Prof. Giacomo P. Comi
Centro Dino Ferrari,Dipartimento di Scienze Neurologiche
Università degli Studi di Milano,Fondazione Ospedale Maggiore
Policlinico,Mangiagalli e Regina Elena,
Milano, Italy
GSD type III: molecular genetics, genotype-phenotype
correlations and the project for an animal model
GENE AND DISEASE
AGL gene (amylo-1,6-glucosidase, 4-α-glucantransferase) encodes for the glycogen debranching enzyme.
AGL is expressed as a single protein containing two distinct catalytic activities:
• 4-α-glucantransferase domain located in the N-terminal half of the protein
• 1,6-glucosidase domain in the C-terminal part
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
GLUCOSIDASE DOMAIN
TRANSFERASE DOMAIN
GLYCOGEN-BINDING DOMAIN
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
TRANSFERASE DOMAIN
GLUCOSIDASE DOMAIN
GLYCOGEN-BINDING DOMAIN
Possible infancy and childhood symptoms:
– Recurrent fasting hypoglycemia
– Seizures– Hepatomegaly– Hypotonus– Growth retardation
Adult symptoms:
– Distal weakness (calves and peroneal muscles)
– Variable degree of proximal muscles weakness
– Fatigue– Back pain – Slow progression– Serum CK increased 5x to
45x– Neuropathy (due to
glycogen storage in Schwann cells and axons)
– Hepatic dysfunction and cardiomyopathy
Clinical manifestations
0
5
10
15
20
25
30
35
1-5 6-10 11-15 16-20 21-25 26-30 >31
Age groups (years)
Num
ber
of p
atie
nts
Age at Diagnosis
0
1
2
3
4
5
6
7
8
9
N. Pts
1 2 3 4 5 6 7 8
Decades
FOCUS ON ITALIAN COHORT OF GSDIII
PATIENTS
Milano
Genova
Bologna
AnconaFirenze
Napoli
Catania
Age Groups
0
200
400
600
800
1000
1200
1400
1600
0 10 20 30 40 50 60 70
Age (yrs)
AST
(UI/
L)
0
200
400
600
800
1000
1200
0 10 20 30 40 50 60 70
Age (Years)
Liver involvement
Serum transaminase
s
Liver ecography0
0,2
0,4
0,6
0,8
1
<10 11-20 21-30 >=31NormalHepatomegaly, diffuse homogeneous hyperechogenicityHepatomegaly, dishomogeneous hyperechogenicityHepatic involution, Cirrhosis - Transplant
0
1000
2000
3000
4000
5000
6000
7000
0 10 20 30 40 50 60
Age (years)
CPK
(IU/
L)
Muscle involvement
Correlation between CK and age
Walton scale
0
1
2
3
4
5
6
7
8
9
10
0 10 20 30 40 50 60 70 80
Age
Walt
on
sco
re
EMG Findings
Normal19%
Myopathic19%
Neurogen48%
Both14%
Normal Myopathic Neurogen Both
Pseudo-myotonic discharges at EMG were found in two patients with myopathic findings and in fiveshowing chronic neurogenic denervation
Electromyography was available for 21 patients Normal 4
Myopathic 4
Neurogen 10
Both 3
Cardiology
Interventricular sept thickness:• normal: <10 mm• mild hypertrophy: 11-12 mm• moderate hypertrophy: 13-14 mm• severe hypertrophy: >14 mm
Heart echography
0
2
4
6
8
10
<10 11-20 21-30 >=31
Normal Mild Hypertrophy Moderate Hypertrophy
ATG TAG
3513 14 15 26 27 31 32 33 34
c.100G>Tc.112A>Tc.276delG
c.293+2T>A c.664+3A>G
c.442delA
c.672insTc.700T>Cc.753_756delGACAc.757G>C
c.853C>T
c.1571G>Ac.1589G>C
c.1264A>Tc.1283G>A
c. 2023C>Tc. 2147delG
c.2590C>T
c.2681+1G>A
c.2728C>T
c. 2929C>T
c.3258_3259AG>CC
c.3355G>Cc.3358G>C
c.3464G>Ac.3512_3549dup+3512_3519del
c.3362+1G>C
c.3652C>T
c.3912insA
c.3980G>Ac.3963delG
c.4193G>A c.4324insA
31
Genetic screening of 57 patients:
• 38 patients (66,7%): 2 mutations
• 7 patients (12,3%): 1 mutation
• 12 patients (21%): no mutations
Mutations are widespread along the whole gene and no hot spot region were found
Private mutations
No mutations were found in exon 3
Transferase domain
Glucosidase domain
Glycogen
binding domain
DNA mutational analysis
ATG TAG
3513 14 15 26 27 31 32 33 34
c.100G>Tc.112A>Tc.276delG
c.293+2T>A c.664+3A>G
c.442delA
c.672insTc.700T>Cc.753_756delGACAc.757G>C
c.853C>T
c.1571G>Ac.1589G>C
c.1264A>Tc.1283G>A
c. 2023C>Tc. 2147delG
c.2590C>T
c.2681+1G>A
c.2728C>T
c. 2929C>T
c.3258_3259AG>CC
c.3355G>Cc.3358G>C
c.3464G>Ac.3512_3549dup+3512_3519del
c.3362+1G>C
c.3652C>T
c.3912insA
c.3980G>Ac.3963delG
c.4193G>A c.4324insA
31
Mutation analysis:
• 39,8% splicing mutations
• 24,1% nonsense mutations
• 16,9% missense mutations
• 9,6% microinsertions
• 8,4% microdeletions
• 1,2% micro-rearrangements
24,1%: c.2681+1G>A (IVS21 +1 G>A)
10,8%: c.664+3A>G (IVS6 +3 A>G)
Challenging points
1. Molecular screening of the coding sequence and exon-intron junction of AGL gene
Mutations in promoter and intron sequences are missed
BUT
this kind of mutations and missense mutations request FUNCTIONAL ANALYSIS
In vitro models (fibroblast and
myoblast)
Mutated AGL cDNA
Feasible for each missense mutation?
4 missense mutations in three different functional domains: • Transferase e glycosidase domain mutations inactivate the specific function of the respective domain and decrease the funtion of the other domain.• Glycogen binding domain mutations impair both binding and enzymatic activities, proabably through an instability at the protein level.
Trasferasicdomain
Glycosidase domain
Glycogen binding domain
2. Missense mutations in our cohort account for < 20%
of total similar of the findings of other groups working
on GSDIII (Goldstein et al., Genet Med 2010)
3. Could it be proper to screen more than 100 control
alleles as usual?
4. Enzyme activity assay on red blood cells does not
discriminate among the different catalytic functions but
avoid patients to undergo liver or muscle biopsy.
5. Genetically undiagnosed patients often lack enzyme
activity assay which should come as a first step. Are
those patients true GSDIII?
MOUSE MODEL
Associazione Italiana Glicogenosi
www.genoway.com
MOUSE Agl: locus and gene organization
1. Constitutive knock-out by deletion of exon 1 containing ATG (corresponding to human exon 3)
The removal of the first ATG should abolish the mRNA translation
Different hypothesis for the knock-out model
2. Constitutive knock-out by deletion of a functional domain
The removal of a catalitic domain would not allow the function of the enzyme 3. Tissue-specific or adult-specific “Safe knock-out™” mouse (targeting exon 1)
This construct would allow the regular expression of the enzyme until the induction of the Knock-out, which could be tissue specific or time specific, by breeding the chimera with Cre-recombinant strain
•We need a model reproducing the human disease
•AGL is expressed in many tissues
•Mutations do not seem to affect fertility and fetal development
Constitutive knock-out model
Criteria for the choice of the best mouse model
Evidences that deletions of the C-terminal of the protein abolish both enzymatic activities (Cheng et al., Genes Dev 2007; Cheng et al., Hum Mol Genet 2009)
Deletion of the last 114aa of the glycogen-binding domain (ex 32-34)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Transferase Domain
Glucosidase Domain
GLYCOGEN-BINDING DOMAIN
Constitutive knock-out model by deletion of the glycogen-binding domain: targeting vector
Constitutive knock-out model by deletion of the glycogen-binding domain
Testing mouse to better understand
• Diet treatment: composition and relation with age
• Physical exercise and metabolism
• Peripheral nerve involvement
• Glycogen deposition vs musculoskeletal impairment
…and more
A good mouse model would allow the development of potential drugs and Enzyme Replacement Therapy
Thanks to
• Patients
• Physicians
• Associazione Italiana Glicogenosi
In collaboration with:
Istituto G. Gaslini, Genova: Mirella Filocamo, Maja Di Rocco
Università degli Studi, Catania: Agata Fiumara
Ist. Pediatrico, Univ. Federico II, Napoli: Daniela Melis
Ospedale S. Gerardo, Monza: Rossella Parini
Pediatria Ospedale San Paolo, Milano: Sabrina Paci
Ospedale Pediatrico Meyer, Firenze: Maria Alice Donati
Università di Pisa: Giuseppe Maggiore
Università Politecnica delle Marche: Anna Ficcadenti
Ospedale S. Orsola-Malpighi, Bologna: Monia Gennari
Università degli Studi, Padova: Corrado Angelini
Dipartimento di Neuroscienze, Messina: Giuseppe Vita, Antonio Toscano
Giacomo P. ComiGiacomo P. Comi
Stefania CortiStefania Corti
Sabrina Sabrina LucchiariLucchiari
Monica NizzardoMonica Nizzardo
Serena Serena PagliaraniPagliarani
Serena GhezziSerena Ghezzi
Dario RonchiDario Ronchi
Sabrina SalaniSabrina Salani
Chiara DonadoniChiara Donadoni
Martina NardiniMartina Nardini
Elisa FassoneElisa Fassone
Francesco FortunatoFrancesco FortunatoAndreina BordoniAndreina BordoniRoberto Del BoRoberto Del BoDomenica Domenica SaccomannoSaccomannoGianna UlziGianna UlziAlessio Di Fonzo Alessio Di Fonzo Isabella GhioneIsabella GhioneDomenico SantoroDomenico SantoroFrancesca MagriFrancesca MagriAlessandra GovoniAlessandra GovoniMichela RanieriMichela Ranieri
Laboratorio di Biochimica e Laboratorio di Biochimica e GeneticaGenetica
Dip. di Scienze NeurologicheDip. di Scienze Neurologiche
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