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