MINIGENE SERVICE
Functional analysis of DNA variants from disease-responsible genes can be carried out by direct patient RNA RT-PCR (usually lymphocyte RNA) or functional assay by hybrid minigenes.
Impact of mutations on splicing. A DNA variant (indicated by an arrow in the sequence) of the donor site of BRCA2 exon 13 induces its loss (skipping) in the mature transcript. This causes an ORF change and the generation of a premature STOP codon.
A high proportion of pathogenic variants in disease-responsible genes alter pre-mRNA processing or splicing (Lopez-Bigas et al 2005; Sanz et al 2010; Acedo et al 2012). In addition, more than 50% of DNA variants of susceptibility genes are of unknown clinical significance, which represent a serious problem for the genetic counselling of such diseases.For obvious reasons, direct analysis of RNA from a patient would the most reliable method of establishing with certainty whether a particular DNA substitution affects splicing but patient RNA is not always available and often difficult to obtain. Splicing reporter plasmids are useful alternative tools to study ex vivo the impact of a variant on splicing. The Splicing and Genetic susceptibility group of the IBGM (CSIC, PI Eladio A. Velasco) has developed a new splicing reporter plasmid (pSAD;
https://docs.google.com/file/d/0B7Ne4lHDwcbubGpfdV9xX2NmQ3M/edit?usp=sharing) to carry out splicing functional assays by hybrid minigenes, which helps to identify variants with impact on splicing.The protocol for a splicing functional analysis of a particular DNA variant of a disease-responsible gene is as simple as follows: Cloning of exon(s) of interest in pSAD. It is recommended to clone flanking exons to keep the genomic context that can influence on the splicing reactions. Alternatively, gene synthesis procedures can be carried out to circumvent this laborious step.
Generation of the DNA variant by site directed-mutagenesis;
Transfection of the wild type and mutant minigenes into eukaryotic cells;
RNA Isolation and RT-PCR with specific primers of the constitutive exons (V1 and V2) that do not amplify RNA transcribed by the host cell;
Electrophoresis and sequencing of RNA isoforms: characterization of anomalous events (e.g. exon skipping, use of alternative splice sites, intron retention, etc).
The full protocol (cloning and functional test) would take 6-12 weeks depending on the number of the cloning steps. Once the wild type minigene has been constructed, it can serve as a template to introduce and test any candidate DNA variant by PCR-mutagenesis so the experimental time is significantly reduced.
Outline of the cloning procedure into the pSAD vector. The insert with exon(s) is generated by PCR with a high fidelity polymerase and primers with tails containing suitable restriction sites to keep the orientation of acceptor and donor sites with those of the constitutive exons of pSAD (V1 and V2). E. coli DH5α cells are transformed and selected with ampicillin and X-Gal/IPTG. The splicing reactions in eukaryotic cells are indicated.
EXONS 21-22
Splicing functional assays of BRCA2 minigene MGBR2_ex19-27: Impact on splicing of DNA variants of the acceptor and donor sites of exons 21 and 22. A) Capillary electrophoresis of fluorescent RT-PCRs
List of constructed minigenes
GeneMinigeneExonsStatusDisease
BRCA1MGBR1_ex2-82 to 8
ValidatedHBOC
MGBR1_ex12-1612 to 16
Validated
MGBR1_ex15-2015 to 20
Validated
BRCA2MGBR2_ex2-92 to 9
Validated
MGBR2_ex14-2014 to 20
Validated
MGBR2_ex19-27119 to 27
Validated
MLH1MGmlh1_EX22
ValidatedLynch Syndrome
COL1A1MGcol1a1_EX6-96 to 9
ValidatedOsteogenesis imperfecta
SERPINA1MGserpa_ex1b-1c21b-1c
Validatedα1-antitrypsin defficiency
CHD7MGchd7_ex26-28326 to 28
ValidatedCharge Syndrome
GRNMGgrn_ex5-105 to 10
ValidatedFrontotemporal Dementia
1 Acedo et al (2015); 2 Lara et al (2014); 3 Villate et al (2018)
Price List (01/04/2017)1 Bioinformatics analysis of DNA variants 20 €
Analysis of patient samples: RNA extraction from blood 25 €
Lymphocyte RT-PCR 225 €
Splicing functional assays with hybrid minigenes: Construction of 1 custom minigene2 350 € (each cloning step, max. 3)
Functional assay of a DNA variant 250 €
1 VAT not included.
2 Any human exon can be cloned.
Dr Eladio A. Velasco Sampedro (
eavelsam@ibgm.uva.es) must be contacted for any functional test.
Members:
Beatriz Díez Gómez, Research Technician CSIC.
Eladio A. Velasco Sampedro, Staff Scientist CSIC.
REFERENCES Villate O; Ibarluzea N; Fraile-Bethencourt E; Valenzuela A; Velasco EA; Grozeva D; Raymond FL; Botella MP; Tejada MI (2018). Functional analyses of a novel splice variant in the CHD7 gene, found by Next Generation Sequencing, confirm its pathogenicity in a Spanish patient and diagnose him with CHARGE syndrome. Frontiers in Genetics 2018, 9: 7. DOI: 10.3389/fgene.2018.00007
Fraile-Bethencourt E, Díez-Gómez B, Velásquez-Zapata V, Acedo A, Sanz DJ, Velasco EA (2017). Functional classification of DNA variants by hybrid minigenes: Identification of 30 spliceogenic variants of BRCA2 exons 17 and 18. PLoS Genetics 13(3):e1006691. doi: 10.1371/journal.pgen.1006691.
http://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1006691Acedo A, Hernández-Moro C, Curiel-García Á, Díez-Gómez B, Velasco EA. (2015). Functional Classification of BRCA2 DNA Variants by Splicing Assays in a Large Minigene with 9 Exons. Human Mutation 36: 210–221.
[PubMed]Lara B, Martínez MT, Blanco I, Hernández-Moro C, Velasco EA, Ferrarotti I, Rodriguez-Frias F, Perez L, Vazquez I, Alonso J, Posada M, Martínez-Delgado B. 2014. Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid. Respiratory Research 15: 125.
[PubMed]Ruiz de Garibay G, Acedo A, García-Casado Z, Gutiérrez-Enríquez S, Tosar A, Romero A, Garre P, Llort G, Thomassen M, Díez O, Pérez-Segura P, Eduardo Díaz-Rubio E, Velasco EA, Caldés T, de la Hoya M. Capillary Electrophoresis Analysis of Conventional Splicing Assays: IARC Analytical and Clinical Classification of 31 BRCA2 Genetic Variants. Human Mutation 35: 53-57.
[PubMed]Acedo A, Sanz DJ, Duran M, Infante M, Perez-Cabornero L, Miner C, Velasco EA. 2012. Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes. Breast Cancer Res 14:R87.
[PubMed]Baralle D, Lucassen A, Buratti E. 2009. Missed threads. The impact of pre-mRNA splicing defects on clinical practice. EMBO Rep 10:810-816.
[PubMed]Lopez-Bigas N, Audit B, Ouzounis C, Parra G, Guigo R. 2005. Are splicing mutations the most frequent cause of hereditary disease? FEBS Lett 579:1900-1903.
[PubMed]Sanz DJ, Acedo A, Infante M, Duran M, Perez-Cabornero L, Esteban-Cardenosa E, Lastra E, Pagani F, Miner C, Velasco EA. 2010. A high proportion of DNA variants of BRCA1 and BRCA2 is associated with aberrant splicing in breast/ovarian cancer patients. Clin Cancer Res 16:1957-1967.
[PubMed]
