Consequences on RNA level - Amazon S3 · • 1.900 –3.800 human miRNAs (Friedländer et al.; 2014...

Potential Consequences on RNA level

Andreas LanerMGZ München

laner@mgz-muenchen.de

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

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

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

A. Variants altering the mRNA structure / integrity

pre-mRNA splicing

Scotti & Swanson. Nature Reviews Genetics 17, 19–32 (2016))

pre-mRNA splicing / Overview

Scotti & Swanson. Nature Reviews Genetics 17, 19–32 (2016))

pre-mRNA splicing / Overview

pre-mRNA splicing

Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457

Variants affecting splicing

pre-mRNA splicing

Variants affecting splicing

pre-mRNA splicing

Variants affecting splicing Effect on RNA

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

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

pre-mRNA splicing / splice sites

Scotti & Swanson. Nature Reviews Genetics 17, 19–32 (2016)

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

pre-mRNA splicing / Prediction

Review of user orientated in silico tools for splicing: Xueqiu et al.; Genetics in Medicine (2013) 16

Method

Neural Networks

Position dependent logic

Maximum entropy principle

http://www.umd.be/HSF3/

HSF3 Pro takes into account the U2 and U12 introns

When to recommend or perform a cDNA study ?

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

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

Houdayer et al. Hum Mutat 2013

MES+SSF: 96% sensitivity and 83% specificity

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

Be carefull with alternative splicing

ENIGMA: > 30% skipped transcript

Be carefull with alternative splicing

ENIGMA (BRCA)

pre-mRNA splicing / splicing regulatory elements: ESE ESS ISE ISS

Wang et al.; RNA 14: 802-813 (2008)

pre-mRNA splicing / splicing regulatory elements: ESE ESS ISE ISS

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

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)

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

B. Variants altering the stability / turnover

mRNA / Possible mechanisms

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

• CYP2D6: synonymous codingvariant causes decreasedmRNA expression by alteringthe secondary structure ofthe mRNA leading to itsdegradation (Toscano et al.; 2006)

mRNA / stability

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)

• 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

• KEGG pathway

• miRNA in cancer

mRNA stability / miRNA binding

mRNA / miRNA prediction

C. Variants altering the translation dynamics

mRNA / Codon usage

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

• Codon Usage Bias: although thegenetic code is degenerate, synonymous codons are NOT used in equal frequencies

mRNA / Codon usage

• 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

Diederichs et al.; 2016. EMBO Molecular Medicine 8(5):442-457Sauna et al.; 2012. Nat Rev Genet 12: 683-691

• 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

• Removal or introduction of a ribosomalpause site can lead to an alternative proteinconformation

mRNA / ribosomal pause sites

• 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)

mRNA / ribosomal pause sites

mRNA / Codon usage examples

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)?

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)?

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

Thank you for listening…

Consequences on RNA level - Amazon S3 · • 1.900 –3.800 human miRNAs (Friedländer et al.; 2014...

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