Journal: Scientific Reports

Article Title: Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies

doi: 10.1038/s41598-018-20894-0

Figure Lengend Snippet: A summary of five patients identified with VUSs from our cohort using the multigene panel sequencing approach. *Clinical significance was labelled according to the ClinVar database from NCBI ( https://www.ncbi.nlm.nih.gov/clinvar/ ). # Variant class was determined according to the HGMD (Human Gene Mutation Database http://www.hgmd.cf.ac.uk/docs/new_help.html ). ^ Variant classification was determined according to the NSEuroNet (European Network on Noonan Syndrome and Related Disorders; https://nseuronet.com/php/about.php ). % The maximum credible population allelic frequency for Noonan syndrome (1.0 × 10 −4 ) was calculated based on disease prevalence, maximum allelic contribution, maximum genetic contribution and penetrance 37 . Allelic frequency data was extracted from ExAC (Exome Aggregation Consortium; http://exac.broadinstitute.org ).

Article Snippet: Expression plasmids for RAF1 (#RC201983), A2ML1 (#RC219615) and RIT1 (#RC220552) were ordered from Origene (MD, USA).

Techniques: Sequencing, Variant Assay, Mutagenesis

Journal: Scientific Reports

Article Title: Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies

doi: 10.1038/s41598-018-20894-0

Figure Lengend Snippet: Dual luciferase assay of the phosphorylation activity changes to ELK1 from 293 T cells transfected with the corresponding expression and reporter plasmids. Statistical significance was derived using an unpaired t-test comparing the mutants with the corresponding WT. ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant. Mutated human transcript with VUSs from ( a ) RAF1 ( b ) RIT1 and ( c ) A2ML1 from this study were compared with the wild-type transcript. The data are the mean ± SD from 3 determinations.

Article Snippet: Expression plasmids for RAF1 (#RC201983), A2ML1 (#RC219615) and RIT1 (#RC220552) were ordered from Origene (MD, USA).

Techniques: Luciferase, Phospho-proteomics, Activity Assay, Transfection, Expressing, Derivative Assay

Journal: Scientific Reports

Article Title: Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies

doi: 10.1038/s41598-018-20894-0

Figure Lengend Snippet: Transient expression of RNA transcripts in zebrafish embryos. The injection dosage of each RNA transcript was optimised for comparison ( RAF1 : 50 pg/embryo; RIT1 : 400 pg/embryo; A2ML1 : 200 pg/embryo). A morphometric analysis was performed to compare the effect of the VUSs compared to the wild-type transcript at three dpf. Statistical significance was derived using a two-sided Fisher’s exact test to compare mutants with the corresponding WT. ** p < 0.01; *** p < 0.001; ns: not significant. NIC: No injection control; ( a ) Proportion of zebrafish embryos with normal or diseased phenotypes. ( b ) Representative zebrafish embryos with a normal phenotype ( c ) craniofacial dysmorphism ( d ) gross malformations and ( e ) cardiac oedema.

Article Snippet: Expression plasmids for RAF1 (#RC201983), A2ML1 (#RC219615) and RIT1 (#RC220552) were ordered from Origene (MD, USA).

Techniques: Expressing, Injection, Comparison, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Integrating Functional Analysis in the Next-Generation Sequencing Diagnostic Pipeline of RASopathies

doi: 10.1038/s41598-018-20894-0

Figure Lengend Snippet: A summary of 20 patients identified with pathogenic or likely pathogenic mutations from our cohort using the multigene panel sequencing approach. *Clinical significance was labelled according to the ClinVar database from NCBI ( https://www.ncbi.nlm.nih.gov/clinvar/ ). # Variant class was determined according to the HGMD (Human Gene Mutation Database http://www.hgmd.cf.ac.uk/docs/new_help.html ). ^ Variant classification was determined according to the NSEuroNet (European Network on Noonan Syndrome and Related Disorders; https://nseuronet.com/php/about.php ).

Article Snippet: Expression plasmids for RAF1 (#RC201983), A2ML1 (#RC219615) and RIT1 (#RC220552) were ordered from Origene (MD, USA).

Techniques: Sequencing, Variant Assay, Mutagenesis, Functional Assay

Journal: Cancer genetics

Article Title: A new NFIA:RAF1 fusion activating the MAPK pathway in pilocytic astrocytoma.

doi: 10.1016/j.cancergen.2016.09.002

Figure Lengend Snippet: Figure 1 Identification of a novel NFIA:RAF1 fusion in pilocytic astrocytoma. (a) Graphic illustration of translocation between the chromosomal regions 1q31.3 and 3p25.2, resulting in fusion between NFIA (NM_001134673.3) exons 1–6 and RAF1 (NM_002880.3) exons 9–17, including the Raf-1 kinase domain. (b) RT-PCR with primers in NFIA exon 6 and RAF1 exon 9 re- sulted in a 406 bp PCR product. An in-house non-CNS tumor was used as negative control. (c) The junction was verified by Sanger sequencing of the RT-PCR product.

Article Snippet: Cells were transfected with empty vector, EV (pCMV6Entry-FLAG, Origene), wild type NFIA (pCMV6-NFIA-FLAG, Origene), wild type RAF1 (pCMV6-RAF1-FLAG, Origene), NFIA:RAF1 (cDNA cloned into pCMV6-Entry-FLAG) (Primer sequences are available on request).

Techniques: Translocation Assay, Reverse Transcription Polymerase Chain Reaction, Negative Control, Sequencing

Journal: Cancer genetics

Article Title: A new NFIA:RAF1 fusion activating the MAPK pathway in pilocytic astrocytoma.

doi: 10.1016/j.cancergen.2016.09.002

Figure Lengend Snippet: Figure 2 Functional characterization and subcellular localization of NFIA:RAF1 fusion. (a) HEK293 cells were transfected with empty vector, EV (pCMV6-Entry-FLAG), wild type NFIA (pCMV6-NFIA-FLAG), wild type RAF1 (pCMV6-RAF1-FLAG), NFIA:RAF1 (cDNA cloned into pCMV6-Entry-FLAG). Western blot analysis was performed using antibodies against Raf-1 and GAPDH. The molecular weight of the fusion protein was calculated to 80 kDa, while the endogenous Raf-1 is 75 kDa as depicted. (b) HEK293 cells were transfected as described above, or stimulated for 10 minutes with 10 ng/ml human recombinant epidermal growth factor (EGF) as positive control for activated MAPK pathway. Antibodies against phospho-Ser221-MEK1/2, MEK1/2 were used. (c) Cell growth of trans- fected HeLa cells was measured 3 days after transfection. Cell growth is calculated relative to EV transfected cells. Error bars shown are standard error of the mean (SEM) and the asterisk denotes statistical significance (P = 0.049) compared to EV. (d) HeLa cells were transfected with EV, wild type RAF1 or NFIA:RAF1 constructs and fractionation into cytoplasmic and membrane fractions was performed as described in the Materials and Methods section. Western blotting was performed using an antibody against FLAG-tag. (e–g) Transfected HeLa cells were fixed and stained for FLAG-tag (green) indicating the FLAG-tagged constructs; NFIA-FLAG, RAF1- FLAG and NFIA:RAF1-FLAG, F-actin (red), reflecting the localization of the cellular membrane and nucleus (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Cells were transfected with empty vector, EV (pCMV6Entry-FLAG, Origene), wild type NFIA (pCMV6-NFIA-FLAG, Origene), wild type RAF1 (pCMV6-RAF1-FLAG, Origene), NFIA:RAF1 (cDNA cloned into pCMV6-Entry-FLAG) (Primer sequences are available on request).

Techniques: Functional Assay, Transfection, Plasmid Preparation, Clone Assay, Western Blot, Molecular Weight, Recombinant, Positive Control, Construct, Fractionation, Membrane, FLAG-tag, Staining