar; Pfiffner, Fatma Kivrak; Berger, Wolfgang; Weener, Marianna E.; Ďuďáková, Ľubica; Lišková, Petra; Sharon, Dror; Salameh, Manar; Offenheim, Ashley; Hèon, Elise; Girotto, Giorgia; Gasparini, Paolo; Morgan, Anna; Bergen, Arthur A.; Brink, Jacoline B. ten; Klaver, Caroline C W; Tranebjærg, Lisbeth; Rendtorff, Nanna Dahl; Vermeer, Sascha; Smits, Jeroen J.; Pennings, Ronald J.E.; Aben, Marco; Oostrik, Jaap; Astuti, Galuh D.N.; Galbany, Jordi Corominas; Kroes, Hester Y.; Phan, Milan; Zelst-Stams, Wendy A. G. van; Thiadens, Alberta A H J; Verheij, Joanne; Schooneveld, Mary J. van; Bruijn, Suzanne E. de; Li, Catherina H Z; Hoyng, Carel B.; Gilissen, Christian; Vissers, Lisenka E.L.M.; Cremers, Frans P.M.; Kremer, Hannie; Wijk, Erwin van; Roosing, Susanne

doi:10.1016/j.xhgg.2023.100181

Cited by 10 publications

(6 citation statements)

References 42 publications

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“…In addition pathogenic deep intronic variants commonly cause splicing aberration 23 and in most studies, in silico analysis was performed first to select candidate variants for targeted functional studies. 14,24 However, several publications have reported the lack of sensitivity of splice in silico tools for deep intronic variants in the context of HA, 10,12,13 which is confirmed herein as positive in silico analysis was found only for a quarter of the pathogenic deep intronic variants identified (de novo DSS creation in each case). This underlines the need to systematically perform functional analyses to assess the splicing impact of the candidate variant.…”

Section: Discussionsupporting

confidence: 67%

“…This emphasises the value of whole F8 gene sequencing compared to the conventional genetic approach in particular in France where deep intronic variants are frequent. In addition pathogenic deep intronic variants commonly cause splicing aberration 23 and in most studies, in silico analysis was performed first to select candidate variants for targeted functional studies 14,24 . However, several publications have reported the lack of sensitivity of splice in silico tools for deep intronic variants in the context of HA, 10,12,13 which is confirmed herein as positive in silico analysis was found only for a quarter of the pathogenic deep intronic variants identified (de novo DSS creation in each case).…”

Section: Discussionmentioning

confidence: 53%

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Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non‐severe haemophilia A

et al. 2023

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IntroductionConventional genetic investigation fails to identify the F8 causal variant in 2.5%‐10% of haemophilia A (HA) patients with non‐severe phenotypes. In these cases, F8 deep intronic variants could be causal.AimTo identify pathogenic F8 deep intronic variants in genetically unresolved families with non‐severe HA analysed in the haematology laboratory of the Hospices Civils de Lyon.MethodsThe whole F8 was analysed by next generation sequencing. The pathogenic impact of candidate variants identified was assessed using both in silico analysis (MaxEntScan and spliceAI) and functional analysis (RNA or minigene assay).ResultsSequencing was performed in 49/55 families included for which a DNA sample from a male propositus was available. In total, 33 candidate variants from 43 propositi were identified. These variants corresponded to 31 single nucleotide substitutions, one 173‐bp deletion, and an 869‐bp tandem triplication. No candidate variant was found in six propositi. The most frequent variants found were the association of [c.2113+1154G>C and c.5374‐304C>T], identified in five propositi, and the c.2114‐6529C>G identified in nine propositi. Four variants had been previously described as HA‐causing. Splicing functional assay found a deleterious impact for 11 substitutions (c.671‐94G>A, c.788‐312A>G, c.2113+1154G>C, c.2114‐6529C>G, c.5999‐820A>T, c.5999‐786C>A, c.5999‐669G>T, c.5999‐669G>A, c.5999‐669G>C, c.6900+4104A>C, and c.6901‐2992A>G). The HA‐causing variant was identified in 33/49 (67%) cases. In total, F8 deep intronic variants caused 8.8% of the non‐severe HA among the 1643 families analysed in our laboratory.ConclusionThe results emphasise the value of whole F8 gene sequencing combined with splicing functional analyses to improve the diagnosis yield for non‐severe HA.

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Section: Discussionsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 53%

Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non‐severe haemophilia A

et al. 2023

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“…We further developed antisense oligonucleotides which successfully suppressed expression of pseudoexon-containing transcripts 4 . In view of the recent progress of antisense oligonucleotide-mediated exon-skipping therapy for monogenic disorders caused by splicing abnormalities 5,6 , our data presented here have provided supportive evidence for in vivo preclinical studies.…”

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confidence: 70%

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration

Yamada,

Maeta,

Suzuki

et al. 2024

Sci Rep

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Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon–intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

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“…GATK was used to call SNVs and small indels. Two individuals underwent whole genome sequencing as described by Fadaie et al 43 Variant analysis was performed as part of a study by Reurink et al 44 The American College of Medical Genetics and Genomics and The Association for Molecular Pathology (ACMG-AMP) criteria for classifying pathogenic variants was utilised for interpretation of candidate variants. 45 Confirmatory testing and further analysis for unresolved cases was carried out at accredited laboratories (Blueprint Genetics, Helsinki, Finland and the Manchester Centre for Genomic Medicine).…”

Section: Methodsmentioning

confidence: 99%

Usher Syndrome on the Island of Ireland: A Genotype-Phenotype Review

Stephenson

Whelan

Zhu

et al. 2023

Invest. Ophthalmol. Vis. Sci.

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Purpose Usher syndrome (USH) is a genetically heterogeneous group of autosomal recessive (AR) syndromic inherited retinal degenerations (IRDs) representing 50% of deaf-blindness. All subtypes include retinitis pigmentosa, sensorineural hearing loss, and vestibular abnormalities. Thorough phenotyping may facilitate genetic diagnosis and intervention. Here we report the clinical/genetic features of an Irish USH cohort. Methods USH patients were selected from the Irish IRD registry (Target 5000). Patients were examined clinically (deep-phenotyping) and genetically using a 254 IRD–associated gene target capture sequencing panel, USH2A exon, and whole genome sequencing. Results The study identified 145 patients (24.1% USH1 [n = 35], 73.8% USH2 [n = 107], 1.4% USH3 [n = 2], and 0.7% USH4 [n = 1]). A genetic diagnosis was reached in 82.1%, the majority (80.7%) being MYO7A or USH2A genotypes. Mean visual acuity and visual field (VF) were 0.47 ± 0.58 LogMAR and 31.3° ± 32.8°, respectively, at a mean age of 43 years. Legal blindness criteria were met in 40.7%. Cataract was present in 77.4%. ADGRV1 genotypes had the most VF loss, whereas USH2A patients had greater myopia and CDH23 had the most astigmatism. Variants absent from gnomAD non-Finnish Europeans and ClinVar represented more than 20% of the variants identified and were detected in ADGRV1, ARSG, CDH23, MYO7A , and USH2A. Conclusions USH is a genetically diverse group of AR IRDs that have a profound impact on affected individuals and their families. The prevalence and phenotype/genotype characteristics of USH in Ireland have, as yet, gone unreported. Understanding the genotype of Irish USH patients may guide clinical and genetic characterization facilitating access to existing/novel therapeutics.

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Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

Cited by 10 publications

References 42 publications

Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non‐severe haemophilia A

Whole F8 gene sequencing combined with splicing functional analyses led to a substantial increase of the molecular diagnosis yield for non‐severe haemophilia A

Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration

Usher Syndrome on the Island of Ireland: A Genotype-Phenotype Review

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