Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

Sharma, Neeraj; Sosnay, Patrick R.; Ramalho, Anabela S.; Douville, Christopher; Franca, Arianna; Gottschalk, Laura; Park, Jeenah; Lee, Melissa; Vecchio-Pagán, Briana; Raraigh, Karen S.; Amaral, Margarida D.; Karchin, Rachel; Cutting, Garry R.

doi:10.1002/humu.22624

Cited by 55 publications

(66 citation statements)

References 60 publications

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“…Thus, c.864+5G>T induced a deletion of 141 nt of exon 1, similar to previously reported variant c.864+1G>C (Gantla et al, 1998), but also a significant proportion of the full-length transcript (29.6%) (Figures 2B-E). Although in some cases splicing does not differ among different cell lines (Sanz et al, 2010;Sharma et al, 2014), these splicing outcomes have been obtained in MCF-7 cells (human breast adenocarcinoma) and should be further confirmed in liver cell lines.…”

Section: Discussionmentioning

confidence: 77%

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

et al. 2020

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A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → −11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an UGT1A1 splicing-reporter minigene with exons 1-4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, (E2) (exon 2 skipping/64%) and (E2q135) (intron retention of 135nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript (E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). (E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate UGT1A1. Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases UGT1A1 expression by 70%, so the fulllength transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.

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Section: Discussionmentioning

confidence: 77%

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

et al. 2020

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“…After performing the minigene assay for the c.419‐43delT variant to verify its pathogenicity in vitro , we did not find any differences concerning transcript processing between wild‐type and mutant constructions, as this variant is not located in a consensus splicing region . Likewise, we performed functional studies, using lymphoblastoid mRNA from carrier patients and patients without the deletion to confirm the minigene assay results.…”

Section: Discussionmentioning

confidence: 86%

Functional assessment of the BMPR2 gene in lymphoblastoid cell lines from Graves’ disease patients

Pousada

Lago-Docampo

Prado

et al. 2017

J Cellular Molecular Medi

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In this study, we analysed the possible influence of the c.419‐43delT BMPR2 variant in patients with Graves’ disease (GD), in a molecular basis, focusing our efforts on possible alterations in the mRNA processing and synthesis. The molecular assessment of this variant in patients with GD would shed light on the association between the BMPR2 gene and the disease. The variant was detected in 18%, 55% and 10% of patients with pulmonary arterial hypertension, GD and in general population, respectively. Patients with GD fold change showed increased BMPR2 expression when matched against the controls, with a mean of 4.21 ± 1.73 (P = 0.001); BMPR2 was overexpressed in the analysed cell cycle stages. Fold change analysis of variant carriers and non‐carriers showed slight overexpression and differences between phases, but none of them were statistically significant. BMPR2 expression was confirmed in the lymphoblastoid cell lines (LCLs) with a molecular weight of 115 kD, and no differences between variant carriers and non‐carriers were detected. To conclude, the BMPR2 variant c.419‐19delT appears in high frequency in patients with GD, and independently of its presence, BMPR2 is overexpressed in the LCLs from the GD patients tested. This increase could be paired with the described decreased expression of transforming growth factor‐β1 in thyroid tissue from patients with GD.

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“…12 As no patient's RNA was available, to confirm its pathogenicity, we functionally characterized the synonymous variant using a β-globin hybrid minigene assay, which has been effectively used to validate potential splicing variants (Figure 2c). 13 RT-PCR analysis from cells transfected with the mutant construct showed an aberrant splicing compared with controls and yielded two bands (Figure 2d): sequencing of the lower fragment revealed that it corresponds to shorter transcripts owing to the activation of a novel canonical AG/GT 5′ donor splice site within SF3B4 exon 3 and the use of two different AG acceptor sites (the first in position c.536_537, the second three nucleotides downstream) (Figure 2e). The corresponding messenger RNAs (mRNAs) result in the loss of 122 or 125 nucleotides, with the consequent formation of prematurely truncated proteins in both the cases (p.Gly139Glufs*6 or p.Asn140Leufs*4).…”

Section: Resultsmentioning

confidence: 99%

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

et al. 2016

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Nager syndrome is a rare preaxial acrofacial dysostosis that is caused by heterozygous loss-of-function variants in SF3B4. This gene encodes for a protein required for the assembly of spliceosomal complexes, being a master gene for splicing regulation. The main clinical features of Nager syndrome include facial-mandibular and preaxial limb malformations, with normal cognitive functioning. Most Nager patients are sporadic, but few familial cases with a highly variable phenotype have been reported. In this work, we report a novel synonymous variant within exon 3 of the SF3B4 gene in a family with three members affected by Nager syndrome. No pathogenic variants have been detected in other 24 genes associated with syndromes characterized by mandibulo-facial anomalies. The pathogenicity of the mutation was demonstrated through a hybrid minigene assay, which confirmed an aberrant splicing with the creation of a cryptic splice site, and showed that this allele is hypomorphic. Our findings emphasize the importance to perform functional analyses to assess the possible consequences of synonymous variants and confirmed that hybrid minigenes represent an effective tool to evaluate the effects of variants on splicing, particularly when RNA is not available.

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Experimental Assessment of Splicing Variants Using Expression Minigenes and Comparison with In Silico Predictions

Cited by 55 publications

References 60 publications

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

Functional assessment of the BMPR2 gene in lymphoblastoid cell lines from Graves’ disease patients

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

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