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Potential Consequences on RNA level
A. Variants altering the structure/ integrity: pre-mRNA splicing
B. Variants altering the stability/ turnover: mRNA (UTRs, 3D, miRNA binding)
C. Variants altering the translation dynamics: mRNA (codon usage, +/- ribosomal PS)
D. Considerations for functional RNA tests: material, strategies
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Speaking about RNA
ENCODE project (Nature 489, 57-74. 2012):
• Most of the genome (>85%) is transcribed
• 60.000 „genes“:~ 20.000 protein coding genes (>90% multiple isoforms)
~ 16.000 long non-coding (lnc) RNAs~ 10.000 small non-coding (snc) RNAs~ 14.000 pseudogenes
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Palazzo et al.; Front. Genet., 26 January 2015
Speaking about RNA
ncRNA, tRNA, rRNA, snRNA, siRNA, hnRNA, scRNA, RNA editing, Ribosome, RNP, mRNA surveillance/ decay
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Potential Consequences on RNA level
A. Variants altering the structure/ integrity: pre-mRNA splicing
B. Variants altering the stability/ turnover: mRNA (UTRs, 3D, miRNA binding)
C. Variants altering the translation dynamics: mRNA (codon usage, +/- ribosomal PS)
D. Considerations for functional RNA tests: material, strategies
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
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Scotti & Swanson. Nature Reviews Genetics 17, 19–32 (2016))
A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Overview
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Scotti & Swanson. Nature Reviews Genetics 17, 19–32 (2016))
A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Overview
99 %
0,6 %
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
Variants affecting splicing
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
Variants affecting splicing
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
Variants affecting splicing Effect on RNA
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
Stump et al.; American Journal of Physiology - January 2011 Vol. 300 no. 1
Multiple splicing defects caused by hERG splice site mutation 2592+1G>A associated with long QT syndrome
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
Pathogenic variants that affect pre-mRNA splicing account for at least 15% of disease-causing mutations (Krawczak et al.; Hum Genet. 1992; 90(1–2): 41–54).
With up to 50% of all pathogenic mutations described in some genes (NF1, ATM) (Teraoka et al.; Am J Hum Genet. 1999; 64(6): 1617–1631 / Ars et al.; Hum Mol Genet. 2000; 9(2): 237–247.)
Most variants affect the canonical (+/- 1, 2) splice sites
Caminsky et al.; F1000Research 2015
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / splice sites
Scotti & Swanson. Nature Reviews Genetics 17, 19–32 (2016)
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing
Exons that were skipped as a result of splicing variants are shorter than average exons
Kralovicova et al.; Nucleic Acids Research 35(19):6399-413 · February 2007
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Prediction
Review of user orientated in silico tools for splicing: Xueqiu et al.; Genetics in Medicine (2013) 16
Method
Neural Networks
Neural Networks
Position dependent logic
Maximum entropy principle
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Prediction
http://www.umd.be/HSF3/
HSF3 Pro takes into account the U2 and U12 introns
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A. Variants altering the mRNA structure / integrity
When to recommend or perform a cDNA study ?
pre-mRNA splicing / Prediction
Pyrimidin to purin change
• Weakening of SA site
• Possible de novo SA site
How to weight different
algorithms?
Important:
Lab should install rules
for prediction / reporting
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A. Variants altering the mRNA structure / integrity
Comprehensive in silico analysis (MES, SSF, NNsplice, HSF; ESEfinder, Rescue-ESE)
Comprehensive in vitro mRNA analysis (cDNA: PAX + cell culture; mini-gene)
Comparison of different in silico tools with regard to specificity and sensitivity
pre-mRNA splicing / Prediction
Houdayer et al. Hum Mutat 2013
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Prediction
MES+SSF: 96% sensitivity and 83% specificity
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BRCA2: c.68-7T>A(IVS2)
Santos (2014) J Mol Diagn 16: 324: Alternative splicing of ex3, no segregation with diseaseHoudayer (2012) Hum Mutat 33: 1228: Increase in delta3 alternative splicingOlfson (2015) PLoS One 10: e013519: Co-occurs with deleterious BRCA2 variants
A. Variants altering the mRNA structure / integrity
Be carefull with alternative splicing
ENIGMA: > 30% skipped transcript
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A. Variants altering the mRNA structure / integrity
Be carefull with alternative splicing
ENIGMA (BRCA)
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / splicing regulatory elements: ESE ESS ISE ISS
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A. Variants altering the mRNA structure / integrity
Wang et al.; RNA 14: 802-813 (2008)
pre-mRNA splicing / splicing regulatory elements: ESE ESS ISE ISS
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / ESE ESS ISE ISS
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / ESE ESS ISE ISS
Gen Variant Disease Effect Ref.
GH 1 c.176A>GFamilial isolated GH
deficiency type IIDisruption of ESE site
Moseley et al.; J ClinEndocrinol Metab. 2002
CFTR c.1966G>T Cystic Fibrosis Disruption of ESE siteAznarez et al.; Hum. Mol. Genet. 2003
ATP6AP2c.345C>T
X-linkedparkinsonism with spasticity (XPDS)
Novel ESS creationKorvatska et al. Hum. Mol. Genet. 2013
DMD c.4250T>ABecker musculardystrophy (BMD)
Novel ESS creationDisset et al.; Hum. Mol. Genet. 2006
MAPT c.892A>G
Frontotemporaldementia w.
parkinsonism chr 17 (FTDP-17)
Disruption of ESSIovino et al.; Acta Neuropathol. 2014
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / ESE ESS ISE ISS
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Branch Point
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Branch Point
Very rarely described (<20)
Possible explanations for the rarity of BPS mutations:
• Compensatory, alternative BPS sequences can be recognized and used
• The weak constraint on the precision of the distance between the BPS and the 3´ (acceptor)
splice site further enables activation of these alternative sites.
• Bias due to technical limitations (-20-50 region was traditionally a preferred sanger primer
location, NGS capture or bioinformatic filter criteria)
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A. Variants altering the mRNA structure / integrity
pre-mRNA splicing / Branch Point
Gene Variant Disease Effect Ref.
FBN2 c.3974-26T>GCongenital contractural
arachnodactyly(CCA)
Skipping of Ex31 in 25% oftranscripts
Maslen et al.; Am J Hum Genet 1997
COL5A1 c.2701-25T>G Ehlers-Danlos syndrome
(EDS)45 bp of exon 33 are “skipped”
in 60% of transcriptsBurrows et al.;
Am J Hum Genet 1998
LCAT c.524-22T>C Fish-eye diseaseComplete loss of function due to
intron retentionLi; et al.;
Biochim Biophys Acta 1998
NPC1 c.882-28A>G Niemann-Pick disease
(NPC) Shorter transcript lacking exon 7
Di Leo et al.;Hum Mutat 2004
KCNH2 c.2399-28A>GLong QT
(LQT)Incorrect identification of the
acceptor site of intron 9 Crotti et al.;
Heart Rhythm 2009
UROS c.661-31T>G Congenital
erythropoietic porphyria100% intron retention without
exon skipping (last exon)Bishop et al.;Blood 2010
NF2 c.516+232G>A Neurofibromatosis 2creates a functional de novo BP
sequence in intron 5De Klein et al.;
Hum Mol Genet 1998
ITGB4 c.1762-25T>Apyloric atresia-junctional
epidermolysis bullosaresulted in two abnormal
transcripts each with a PTCMasunaga et al.;
J Dermatol Sci 2015
BRAF c.1141-51C>G Melanoma (cell line)BRAF exon 4–8∆; also referred to
as BRAF3–9Salton et al.;
Nat Commun 2015
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Potential Consequences on RNA level
A. Variants altering the structure/ integrity: pre-mRNA splicing
B. Variants altering the stability/ turnover: mRNA (UTRs, 3D, miRNA binding)
C. Variants altering the translation dynamics: mRNA (codon usage, +/- ribosomal PS)
D. Considerations for functional RNA tests: material, strategies
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B. Variants altering the stability / turnover
mRNA
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B. Variants altering the stability / turnover
mRNA / Possible mechanisms
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
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B. Variants altering the stability / turnover
mRNA / stability
GFP library: 154 copies of GFP with random changes at synonymous sites:
• >250-fold variation in protein levels
• stability of mRNA secondary structure near ATG explained > 50%
Kudla et al.; 2006. PLoS Biol. 4:933-942
Sauna et al.; 2012. Nat Rev Genet 12: 683-691
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• CYP2D6: synonymous codingvariant causes decreasedmRNA expression by alteringthe secondary structure ofthe mRNA leading to itsdegradation (Toscano et al.; 2006)
B. Variants altering the stability / turnover
mRNA / stability
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
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B. Variants altering the stability / turnover
mRNA / miRNA binding
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B. Variants altering the stability / turnover
mRNA / miRNA binding
• 1.900 – 3.800 human miRNAs (Friedländer et al.; 2014 Genome Biology / Telonis et al.; 2015 Nucleic Acids Res.)
• ~ 60% (30-80%) of human genes are regulated by miRNAs (Friedmann et al.; 2008 Genome Res)
• 1 miRNA usually targets more than 100 human genes
• A gene may, in turn, be regulated by multiple miRNAs
Melanoma: synonymous codingvariant causes increased mRNAstability of the oncogene BCL2L12 due to loss of the mi-R-671-5p target site(Gartner et al.; 2013)
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
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B. Variants altering the stability / turnover
mRNA / miRNA binding
• 1.900 – 3.800 human miRNAs (Friedländer et al.; 2014 Genome Biology / Telonis et al.; 2015 Nucleic Acids Res.)
• ~ 60% (30-80%) of human genes are regulated by miRNAs (Friedmann et al.; 2008 Genome Res)
• 1 miRNA usually targets more than 100 human genes
• A gene may, in turn, be regulated by multiple miRNAs
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• KEGG pathway
• miRNA in cancer
B. Variants altering the stability / turnover
mRNA stability / miRNA binding
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B. Variants altering the stability / turnover
mRNA / miRNA prediction
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B. Variants altering the stability / turnover
mRNA / miRNA prediction
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Potential Consequences on RNA level
A. Variants altering the structure/ integrity: pre-mRNA splicing
B. Variants altering the stability/ turnover: mRNA (UTRs, 3D, miRNA binding)
C. Variants altering the translation dynamics: mRNA (codon usage, +/- ribosomal PS)
D. Considerations for functional RNA tests: material, strategies
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C. Variants altering the translation dynamics
mRNA / Codon usage
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Silent vs Synonymous
• Silent: outside coding regions, UTR´s and synonymous
• Synonymous: in coding region NOT altering aminoacid sequence due toredundancy of the genetic code
• Synonymous variants and disease: Supek 2014: 20% of all mutations in cancer are synonymous, and 6-8% of these are selected for (drivermutations)
• Mechanisms: changes in protein abundance and structure bydisruption/creation of splicing regulatory sites, alterations of RNA stability, gain/loss of mi-RNA binding sites and changes in translation efficiency
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C. Variants altering the translation dynamics
• Codon Usage Bias: although thegenetic code is degenerate, synonymous codons are NOT used in equal frequencies
mRNA / Codon usage
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C. Variants altering the translation dynamics
• Codon Usage Bias: although thegenetic code is degenerate, synonymous codons are NOT used in equal frequencies
mRNA / Codon usage
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C. Variants altering the translation dynamics
• Codon Usage Bias: although thegenetic code is degenerate, synonymous codons are NOT used in equal frequencies
mRNA / Codon usage
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• Codon Usage Bias: although thegenetic code is degenerate, synonymous codons are NOT used in equal frequencies
• Variants can alter translationalspeed (if a abundant codon ischanged to a rare one) leading toa change in cotranslationalprotein folding (Yu et al, 2015)
C. Variants altering the translation dynamics
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457
mRNA / translational speed
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C. Variants altering the translation dynamics
Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457Sauna et al.; 2012. Nat Rev Genet 12: 683-691
• Codon Usage Bias: although thegenetic code is degenerate, synonymous codons are NOT used in equal frequencies
• Variants can alter translationalspeed (if a abundant codon ischanged to a rare one) leading toa change in cotranslationalprotein folding (Yu et al, 2015)
mRNA / translational speed
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• Removal or introduction of a ribosomalpause site can lead to an alternative proteinconformation
C. Variants altering the translation dynamics
Sauna et al.; 2012. Nat Rev Genet 12: 683-691
mRNA / ribosomal pause sites
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• Removal or introduction of a ribosomalpause site can lead to an alternative proteinconformation
• Domains can fold differently byexperiencing stabilization from neighboringdomains (Sauna & Kimchi-Sarfaty, 2011)
C. Variants altering the translation dynamics
Sauna et al.; 2012. Nat Rev Genet 12: 683-691
mRNA / ribosomal pause sites
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C. Variants altering the translation dynamics
mRNA / Codon usage examples
Sauna et al.; 2012. Nat Rev Genet 12: 683-691
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Potential Consequences on RNA level
A. Variants altering the structure/ integrity: pre-mRNA splicing
B. Variants altering the stability/ turnover: mRNA (UTRs, 3D, miRNA binding)
C. Variants altering the translation dynamics: mRNA (codon usage, +/- ribosomal PS)
D. Considerations for functional RNA tests: material, strategies
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D. Considerations for RNA functional testsM. Genuardi, L. Valle, CRC-Book 2017; Springer
Is blood the right tissue?
Is the gene of interest expressed in lymphocytes (PBMC)?
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D. Considerations for RNA functional testsM. Genuardi, L. Valle, CRC-Book 2017; Springer
Is blood the right tissue?
Is the gene of interest expressed in lymphocytes (PBMC)?
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D. Considerations for functional RNA tests
Sources of RNA material suitable for analysis• cell culture• blood (heparin, citrate, or EDTA) • tissue samples (FFPE)
Dealing with NMD• Cycloheximide: concentration between 100-250 mg/ml and an incubation time of at least 4 hours• Puromycin: concentration of 10-20 mg/ml and a 5-hour incubation time is commonly used
In addition to at least 10 wildtype controls in the same assay—to facilitate the interpretation of the relevance of naturally occurring isoforms—the inclusion of a cell line with a validated NMD-prone variant is highly recommended in order to verify the fidelity of the inhibition process (positive control)
Strategies depending on expected effect• (RT) PCR-amplified cDNA fragments from fresh blood, PAX RNA, or lymphocyte cultures
• ASE-assay: The determination of allele-specific expression (ASE) is a powerful tool for assessing the relevance of suspected pathogenic alleles. In single-nucleotide extension assays such as SNuPE, SNaPshot and pyrosequencing or in MALDI-ToF mass spectrometry, ASE analysis takes advantage of a previously detected germline single-nucleotide variant (SNV) as a proxy for allelic expression.
• Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) or allele-specific expression (ASE) for promotor variants.
M. Genuardi, L. Valle, CRC-Book 2018; Springer
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Thank you for listening…
Potential Consequences on RNA level