hereditary spastic paraplegia · reflux, and spastic paraparesis with amyotrophy am. j. hum. genet....

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


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


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?


G93

Glu

*

*

*

*

* * ADP

PUA domain

Su

bu

nit 2

Subunit 4

V243

V120

Why dominant and recessive mutations exist?


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

hereditary spastic paraplegia · reflux, and spastic paraparesis with amyotrophy am. j. hum. genet....

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