High-throughput splicing assays identify missense and silent splice-disruptive<i>POU1F1</i>variants underlying pituitary hormone deficiency

Gergics, Péter; Smith, Cathy; Bando, Hironori; Jorge, Alexander A L; Rockstroh-Lippold, Denise; Vishnopolska, Sebastián Alexis; Castinetti, Frédéric; Maksutova, Mariam; Carvalho, Luciani R.; Hoppmann, Julia; Mayer, Julián Martínez; Braslavsky, Débora; Keselman, Ana; Bergadá, Ignacio; Martí, Marcelo A.; Saveanu, Alexandru; Barlier, Anne; Jamra, Rami Abou; Guo, Michael H.; Dauber, Andrew; Nakaguma, Marilena; Mendonça, Berenice Bilharinho; Ozel, Ayse Bilge; Fang, Qing; Ma, Qianyi; Li, Jun Z.; Brue, Thierry; Millán, María Inés Pérez; Kitzman, Jacob O.; Arnhold, Ivo J.P.; Pfaeffle, Roland; Camper, Sally A.

doi:10.1101/2021.02.04.21249469

Cited by 2 publications

(2 citation statements)

References 74 publications

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“…Therefore, combining assays that assess splicing with those measuring effects on the protein level will be essential to achieve optimal clinical accuracy. Recently, >1000 variants in POU1F1 were assayed using a minigene reporter, and 113 were deemed splice-disruptive ( 90 ). Two of these co-segregated with disease in unsolved families with familial hypopituitarism.…”

Section: Emerging Themes: Integration Of Readouts From Multiple Functional Assays Achieves Greater Phenotypic Depthmentioning

confidence: 99%

Linking genome variants to disease: scalable approaches to test the functional impact of human mutations

Findlay

2021

Human Molecular Genetics

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The application of genomics to medicine has accelerated the discovery of mutations underlying disease and enhanced our knowledge of the molecular underpinnings of diverse pathologies. As the amount of human genetic material queried via sequencing has grown exponentially in recent years, so too has the number of rare variants observed. Despite progress, our ability to distinguish which rare variants have clinical significance remains limited. Over the last decade, however, powerful experimental approaches have emerged to characterise variant effects orders of magnitude faster than before. Fuelled by improved DNA synthesis and sequencing, and more recently by CRISPR/Cas9 genome editing, multiplex functional assays provide a means of generating variant effect data in wide-ranging experimental systems. Here, I review recent applications of multiplex assays that link human variants to disease phenotypes and describe emerging strategies that will enhance their clinical utility in coming years.

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Section: Emerging Themes: Integration Of Readouts From Multiple Functional Assays Achieves Greater Phenotypic Depthmentioning

confidence: 99%

Linking genome variants to disease: scalable approaches to test the functional impact of human mutations

Findlay

2021

Human Molecular Genetics

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“…An alternative approach is highthroughput multiplexed assays that use deep sequencing to read out pools of splicing outcomes. Several successful implementations of this approach have enabled splicing investigation through saturation mutagenesis of specific genes 16,17 and medium to large variant libraries by pooled oligonucleotide synthesis 6,[18][19][20][21] . Building upon these seminal contributions, we sought to develop a versatile, accurate, multiplexed assay of putative splice-altering variants that could facilitate clinical classification of variants 2 .…”

Section: Introductionmentioning

confidence: 99%

ParSE-seq: A Calibrated Multiplexed Assay to Facilitate the Clinical Classification of Putative Splice-altering Variants

O’Neill,

Yang,

Laudeman

et al. 2023

Preprint

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BackgroundInterpreting the clinical significance of putative splice-altering variants outside 2-base pair canonical splice sites remains difficult without functional studies.MethodsWe developed Parallel Splice Effect Sequencing (ParSE-seq), a multiplexed minigene-based assay, to test variant effects on RNA splicing quantified by high-throughput sequencing. We studied variants in SCN5A, an arrhythmia-associated gene which encodes the major cardiac voltage-gated sodium channel. We used the computational tool SpliceAI to prioritize exonic and intronic candidate splice variants, and ClinVar to select benign and pathogenic control variants. We generated a pool of 284 barcoded minigene plasmids, transfected them into Human Embryonic Kidney (HEK293) cells and induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), sequenced the resulting pools of splicing products, and calibrated the assay to the American College of Medical Genetics and Genomics scheme. Variants were interpreted using the calibrated functional data, and experimental data were compared to SpliceAI predictions. We further studied some splice-altering missense variants by cDNA-based automated patch clamping (APC) in HEK cells and assessed splicing and sodium channel function in CRISPR-edited iPSC-CMs.ResultsParSE-seq revealed the splicing effect of 224SCN5Avariants in iPSC-CMs and 244 variants in HEK293 cells. The scores between the cell types were highly correlated (R2=0.84). In iPSCs, the assay had concordant scores for 21/22 benign/likely benign and 24/25 pathogenic/likely pathogenic control variants from ClinVar. 43/112 exonic variants and 35/70 intronic variants with determinate scores disrupted splicing. 11 of 42 variants of uncertain significance were reclassified, and 29 of 34 variants with conflicting interpretations were reclassified using the functional data. SpliceAI computational predictions correlated well with experimental data (AUC = 0.96). We identified 20 uniqueSCN5Amissense variants that disrupted splicing, and 2 clinically observed splice-altering missense variants of uncertain significance had normal function when tested with the cDNA-based APC assay. A splice-altering intronic variant detected by ParSE-seq, c.1891-5C>G, also disrupted splicing and sodium current when introduced into iPSC-CMs at the endogenous locus by CRISPR editing.ConclusionsParSE-seq is a calibrated, multiplexed, high-throughput assay to facilitate the classification of candidate splice-altering variants.

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High-throughput splicing assays identify missense and silent splice-disruptivePOU1F1variants underlying pituitary hormone deficiency

Cited by 2 publications

References 74 publications

Linking genome variants to disease: scalable approaches to test the functional impact of human mutations

Linking genome variants to disease: scalable approaches to test the functional impact of human mutations

ParSE-seq: A Calibrated Multiplexed Assay to Facilitate the Clinical Classification of Putative Splice-altering Variants

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