hereditary spastic paraplegia - vips onlus panza hsp.pdf · 2017-07-18 · euro hsp annual meeting...
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Euro HSP Annual Meeting
17/6/2017
Hereditary Spastic Paraplegia:
the complex form SPG9
Emanuele Panza, PhD
Medical Genetics Unit
Department of Medical and Surgical Sciences
University of Bologna
COMMON FEATURES OF HEREDITARY SPASTIC PARAPLEGIA
• Hereditary neurodegeneration disease
• Clinically and genetically heterogeneous group of disease
• Main neurological signs: spasticity and wakness of the lower limbs
• 80 forms reported (Mendelian inheritance and non)
• Common pathological feature:
retrograde distal axonopathy of the long discending motor fibers
• Several intracellular pathogenetic mechanisms
• No therapies available, only symptomatic treatments
Blackstone et al., Ann Rev Neurosci 2012
Blackstone, Annu. Rev. Neurosci. 2012
Common Pathogenetics Themes in HSPs
Genetic Mapping to 10q23.3-q24.2, in a Large Italian Pedigree,
of a New Syndrome Showing Bilateral Cataracts, Gastroesophageal
Reflux, and Spastic Paraparesis with Amyotrophy
Am. J. Hum. Genet. 64:586-593, Seri Marco et al.,1999.
Main Clinical Features:
spastic paraplegia with incomplete dominance and/or variable expressivity
early bilateral cataracts
persistent vomiting
autosomal dominant inheritance
spastic paraplegia arises in the first and third decade of life
25 422 4 3 2 2
I 1 2
II
III
IV
1 2 3 4 5 7 8 9 10 11
14 15 16 17 18 20 21 32 33 34 38 39 40
4
Markers
4 2
2 1
0 0
2 4
4 1
3 4
2 3
2 3
5 1
0 0
0 0
1 5
3 3
3 3
3 3
0 0
2 2
4 4
1 1
1 1
3 3
1 1
2 2
2 2
3 3
1 1
2 3
2 3
1 2
2 2
3 5
2 2
1 3
4 3
4 1
4 3
1 2
5 5
1 5
3 3
3 3
0 0
2 2
4 4
1 1
1 1
3 3
1 1
0 0
0 0
3 3
1 1
3 2
3 1
2 3
2 4
5 1
2 4
3 3
3 3
1 1
0 0
0 0
5 5
5 3
2 4
2 2
1 2
2 2
3 4
2 3
1 2
4 2
4 5
0 0
0 0
5 1
1 3
2 2
2 3
3 4
2 3
7 7
3 5
3 4
5 4
2 3
0 0
0 0
4 5
4 2
3 2
3 1
2 3
2 4
5 1
2 4
3 3
3 3
1 1
0 0
0 0
5 5
5 3
2 1
1 4
0 0
2 1
6 2
5 2
3 2
1 3
4 1
0 0
0 0
5 2
6 6
2 3
2 3
1 2
2 2
3 5
2 2
1 3
4 3
4 1
0 0
0 0
5 5
1 5
2 2
1 2
2 1
2 2
6 3
5 2
3 1
1 4
4 4
0 0
0 0
5 5
6 1
2 2
1 2
2 1
2 2
6 3
5 2
3 1
1 4
4 4
0 0
0 0
5 5
6 1
2 2
1 2
2 1
2 2
6 3
5 2
3 1
1 4
4 4
0 0
0 0
5 5
6 1
2 2
2 3
1 4
2 3
4 7
3 5
2 4
2 4
5 3
0 0
0 0
1 5
3 2
2 2
2 2
1 3
2 2
3 7
2 3
1 3
4 5
4 2
0 0
0 0
5 4
1 4
4 2
2 3
2 4
2 3
4 7
3 5
2 4
2 4
5 3
0 0
0 0
1 5
3 2
3 2
3 2
0 0
2 2
4 3
1 2
1 1
3 4
1 4
0 0
0 0
3 5
1 1
3 2
3 2
0 0
2 2
4 3
1 2
1 1
3 4
1 4
2 4
2 1
3 5
1 1
3 2
3 2
0 0
2 2
4 3
1 2
1 1
3 4
1 4
2 3
2 2
3 5
1 5
2 2
2 3
0 0
3 2
2 6
5 1
3 2
3 4
1 1
0 0
0 0
4 5
1 1
3 2
3 2
0 0
2 2
4 3
1 2
1 1
3 4
1 4
2 4
2 1
3 5
1 1
3 3
3 3
0 0
2 2
4 5
1 2
1 3
3 3
1 1
2 3
2 2
3 5
1 5
3 3
3 3
0 0
2 2
4 5
1 2
1 3
3 3
1 1
2 3
2 2
3 5
1 5
2 2
2 2
0 0
2 3
3 2
2 5
1 3
4 3
4 1
0 0
0 0
5 4
1 1
D10S536
pol-430L14
pol-360G10
pol-543N17
D10S583
D10S1755
D10S1680
D10S574
D10S1736
pol-153G4
pol-208M2
D10S1758
D10S603
bA360G10 n.i.
bA56M3
bA366I13
bA543N17
bA3N15
bA153G4
bA208M2
SPG9
critical
region
5 Mb
bA430L14
Redefinition of the SPG9 locus
SPG9 critical
region
4.8 Mb
The SPG9 critical region
contains 52 genes
NCBI Build 36.2
OMIM 138250
iperammonemia, ipoornitinemia, ipocitrullinemia,
ipoarginemia, ipoprolinemia
Deficit ∆1-pyrrolin-5-carbossilato-sintasi
Chronic vomiting
(due to gastro-esophageal reflux)
Joint laxity resulting in severe pes planus
and dislocated hips.
Slight dysmorphic features
(short neck long fingers and toes)
Hyperelasticity of the skin
Mental deterioration and abnormal
behaviour
Bilateral zonular cataracts
Severe hypotonia, muscular wasting of
the limbs,dystonia of the hands and feet
Pyramidal syndrome and peripheral,
predominantly axonal, neuropathy
Persistent vomiting,
gastroesophageal reflux
Skeletal abnormalities,
Short stature
Learning disabilities
Bilateral cataracts
Amyotrophy
Motor system disorder
Spastic Paraparesis
………………
…………………
……………
………
…
SPG9 Deficit ∆1-pyrrolin-5-
carbossilato-sintetasi
……………………………….
wt/wt wt/wt
wt/wt wt/wt wt/wt V243L /wt
wt/wt wt/wt wt/wt wt/wt
wt/wt wt/wt
wt/wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
V243L /wt
Italian family
wt/wt
wt/wt wt/wt
R252Q/wt
R252Q/wt R252Q/wt
R252Q/wt
British family
R252Q/wt R252Q/wt R252Q/wt
Sanger sequencing and NextGen sequencing confirm the presence of the mutation p.V243L in the Italian family and the mutation
p.R252Q in a second English family.
The mutations segregate only in affected patients of the families and are not present in a panel of 466 chromosomes
Geographically matched, or in polymorphisms databases (dbSNPs, 1000 Genome Project, Exac).
P5CS protein
ALDH18A1 gene
1 67 354 362 795
G5K domain G5PR
catalytic
C612p.Val243Leu p.Arg252Glnalternative splicing
V238N239
Mitoch.target.
E3 E4 E5 E6 E7 E8 E12E11
E13 E14 E15 E16 E18E17
E1 E9
c.727G>C c.755G>A
E2
ATG TGA
E10
Italian patient
British patient
L-glutamate-5-semialdehyde
ATP ADP NADPH NADP+ + Pi
L-glutamate
L-citrulline,L-arginine,
urea....
1-pyrroline-5-carboxylate
L-glutamate2-oxoglutarate
OAT
spontaneousH2O
L-proline
NADPH NADP+
PYCR1
L-glutamate-5-phosphate
L-ornithine
Inhibition (short form)
1 67 354 362 795
Glutamate-5-kinase (G5K) Glutamate-5-phosphate reductase (G5PR)
R84QG93R
H784Y
catalytic
C612
S742I
R425C
alt. splicing
V238N239
Mitoch.target.
M586-S657del
V601I fs*26
V601G fs*24
Y782C
L711C fs*3
R765QR749Q
V120AR128H
R665LS652FL637P
D715H
R252QV243L
P5CS is a byfunctional enzyme catalyzing the first two steps of de novo syntheisis of proline,
ornithine, citrulline and arginine
Homo sapiens -NVISVKDNDSLAARLAVEMKTDL 261
Mus musculus -NVISVKDNDSLAARLAVEMKTDL 261
Canis familiaris -NVISVKDNDSLAARLAVEMKTDL 261
Gallus gallus -NVISVKDNDSLAARLAVEMKTDL 266
Xenopus laevis --VISIKDNDSLAARLAVEMKADL 260
Tetraodon nigroviridis --VISIKDNDSLAARLAVEMKADL 211
Dario rerio -NVISIKDNDSLAARLAVEMRADL 241
Branchiostoma floridae -GVISVKDNDSLAARLAAEVQADL 294
Drosophila melanogaster RRGIPIKDNDSLSAMLAAEVQADL 248
Caenorbabditis elegans ---MHISDNDSLAARLSAEIEAEL 195
Nematostella vectensis -GVISLKDNDSLAALLAVEIRADL 219
Strongylocectotus purpuratus -GVISIKDNDSLAARLAIEINADL 214
Hydra magnipapillata -DEIKFGDNDTLGALVANLVEADA 173
Triticum aestivum ----IFWDNDSLAGLLALELKADL 191
Zea mays ----IFWDNDSLAGLLAIELKADL 192
Glycin max ----IFWDNDSLSALLALELKADL 191
Medicago sativa ----IFWDNDSLSALLALELKADL 191
Vigna vinifera ----IFWDNDSLAGLLALQLKADL 193
Vigna unguiculata ----IFWDNDSLAGLLALELKADL 225
Brassica napus ----IFWDNDSLAALLALELKADL 191
Arabidopsis thaliana ----IFWDNDSLAALLSLELKADL 191
Picea sitchensis ----IFWDNDSLAALLALELRADI 188
Physcomitella patens ----IFWDNDSLAALLALELQADL 191
Saccharamyces cerevisiae -REIKFGDNDTLSAITSALIHADY 170
Escherichia coli -AEIKVGDNDNLSALAAILAGADK 164
Campylobacter jejuni -EEIVFGDNDSLSAYATHFFDADL 157
V243LLL
R252Q
Protein sequence of the mutated region in comparison with the same region in other species
da Panza et al., Brain 2016
Mutations in SPG9 cause «Loss of Function»
Plasma levels of selected aminoacids in SPG9 patients
da Panza et al., Brain 2016
Mutations in SPG9 cause «Loss of Function»
Mutations in the Human recombinant protein P5CS abolish the activity of the mutated G5K domain
Anti-P5CS Anti-PYCR1 Merge
Immunofluorescence:
Patient’s fibroblasts in a patient of the Italian family bearing the p.V243L mutation C
on
tro
l V
24
3L
Merge
Mutant cDNAs are expressed with a pattern similar to the Wild type
and are not associated with abnormalities in the localization nor a premature degradation
Western blot on patient’s fibroblasts and controls show a roughly 45% reduction of P5CS in p.V243L cells
Control V243L
P5CS
Actin
Co
ntr
ol
V24
3L
p<0.05
da Panza et al., Brain 2016
Elution volume (ml)
A280 (
rela
tive
)
Wild type Val243Leu Arg252Gln
Fresh Fresh Fresh
1 day 1 day 1 day
2 days 2 days 2 days
Hexamer
Dimer
Hexamer
Dimer
Hexamer Dimer
Why a dominant inheritance for «Loss of Function» mutations?
da Panza et al., Brain 2016
G93
Glu
*
*
*
*
* * ADP
PUA domain
Su
bu
nit 2
Subunit 4
V243
V120
Why dominant and recessive mutations exist?
da Panza et al., Brain 2016
ARCL3A OMIM 219150 ADCL3 OMIM 616603
Recessive and dominant mutations in ALDH18A1 have been identified in forms of
HSP and of cutis laxa
Towards the generation of a mouse model for «ALDH18A1-related disease»
To investigate the pathogenetic mechanism of this disease
To test new therapies
Rosa26 ALDH18A1V243L
STOP ALDH18A1V243L
LoxP LoxP
X
Generated in Mario Capecchi Laboratory
SPG9 is due to the non-synonimous single nucleotide changes c.727G>C or c.755G>A in exon 7 of the ALDH18A1 gene that
affect proximate amino acids of the G5K domain (p.Val243Leu and p.Arg252Gln, respectively).
These mutations do not prevent production nor cause cellular mislocalization of P5CS.
They are loss-of function mutations as evidenced by plasma amino acid analysis and by enzyme activity studies in
recombinantly produced human P5CS.
They selectively inactivate the domain where they map (G5K).
P5CS is a high oligomer (possibly an hexameric trimer of dimers).
SPG9 mutations disturb the architecture of the P5CS oligomer, making it prone to dissociate to dimers.
In silico structural analysis suggests that P5CS mutations can be dominant or recessive depending on whether they affect or
not residues involved in intersubunit or interdomain interactions, disturbing or not disturbing the architecture of the oligomer,
thus supporting a dominant negative disease-causing mechanism
Recessive and dominant mutations have been identified in HSP (SPG9A MIM601162, SPG9B MIM616586) and in forms of
cutis laxa (ADCL3 MIM616603, ARCL3A MIM219150)
The generation of a mouse model will be essential to dissect the pathogenetic mechanisms of SPG9 and to test new
therapies
Summary
Ackwnoledgments
Professor Marco Seri Unità di Genetica Medica
Azienda Universitaria Ospedaliera di Bologna
Professor Vicente Rubio Zamora Instituto de Biomedicina de Valencia of the CSIC, Valencia, Spain Group 739
Centro para Investigacio´n Biome´dica en Red sobre Enfermedades Raras
CIBERER-ISCIII, Valencia-Spain
Professor Giuseppe de Michele Department of Neurosciences and Reproductive and Odontostomatologic Sciences
University Federico II - Napoli
Professor Rocco Liguori IRCCS Istituto delle Scienze Neurologiche di Bologna
Department of Biomedical and NeuroMotor Sciences, University of Bologna
Professor Jane Hurst Department of Clinical Genetics, Great Ormond Street Hospital, London, UK.
Professor Mario Capecchi Howard Hughes Medical Institute, University of Utah School of Medicine
Department of Human Genetics
Salt Lake City Utah, USA