Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants

Bournazos, Adam; Riley, Lisa G.; Bommireddipalli, Shobhana; Adès, Lesley C.; Akesson, Lauren S; Al-Shinnag, Mohammad; Alexander, Stephen I.; Archibald, Alison D; Balasubramaniam, Shanti; Berman, Yemima; Beshay, Victoria; Boggs, Kirsten; Bojadzieva, Jasmina; Brown, Natasha J; Bryen, Samantha J; Buckley, Michael F.; Chong, Belinda; Davis, Mark; Dawes, Ruebena; Delatycki, Martin B.; Donaldson, Liz; Downie, Lilian; Edwards, Caitlin E.; Edwards, Matthew; Engel, Amanda; Ewans, Lisa; Faiz, Fathimath; Fennell, Andrew; Field, Michael; Freckmann, Mary‐Louise; Gallacher, Lyndon; Gear, Russell; Goel, Himanshu; Goh, Shuxiang; Goodwin, Linda; Hanna, Bernadette; Harraway, James; Higgins, Megan; Ho, Gladys; Hopper, Bruce; Horton, Ari; Hunter, Matthew F.; Huq, Aamira; Josephi-Taylor, Sarah; Joshi, Himanshu; Kirk, Edwin P.; Krzesinski, Emma I.; Kumar, Kishore R.; Lemckert, Frances A.; Leventer, Richard J.; Lindsey-Temple, Suzanna; Lunke, Sebastian; Ma, Alan; Macaskill, Steven; Mallawaarachchi, Amali; Marty, Melanie A.; Marum, Justine E; McCarthy, Hugh; Menezes, Manoj P.; McLean, Alison; Milnes, Di; Mohammad, Shekeeb S; Mowat, David; Niaz, Aram; Palmer, Elizabeth E.; Patel, Chirag; Patel, Shilpan G.; Phelan, Dean; Pinner, Jason; Rajagopalan, Sulekha; Regan, Matthew; Rodgers, Jonathan; Rodrigues, Miriam; Roxburgh, Richard; Sachdev, Rani; Roscioli, Tony; Samarasekera, Ruvishani; Sandaradura, Sarah A.; Savva, Elena; Schindler, Tim; Shah, Margit; Sinnerbrink, Ingrid B.; Smith, Janine; Smith, Richard J.; Springer, Amanda; Stark, Zornitza; Strom, Samuel P.; Sue, Carolyn M.; Tan, Kenneth; Tan, Tiong Yang; Tantsis, Esther; Tchan, Michel; Thompson, Bryony A.; Trainer, Alison H.; Spaendonck‐Zwarts, Karin van; Walsh, Rebecca; Warwick, Linda; White, Stephanie; White, Susan; Williams, Mark; Wilson, Meredith; Wong, Wui‐Kwan; Wright, Dale; Yap, Patrick; Yeung, Alison; Young, Helen; Jones, Kristi; Bennetts, Bruce; Cooper, Sandra T.; Abdulrasool, Ghusoon; Al-Eryani, Ghamdan; Arts, Peer; Bagnall, Richard D.; Baker, Naomi L.; Barnett, Christopher; Beecroft, Sarah J.; Berbic, Marina; Black, Michael W.; Blackburn, Jim; Blombery, Piers; Branford, Susan; Breen, Jimmy; Burnett, Leslie; Canson, Daffodil M.; Cheong, Pak Leng; Chew, Edward; Christodoulou, John; Chung, Seo‐Kyung; Clark, Mike; Cliffe, Corrina; Cole, Melissa; Collins, Felicity; Compton, Alison G.; Cooper, Antony A.; Corbett, Mark; Cowley, Mark J.; Dudding, Tracy; Eggers, Stefanie; Eyras, Eduardo; Fernandez, Miriam Fanjul; Fellowes, Andrew; Fleischer, Ronald; Folland, Chiara; Fox, Lucy; Gaff, Clara; Galea, Melanie; Ghaoui, Roula; Gornanitis, Ilias; Ha, Thuong; Hayashi, Rippei; Hayes, Ian; Henderson, Alex; Hesson, Luke B.; Heyer, Erin E.; Hildebrand, Michael S.; Hipwell, Michael; Hoskins, Cass; Jackson, Matilda R.; James, P.; Wong, Justin Jong-Leong; Kassahn, Karin S.; Kaub, Peter A.; Kevin, Lucy; Kumble, Smitha; Kummerfeld, Sarah; Laing, Nigel G.; Lau, Chiyan; Lee, Eric; Leighton, Sarah; Lundie, Ben; Mayoh, Chelsea; McGaughran, Julie; McPhillips, Mary; Meldrum, Cliff; Middleton, Edwina; Mina, Kym; Nisselle, Amy; Oates, Emily C.; Oshlack, Alicia; Parasivam, Gayathri; Parsons, Michael T.; Quinn, Michael C.; Rasko, John E.J.; Ravenscroft, Gianina; Ravine, Anja; Recsei, Krista; Rehn, Jacqueline; Robertson, Stephen; Ronan, Anne; Ryland, Georgina L; Sadedin, Simon; Schreiber, Andreas; Scott, Hamish S.; Scott, Rodney; Semsarian, Christopher; Simons, Cas; Singer, Emma S.; Smyth, Renee; Spurdle, Amanda B.; Sullivan, Patricia; Sundercombe, Samantha; Thorburn, David R.; Toubia, John; Trent, Ronald J.; Tudini, Emma; Voneague, Irina; Waddell, Leigh B.; Walker, Logan C.; Wallis, Mathew; Warnock, Nick; Weatheritt, Robert J.; White, Deborah; Winship, Ingrid; Worgan, Lisa; Wu, Kathy H C; Ziolowski, Andrew

doi:10.1016/j.gim.2021.09.001

Cited by 51 publications

(43 citation statements)

References 41 publications

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“…While many computational tools are available for use in the clinical setting, they may generate conflicting or inaccurate predictions that can result in overlooking the true cause of a genetic disorder, especially when a synonymous variant is relatively common in controls, as seen with the c.327C>T, p.(Gly109=) variant. The synonymous variant in this study is predicted to introduce a cryptic splice donor site 2 bp upstream of the variant; however, the predicted score provided by an individual bioinformatics tool is still insufficient to satisfy the PP3 criteria (computational evidence of pathogenicity), even for the SpliceAI tool, which outperforms other tools in sensitivity and specificity (Bournazos et al, 2022; Jaganathan et al, 2019; Wai et al, 2020). Of note, SpliceAI predicts no impact of c.5331G>A, p.(Leu1777=) on splicing (Collin et al, 2008).…”

Section: Family‐individuala Hearing Loss Featuresb Genotypec Ethnicit...mentioning

confidence: 87%

“…The variant segregates with the disease in two affected relatives (PP1_supporting). Furthermore, in vitro minigene analysis suggests that this variant likely creates a cryptic donor site, leading to an out-of-frame transcript (PS3_supporting predicted to introduce a cryptic splice donor site 2 bp upstream of the variant; however, the predicted score provided by an individual bioinformatics tool is still insufficient to satisfy the PP3 criteria (computational evidence of pathogenicity), even for the SpliceAI tool, which outperforms other tools in sensitivity and specificity (Bournazos et al, 2022;Jaganathan et al, 2019;Wai et al, 2020).…”

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confidence: 99%

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Characterization of a possible founder synonymous variant in TECTA in multiple individuals with autosomal recessive hearing loss

et al. 2022

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Synonymous variants have been shown to alter the correct splicing of pre-mRNAs and generate disease-causing transcripts. These variants are not an uncommon etiology of genetic disease; however, they are frequently overlooked during genetic testing in the absence of functional and clinical data. Here, we describe the occurrence of a synonymous variant [NM_005422.4 (TECTA):c.327C>T, p.(Gly109=)] in seven individuals with hearing loss from six unrelated families. The variant is not located near exonic/intronic boundaries but is predicted to impact splicing by activating a cryptic splicing donor site in exon 4 of TECTA. In vitro minigene assays show that the variant disrupts the reading frame of the canonical transcript, which is predicted to cause a premature termination codon 48 amino acids downstream of the variant, leading to nonsense-mediated decay. The variant is present in population databases, predominantly in Latinos of African ancestry, but is rare in other ethnic groups. Our findings suggest that this synonymous variant is likely pathogenic for TECTA-associated autosomal recessive hearing loss and seems to have arisen as a founder variant in this specific Latino subpopulation. This study

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Section: Family‐individuala Hearing Loss Featuresb Genotypec Ethnicit...mentioning

confidence: 87%

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confidence: 99%

Characterization of a possible founder synonymous variant in TECTA in multiple individuals with autosomal recessive hearing loss

et al. 2022

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“…At least 50% of disease-causing variations are estimated to be splice site variants. Functional analyses of the spliced isoforms such as the minigene assay and RT-PCR can reclassify up to 75% of putative splicing variants [ 29 ]. The c.-1-2A>T and c.2247-2A>G variants belong to special types of PVS1 null variants.…”

Section: Discussionmentioning

confidence: 99%

Prenatal Detection of Novel Compound Heterozygous Splice Site Variants of the KIAA0825 Gene in a Fetus with Postaxial Polydactyly Type A

Yao

Deng

Zhu

2022

Genes

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Postaxial polydactyly (PAP) is a common abnormality characterized by extra digits on hands and/or feet. To date, sequence variants in seven genes have been identified in non-syndromic PAP. In the present study, a fetus manifesting non-syndromic postaxial polydactyly type A (PAPA) was found by fetal ultrasonography. To better evaluate fetal prognosis, SNP array analysis and trio whole-exome sequencing (trio-WES) were performed to identify the underlying etiology. Although SNP array analysis revealed no abnormality, trio-WES identified compound heterozygous splice site variants in KIAA0825, c.-1-2A>T and c.2247-2A>G in intron 2 and intron 12, respectively. These two splice site variants were absent in control databases and were predicted to influence splicing by in silico analysis. To confirm the potential pathogenicity of the variants, in vitro splicing assays using minigene and RNA from peripheral leukocytes of the heterozygous parents were conducted. Minigene and RT-PCR assays demonstrated that the c.-1-2A>T variant led to the loss of the initiation codon, and the c.2247-2A>G variant mainly resulted in exon 13 skipping. Prenatal WES and subsequent functional studies are important approaches for defining the genetic etiology of fetuses with PAPA and are also essential for accurate genetic counseling and decision making. Taken together, this study expands the spectrum of KIAA0825 variations in PAPA patients and increases the knowledge of the molecular consequences of KIAA0825 splice site variants.

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“…LRS of messenger RNA has demonstrated the ability to detect many novel reading frames and isoforms previously unknown, in addition to allele-specific expression and variations in posttranscription RNA modifications. This knowledge will have utility in sequence variant interpretation in genetic testing (Bournazos et al, 2022). DNA methylation alterations have long been known as an underlying etiology in diseases such as imprinting defect conditions.…”

Section: Applications Of Lrs In Emerging Areas Of Molecular Diagnosticsmentioning

confidence: 99%

Long‐read sequencing for molecular diagnostics in constitutional genetic disorders

et al. 2022

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Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS Chromosomal microarrays (SNP arrays and array CompetitiveGenomic Hybridization [aCGH]) have been considered the gold standard to detect submicroscopic deletions and duplications greater than 50 kb in length for over a decade. Currently, CMA is a first-tier diagnostic test for many congenital disorders, including developmental delay, autism, and multiple congenital anomalies (D. T. Miller et al., 2010). Targeted arrays, often focusing on exonic regions, are also companions for panel-based or targeted gene testing. While

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Standardized practices for RNA diagnostics using clinically accessible specimens reclassifies 75% of putative splicing variants

Cited by 51 publications

References 41 publications

Characterization of a possible founder synonymous variant in TECTA in multiple individuals with autosomal recessive hearing loss

Characterization of a possible founder synonymous variant in TECTA in multiple individuals with autosomal recessive hearing loss

Prenatal Detection of Novel Compound Heterozygous Splice Site Variants of the KIAA0825 Gene in a Fetus with Postaxial Polydactyly Type A

Long‐read sequencing for molecular diagnostics in constitutional genetic disorders

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