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

Cassina, Matteo; Cerqua, Cristina; Rossi, Silvia; Salviati, Leonardo; Clementi, Maurizio; Trevisson, Eva

doi:10.1038/ejhg.2016.176

Cited by 22 publications

(15 citation statements)

References 13 publications

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“…Direct sequencing of the RT‐PCR amplicon containing exon 13‐27 did not detect any aberrant splicing. Since mutant mRNA may undergo NMRD, we also employed a minigene assay (Figure c) to exclude an effect on transcript maturation using a β‐globin hybrid construct as previously reported (Cassina et al, ). As showed in Figure d, cells transfected with the mutant minigene construct show the same splicing pattern as those expressing the WT plasmid, suggesting that the nucleotide change does not affect splice site recognition.…”

Section: Introductionmentioning

confidence: 99%

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas

Trevisson¹,

Morbidoni²,

Forzan³

et al. 2019

Molec Gen & Gen Med

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Background Neurofibromatosis type 1 (NF1) is an autosomal dominant condition caused by inactivating mutations of the NF1 gene. The wide allelic heterogeneity of this condition, with more than 3,000 pathogenic variants reported so far, is paralleled by its high clinical variability, which is observed even within the same family. The definition of genotype–phenotype correlations has been hampered by the complexity of the NF1 gene and, although a few exceptions have been recognized, the clinical course remains unpredictable in most patients. Methods Sequencing of NF1 in patients with cafè‐au‐lait spots identified the c.3112A>G variant. RNA analysis and a minigene assay were employed to investigate splicing. Results Here we report a novel genotype–phenotype correlation in NF1: the identification of the missense variant NM_000267.3:c.3112A>G p.(Arg1038Gly) in seven individuals from two unrelated families with a mild phenotype. All the patients manifest cafè‐au‐lait spots without neurofibromas or other NF1–associated complications, and Noonan syndrome features in most cases. The missense variant was not previously reported in available databases, segregates with the phenotype and involves a highly conserved residue. Both a minigene assay and patient's RNA analysis excluded an effect on splicing. Conclusion Our data support the correlation of the p.Arg1038Gly missense substitution with the cutaneous phenotype without neurofibromas or other complications. This finding may have relevant implications for patients and genetic counseling, but also to get insights into the function of neurofibromin.

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Section: Introductionmentioning

confidence: 99%

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas

Trevisson¹,

Morbidoni²,

Forzan³

et al. 2019

Molec Gen & Gen Med

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“…Splicing events are generated through the synergetic actions of multi-subunit complexes. Although SF3B4 mutations frequently result in the Nager syndrome [7,8], the biological roles of SF3B4 in human cancer are only just beginning to be perceived. Prior studies have shown that SF3B4 expression is upregulated in HCC [9,10].…”

Section: Introductionmentioning

confidence: 99%

SF3B4 is regulated by microRNA-133b and promotes cell proliferation and metastasis in hepatocellular carcinoma

Liu

Pang

et al. 2018

eBioMedicine

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BackgroundSplicing factor 3b subunit 4 (SF3B4) is a splicing factor and potential oncogene in hepatocellular carcinoma (HCC); however, its regulatory mechanism is yet unclear. We aimed to determine the role of SF3B4 in HCC and the underlying mechanism.MethodsTo investigate the association between alternative splicing events and miRNAs, putative miRNAs were screened using TargetScan. Expression levels of and prognostic information for SF3B4 and miRNAs were determined based on public genomic data and clinical samples. Then, we examined the possible roles of SF3B4 and miRNA-133b in HCC cells and a xenograft mouse model. Pearson correlation analysis and in vitro experiments verified SF3B4 as a miRNA-133b target. Protein levels of key targets from the SF3B4 signaling pathway were estimated using western blotting.FindingsThe expression of SF3B4 was upregulated in HCC tissues and cell lines whereas, the expression of miRNA-133b was downregulated. MiRNA-133b negatively regulated the expression of SF3B4. Effects of SF3B4 overexpression were partially abolished by miRNA-133b mimics, confirming that SF3B4 is a target of miRNA-133b. Moreover, molecules associated with SF3B4, including KLF4, KIP1, and SNAI2, were also modulated by miRNA-133b.InterpretationSF3B4 plays a crucial role in HCC and is negatively regulated by miRNA-133b. The miRNA-133b/ SF3B4 axis may serve as a new therapeutic target for HCC treatment.FundChina National Funds for Distinguished Young Scientists (No.81425019), the State Key Program of National Natural Science Foundation of China (No.81730076), Shanghai Science and Technology Committee Program (No.18XD1405300) and Specially-Appointed Professor Fund of Shanghai (GZ2015009). China National Funds for National Natural Science Fund (No.81672899).

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“…For the remaining 14 non-canonical variants (including exonic changes or those involving intronic residues outside the conserved dinucleotides at the splice site boundaries), concordant results were obtained for 12 substitutions, whereas in two cases (c.7250_7252delACT and c.278G > A) different programs yielded conflicting results. Therefore, we decided to examine all these variants using a classical splicing minigene assay, which we previously successfully employed to analyze ASL , CFTR , and SF3B4 variants [ 37 , 38 , 39 , 40 ] and that has been already used by other groups for validating NF1 changes [ 8 , 36 ].…”

Section: Resultsmentioning

confidence: 99%

“…PCR fragments including the exon adjacent to each NF1 variant and at least 100 bp of the upstream and downstream introns were amplified from patients’ genomic DNA and cloned into the beta -globin vector, as previously described [ 40 ]. In presence of short introns, a bigger portion of sequence including two adjacent exons was amplified and cloned in the minigene construct.…”

Section: Methodsmentioning

confidence: 99%

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

Morbidoni

Baschiera

Forzan

et al. 2021

Cancers

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Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA.

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

Cited by 22 publications

References 13 publications

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas

The Arg1038Gly missense variant in the NF1 gene causes a mild phenotype without neurofibromas

SF3B4 is regulated by microRNA-133b and promotes cell proliferation and metastasis in hepatocellular carcinoma

Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

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