High-throughput splicing assays identify missense and silent splice-disruptive POU1F1 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; Jayakody, Sajini; Ozel, Ayse Bilge; Fang, Qing; Ma, Qianyi; Li, Jun Z.; Brue, Thierry; Millán, María Inés Pérez; Arnhold, Ivo J.P.; Pfaeffle, Roland; Kitzman, Jacob O.; Camper, Sally A.

doi:10.1016/j.ajhg.2021.06.013

Cited by 30 publications

(34 citation statements)

References 73 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…After applying in silico predictive tools to 41 variants previously classified as missense variants, the authors demonstrated that over 45% of them were shown to affect splicing and claimed that such variants have been frequently underscored if not analysed in depth. These observations were further supported by studies using next-generation sequencing technology [32] and highthroughput splicing reporter assays [35] to unravel the effect on splicing of single nucleotide variants, including missense, in the von Willebrand factor gene and POU1F1, respectively. Altogether, there is a need for improvement of in silico tools to predict cryptic splicing, and for the development of guidelines for formal analysis of splicing defects induced by SNVs, particularly synonymous and missense variants.…”

Section: Discussionmentioning

confidence: 82%

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing

Barbosa-Matos

Silva

Garrido

et al. 2021

Cancers

View full text Add to dashboard Cite

Hereditary diffuse gastric cancer (HDGC) caused by CDH1 variants predisposes to early-onset diffuse gastric (DGC) and lobular breast cancer (LBC). In Northern Portugal, the unusually high number of HDGC cases in unrelated families carrying the c.1901C>T variant (formerly known as p.A634V) suggested this as a CDH1-founder variant. We aimed to demonstrate that c.1901C>T is a bona fide truncating variant inducing cryptic splicing, to calculate the timing of a potential founder effect, and to characterize tumour spectrum and age of onset in carrying families. The impact in splicing was proven by using carriers’ RNA for PCR-cloning sequencing and allelic expression imbalance analysis with SNaPshot. Carriers and noncarriers were haplotyped for 12 polymorphic markers, and the decay of haplotype sharing (DHS) method was used to estimate the time to the most common ancestor of c.1901C>T. Clinical information from 58 carriers was collected and analysed. We validated the cryptic splice site within CDH1-exon 12, which was preferred over the canonical one in 100% of sequenced clones. Cryptic splicing induced an out-of-frame 37bp deletion in exon 12, premature truncation (p.Ala634ProfsTer7), and consequently RNA mediated decay. The haplotypes carrying the c.1901C>T variant were found to share a common ancestral estimated at 490 years (95% Confidence Interval 445–10,900). Among 58 carriers (27 males (M)–31 females (F); 13–83 years), DGC occurred in 11 (18.9%; 4M–7F; average age 33 ± 12) and LBC in 6 females (19.4%; average age 50 ± 8). Herein, we demonstrated that the c.1901C>T variant is a loss-of-function splice-site variant that underlies the first CDH1-founder effect in Portugal. Knowledge on this founder effect will drive genetic testing of this specific variant in HDGC families in this geographical region and allow intrafamilial penetrance analysis and better estimation of variant-associated tumour risks, disease age of onset, and spectrum.

show abstract

Section: Discussionmentioning

confidence: 82%

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing

Barbosa-Matos

Silva

Garrido

et al. 2021

Cancers

View full text Add to dashboard Cite

show abstract

“…Interestingly, exonic synonymous variants—which do not alter protein sequences—or even non-synonymous single nucleotide variants have been demonstrated to contribute to human diseases by affecting transcription and splicing regulatory factors within protein-coding regions ( 33 ). Particularly, synonymous variants were reported as a disease-causing mechanism in several endocrine disorders including familial pheochromocytoma ( 34 ), combined pituitary hormone deficiency ( 35 , 36 ), pseudohypoparathyroidism type 1 ( 37 ) and maturity-onset diabetes of the young ( 38 ). Therefore, synonymous variants should not be neglected in gene variant prioritization pipelines as they may produce abnormal mRNAs and dysfunctional proteins.…”

Section: Discussionmentioning

confidence: 99%

Silent but Not Harmless: A Synonymous SLC5A5 Gene Variant Leading to Dyshormonogenic Congenital Hypothyroidism

et al. 2022

View full text Add to dashboard Cite

BackgroundCongenital iodide transport defect (ITD) is an uncommon cause of dyshormonogenic congenital hypothyroidism characterized by the absence of active iodide accumulation in the thyroid gland. ITD is an autosomal recessive disorder caused by loss-of-function variants in the sodium/iodide symporter (NIS)-coding SLC5A5 gene.ObjectiveWe aimed to identify, and if so to functionally characterize, novel ITD-causing SLC5A5 gene variants in a cohort of five unrelated pediatric patients diagnosed with dyshormonogenic congenital hypothyroidism with minimal to absent 99mTc-pertechnetate accumulation in the thyroid gland.MethodsThe coding region of the SLC5A5 gene was sequenced using Sanger sequencing. In silico analysis and functional in vitro characterization of a novel synonymous variant were performed.ResultsSanger sequencing revealed a novel homozygous synonymous SLC5A5 gene variant (c.1326A>C in exon 11). In silico analysis revealed that the c.1326A>C variant is potentially deleterious for NIS pre-mRNA splicing. The c.1326A>C variant was predicted to lie within a putative exonic splicing enhancer reducing the binding of splicing regulatory trans-acting protein SRSF5. Splicing minigene reporter assay revealed that c.1326A>C causes exon 11 or exon 11 and 12 skipping during NIS pre-mRNA splicing leading to the NIS pathogenic variants p.G415_P443del and p.G415Lfs*32, respectively. Significantly, the frameshift variant p.G415Lfs*32 is predicted to be subjected to degradation by nonsense-mediated decay.ConclusionsWe identified the first exonic synonymous SLC5A5 gene variant causing aberrant NIS pre-mRNA splicing, thus expanding the mutational landscape of the SLC5A5 gene leading to dyshormonogenic congenital hypothyroidism.

show abstract

“…The minigene remains a standard assay to address the fidelity of splicing and has been deployed in many applications 34,35 . Although a few reports of high-throughput adaptations of the minigene system have been published, these often remain restricted to a small genomic area 36 or are limited by in-frame exon triplet cassettes 37 . A complementary tool is RT-PCR of primary patient tissue or a secondary tissue developed from patient-derived iPSCs when primary tissue is unavailable.…”

Section: Discussionmentioning

confidence: 99%

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

O’Neill

Wada

Hall

et al. 2022

Preprint

View full text Add to dashboard Cite

Background: Rare protein-altering variants in SCN5A, KCNQ1, and KCNH2 are major causes of Brugada Syndrome (BrS) and the congenital Long QT Syndrome (LQTS). While splice-altering variants lying outside 2-bp canonical splice sites can cause these diseases, their role remains poorly described. Objective: We implemented two functional assays to assess 12 recently reported putative splice-altering variants of uncertain significance (VUS) and 1 likely pathogenic (LP) variant without functional data observed in BrS and LQTS probands. Methods: We deployed minigene assays to assess the splicing consequences of 10 variants. Three variants incompatible with the minigene approach were introduced into control induced pluripotent stem cells (iPSCs) by CRISPR genome editing. We differentiated cells into iPSC-derived cardiomyocytes (iPSC-CMs) and studied splicing outcomes by reverse transcription-polymerase chain reaction (RT-PCR). We used the American College of Medical Genetics and Genomics functional assay criteria (PS3/BS3) to reclassify variants. Results: We identified aberrant splicing, with presumed disruption of protein sequence, in 8/10 variants studied using the minigene assay and 1/3 studied in iPSC-CMs. We reclassified 9 VUS to LP, 1 VUS to Likely Benign, and 1 LP variant to pathogenic. Conclusions: Functional assays reclassified splice-altering variants outside canonical splice sites in BrS- and LQTS-associated genes.

show abstract

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

Cited by 30 publications

References 73 publications

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing

The CDH1 c.1901C>T Variant: A Founder Variant in the Portuguese Population with Severe Impact in mRNA Splicing

Silent but Not Harmless: A Synonymous SLC5A5 Gene Variant Leading to Dyshormonogenic Congenital Hypothyroidism

Functional Assays Reclassify Suspected Splice-Altering Variants of Uncertain Significance in Mendelian Channelopathies

Contact Info

Product

Resources

About