RNA splicing mutations and human disease: Pompe disease - Emanuele Buratti Convegno del 25 novembre "Diagnosi e management della glicogenosi tipo 2" - Centro di coordinamento regionale malattie rare FVG

1. Emanuele Buratti
RNA splicing mutations and
human disease: Pompe
disease.
Author
INTERNCAETINOTNRAEL ENGINFEOERR IGNEGN AENTDIC BIOTECHNOLOGY

2. One CENTRE made of
three Components, two Outstations and a Network of
38 Affiliated Centres in 63 Member States and 83
Signatory Countries
Trieste Component
Inherited genetic disorders
Gene and cell therapy of cardiovascular disorders
Basic molecular biology
Regulation of mRNA processing
Mammalian DNA replication,
Chromosomal stability in yeast
Mechanisms of DSB repair
Genetics of antibodies
Molecular biology of viral infections
Protein structure and bioinformatics
Quorum sensing in bacteria
Production of recombinant proteins for human therapy
ICGEB

3. Pompe disease, Glycogenosis type II
or acid maltase deficiency
Autosomal recessive lysosomal storage
disease (1:40.000 live births)
Due to the deficiency of a-glucosidase (GAA)
or acid maltase
Impaired glycogen degradation and
accumulation within the lysosomes leads to
enlargement of cardiac and skeletal muscle.
Phenotypic continuum:
Infantile onset
•Manifests soon after birth
•Rapidly progressive disease course
•Progressive muscle weakness
•Cardiomegaly and cardiomyopathy
•Hypotonia
•Respiratory insufficiency
•Feeding difficulties
•Moderate hepatomegaly
•Markedly elevated CK
•no GAA activity
Late onset
•Manifests in children or adults
•Progressive muscular
weakness
•No cardiac involvement
•Respiratory insufficiency
•Exercise intolerance
•Swallowing difficulty
•Moderate hepatomegaly
•Elevated CK
•Residual GAA activity
http://www.unitedpompe.ccoomm//aabboouuttppoomm
ppee..ccffmm

4. PolII PolII PolII
PolII
3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’
AAAAAAAAAA
aug uag
5’UTR
ORF
3’UTR
AAAAAAAAAA
pre-mRNA splicing:
5’cap Poly-A
Splicing
aug uag
Export and Translation
DNA
pre-mRNA
mRNA
protein
DDiisseeaassee--ccaauussiinngg mmuuttaattiioonnss ccaann ooccccuurr
iinn aallll tthheessee ttyyppeess sspplliicciinngg ccoonnttrroolllliinngg
eelleemmeennttss::

5. What happens when one of the
basic elements is altered?
Splicing mutations can be found in
virtually any intron-containing gene.
The frequency depends on overall
length and individual susceptibilities
Baralle D. et al. EMBO Rep 2009; 10:810-816.

6. Focus on the splicing mutations:

7. Molecular analysis
ATG TAG
Close to 200 mutations have been described; most of them are private.
Some mutations are common in different ethnic groups (p.R854X among
African-Americans; p.D645E among Asians and del525T in Dutchs)
The mutation profile is very heterogeneous.
In late onset patients the leaky c.-32-13T>G is present in about 40-
70% of the alleles.
Infantile GSDII
c.525delT
(11.8%)
c.1064T>C
(7.9%)
c.1655T>C
(10.5%)
mutation profile highly heterogeneous
all mutations described are considered to be severe
Late onset GSDII
c.2237G>A
(10.3%)
c.-32-13T>G
(42.3%)
c.-32-13T>G is the most frequent GAA
mutation (allelic frequency: 42.3%).
Combination of severe with mild mutations
correlates with late onset of the disease

8. Age at onset of symptoms and the current age of
a cohort of 36 patients with GSDII disease
Each horizontal bar represents the disease duration of an individual patient. *Patients have died. van der
Ploeg and Reuser, Pompe's disease The Lancet, Volume 372, Issue 9646, 2008, 1342 - 1353
Despite the common genotype, patients present with a great
variability in residual enzyme activity, age of appearance of clinical
signs and rate of disease progression
GSDII in Italy:

9. exon 1
5’ss(c1) 3’ss(c2)
exon 2 (578 bp)
exon 3
gcg/gtaaca
35 bp
cgggtgaga
-13T>G
tcttctcccgcaggc….
60 bp
….acggtgggc catctcttctagat
g
tcttccccaag/ga
Why is the -13T>G mutation so harmful?:
Exon 2 is very long with
respect to the majority of
normal human exons
Sakharkar et al., 2005
The 5’splice site is poorly
defined according to
consensus
guccauucauapppG
5’ CAGGURAGU 3’
u
u u u
a
c
c
c
g
3’ U5
gagaca
U6
3’
U1
3’ m3
guccauucauapppG
5’ ACGGUGGGC 3’
u
u u u
a
c
c
c
g
3’ U5
gagaca
U6
3’
U1
3’
m3
Normal interactions GAA exon 2

10. What was already known regarding the effects of this mutation:
exon 1
5’ss(c1) 3’ss(c2)
exon 2 (578 bp)
exon 3
gcg/gtaaca
35 bp
cgggtgaga
-13T>G
tcttctcccgcaggc….
60 bp
….acggtgggc catctcttctagat
g
tcttccccaag/ga
N
SV1
SV2
SV3
c1
c2
Huie ML et al.,, HMG, 1994
Analysis of two patients that express ONLY the allele carrying the -13T/G mutation:

11. Pulldown analysis showed that weakening of the
splice site was associated to loss of binding of one
of the basic splicing factors, U2AF65:
-13u gccucccugcugagcccgcuuucuucucccgcagGCCUGUAugugugug
-13g gccucccugcugagcccgcuugcuucucccgcagGCCUGUAugugugug
-13g -13u Beads
kDa
U2AF65
TDP-43
-13 3‘ss
ug-tail
BP
(yncuray)
MW -13g -13u Beads
83
58
47.5
32.5
P. red
Western
In vitro splicing assay confirmed that 3’ss
was indeed weakened:
SP6/T7
promoter
Labelled RNA is transcribed
in vitro and incubated in nuclear
extract for 2-4 hours.
T7
Splicing products
are separated
in a denaturing gel
or amplified by
RT-PCR
cccgcuuucuucucccgcagGCCUGUAGGAGCUGUCCAGG
cccgcuugcuucucccgcagGCCUGUAGGAGCUGUCCAGG
-13u -13g
1 30 90 1 30 90 minutes
pre-mRNA
mRNA
85 nt. 3‘ss GAA exon 2
In vitro splicing

12. Making a minigene to mimic the effects of the -13T>G
Nde1
SV40 Exon 2
WT Mut
N
SV3
SV2
WT
Mut
GAA
SRSF5 (SRp40)
SRSF6 (SRp55)
N
mutation
SV3
SV2
N
SV3
SV2
-
SRSF1 (ASF/SF2)
SRSF3 (SRp20)
SRSF9 (SRp30c)
SRSF2 (SC-35)
SRSF4 (SRp75)
hnRNP F
N
SV3
WT SV2
SV3
SV2
hnRNP C2
hnRNP A2
-
hnRNP A1
TDP-43
TIA-1
YB-1
DAZAP
hnRNP H
N
Mut
Nde1
50nt 50nt
pA
a2-3 Bra2
Looking for the factors that can influence exon 2 inclusion
both in the wild-type and mutant version
Overexpression of SRSF4 in patient fibroblasts:
*
*
Relative normal spliced mRNA expression (% of mock
Knockdown of SRSF4 in patient fibroblasts:
SRSF4
Tubulin
scramble
siRNA
transfected cells)
*
Relative normal spliced mRNA
expression (% of scramble)
Relative normal spliced mRNA
expression (% of WT)

13. Resveratrol can improve mRNA and enzyme production in the fibroblasts of patient cells:

14. No other HDAC inhibitors except for SAHA can act like Resveratrol to improve normal splicing levels:

15. Highthroughput drug screening for
compounds capable of increasing GAA exon
2 inclusion:
Future directions 1: setting up an HTS assay to test
for compounds/factors capable of increasing exon 2
inclusion:
Nde1
EGFP
GAA
Exon 2
50nt 50nt
SV40
pA
Nde1
CMV
CMV SV40 pA
pEGFP-N1
pEGFP-N1 WT
50nt 50nt and MUT (-13T>G)
Transfection in HeLa cells
Exon inclusion or Exon skipping
cryptic 3’ss activation
5’ 3’
5’ 3’
WT
MUT (-13u/g)
Cont
83
62
47.5
32.5
25
62
47.5
a-EGFP
a-Tubulin
kDa
EGFP
578nt
WT MUT (-13u/g)
HeLa HeLa

16. Approaches to find new therapeutic tragets and strategies:
exon 1
5’ss(c1) 3’ss(c2)
exon 2 (578 bp)
exon 3
gcg/gtaaca
35 bp
cgggtgaga
-13T>G
tcttctcccgcaggc….
60 bp
….acggtgggc catctcttctagat
g
tcttccccaag/ga
1) Search for silencer elements:
-13T>G
tcttctcccgcaggc…. exon 2 (578 bp)
3’ss(c2)
60 bp
….acggtgggc
g
tcttccccaag/ga
Set of overlapping deletions
Once identified,
use of ASO
technology to
improve inclusion

17. 3) Use of U7snRNA to block cryptic site usage:
Sense1 TTCTTCCCCAAGGACATCCTGA
Antisense TCAGGATGTCCTTGGGGAAGAA
Sense2 CCCCACCTTCTTCCCCAAGGAC
Antisense GTCCTTGGGGAAGAAGGTGGGG
Sense3 CCAAGGACATCCTGACCCTGCG
Antisense CGCAGGGTCAGGATGTCCTTGG
U7snRNA1
U7snRNA2
U7snRNA3

18. 2) Improvement of 5’ss recognition: making a mutant U1snRNP that can recognize the poorly defined
5’splice site:
guccauucauapppG
U1
3’
m3
5’ ACGGUGGGC 3’
u
u u u
a
c
c
c
g
3’ U5
gagaca
U6
3’
Previous examples have worked very well:
ugccacucguapppG
5’ ACGGUGGGC 3’
u
u u u
a
c
c
c
g
3’ U5
gagaca
U6
3’
U1
3’
m3
mutant
GAA exon 2 GAA exon 2+mutant U1snRNP
Normal exon

19. Future directions 2: test these compounds on
skeletal muscle cells obtained through
differentiation of hSKIN-Multipotent Adult Stem Cells
(MASC) isolated from LO patients bearing the c.-32-
13T>G mutation
Skeletal muscle differentiation:

20. Acknowledgements:
Elisa Goina
Cristiana Stuani
Maurizio Romano
Francisco E. Baralle
Andrea Dardis
Irene Zanin
Stefania Zampieri
Bruno Bembi
Project detail:
TTNumber: GGP14192
Durata: 3 years
Data inizio: 30/10/2014
Conclusions:
a)The -13T>G is a common splicing mutation in late onset GSDII
disease.
b)The functional effects of this mutation are to lower
recognition of the 3’ss of exon 2.
c)This exon is very long and poorly defined even in its normal
status, and this is the reason why this mutation has a huge
effect on its recognition.
d)Several RNA-based strategies can be made available to
rescue exon 2 recognition in the presence of -13T>G.
e)These strategies may involve the use of antisense nucleotides
to inhibit splicing regulatory regions, modified U1snRNPs to
favour 5’ss recognition, or the discovery of small molecules
capable of increasing the expression of positive factors.
f)At the same, however, it is important to develop suitable
cellular models to study the efficacy of these strategies.