Linfedema torino 4 e 5 marzo gaal palma [modalità compatibilità]
Master Spada 18 Gen 08
1. malattie lisosomiali da accumulo
Marco Spada
Clinica Pediatrica, Università di Torino
Ospedale Infantile Regina Margherita, Torino
Master Universitario di 2° Livello in Malattie Rare
Università degli Studi di Torino, Facoltà di Medicina e Chirurgia
Dipartimento di Medicina e Oncologia Sperimentale
Torino, 18 Gennaio 2008
2. La medicina moderna
Patologia esogena =
1) patologia infettiva
2) malnutrizione
Patologia endogena =
Malattie cronico-degenerative
5. Malattie ereditarie del metabolismo
altre malattie
Malattie genetiche:
5000 malattie
malattie ereditarie del metabolismo:
Difetto proteine enzimatiche
Difetto di proteine di trasporto
Difetto di proteine strutturali
500 malattie monogeniche
a) difetto di una substrato
b) accumulo di un substrato
19. Le malattie lisosomiali: una storia
Prime descrizioni cliniche
1882: Gaucher (MD Thesis, Faculté de Médicine, Paris)
1898: Fabry (Arch Dermat Syph)/Anderson (Br J Dermatol)
Biochimica dei lisosomi
1955: de Duve (Biochem J): scoperta dei lisosomi (Premio Nobel)
1963: Hers (Biochem J): primo difetto enzimatico lisosomiale descritto
(deficit di maltasi acida nella M. di Pompe)
2006: 40 malattie lisosomiali definite biochimicamente
Genetica LSD (1980-1990)
1985: Sorge et al (Proc Natl Acad Sci USA) : cloning gene glucocerebrosidasi
1986: Bishop et al (Proc Natl Acad Sci USA): cloning gene α-gal-A
Terapia LSD
1984: trapianto di midollo in Gaucher disease-BMT (Rappeport, N Engl J Med)
1991: terapia enzimatica-ERT (Beutler et al, N Engl J Med)
2000: terapia di riduzione del substrato-SRT (Cox et al, Lancet)
1999: terapia di stimolazione enzimatica : EET-chaperon (Fan et al, Nat Med)
Screening-Prevenzione LSD
2006: Screening neonatale LSD (Spada, Pagliardini et al, Am J Hum Gen)
23. Malattia di Gaucher
La diagnosi biochimica
- β-glucosidasi: leucociti/fibroblasti
- chitotriossidasi)
24. Malattia di Gaucher
La diagnosi genetica
1) omogeneità molecolare
N370S, L444P, 84GG, IVS2
80 % alleli mutati*
(* Gaucher Registry, 2000,
766 pazienti 1532 alleli)
2) Genotipo e valore predittivo
a) N370S/…. :
forma viscerale
b) L444P/L444P:
forma neurologica
28. La malattia di Gaucher
Esperienza della Clinica Pediatrica di Torino
pazienti Età Inizio Evoluzione clinica Età alla Inizio Risposta
sintomi diagnosi terapia alla terapia
Mar. G. 56 aa 50 aa Piastrinopenia, 53 aa 53 aa buona
Sospetta leucosi
Boc. M. 45 aa 7 aa Splenomegalia, 8 aa 35 aa buona
Splenectomia 26 aa
Ant. G. 30 aa 10 aa Splenomegalia 29 aa 29 aa 6 mesi
piastrinopenia
Mar.C. 27 aa 5 aa splenectomia 6 aa 25 aa buona
Rub.P 25 aa 3 aa Splenectomia 5 aa 16 aa buona
maattia ossea,
Scompenso cardiaco
Mas. A. 17 aa 3 aa Epato-splenomegalia 6 aa 7 aa buona
Bro. S. 11 aa 4 aa Splenomegalia, dolori ossei 10 aa 11 aa 6 mesi
31. Fisiopatologia :
modello Gaucher e modello Fabry
Malattia di Gaucher = malattia da accumulo “classica”
Patologia del macrofago malattia da accumulo parenchimale
(visceromegalie/infiltrazioni: milza, fegato, midollo osseo)
. Malattia di Fabry = malattia da accumulo “peculiare”
. Patologia dell’endotelio malattia vascolare (ischemia/infarto)
. “end-stage organ disease” (rene, encefalo, coronarie)
37. Mucopolissacaridosi tipo I
(difetto di L-iduronidasi)
L-iduronidasi
Accumulo progressivo di GAG
Patologia progressiva multisistemica
38. Mucopolissacaridosi tipo I
(difetto di L-iduronidasi)
Accumulo progressivo di GAG
Patologia progressiva multisistemica
Fenotipi clinici
1) Hurler
1) Forma neuropatica
2) Hurler-Scheie
2) Forma non neuropatica
3) Scheie
39. Mucopolissacaridosi tipo I
(difetto di L-iduronidasi)
CLINICA
- encefalopatia progressiva
- facies grossolana
- macroglossia
- sordità (tipo misto)
- epato-splenomegalia
- opacità corneali
- malattia cardiaca: valvulopatia/CMP
- malattia respiratoria ostruttiva
- malattia scheletrica :
a) deformità ossee (disostosi multipla, cifo-scoliosi)
b) artropatia cronica progressiva
43. La Malattia di Pompe
(Glicogenosi II)
Malattia genetica autosomica recessiva
difetto dell’enzima lisosomiale
α-glucosidasi acida (maltasi acida)
accumulo progressivo tissutale di glicogeno
a prevalente espressione cardio-muscolare
Organi bersaglio:
cuore, muscolo scheletrico, muscolo liscio
44. La malattia di Pompe
La Malattia di Pompe
(glicogenosi II) II)
(Glicogenosi
Basi biochimiche
45. La Malattia di Pompe
(Glicogenosi II)
Popolazioni a rischio
- ipotonia del neonato e del lattante
- cardiomiopatia ipetrofica del neonato e del lattante
- cardiomiopatia ipertrofica e ipotonia del lattante
- miopatia ad esordio clinico variabile (bambino adulto)
- iperCPKemia isolata
52. La Malattia di Pompe
(Glicogenosi II)
Diagnosi biochimica
- muscolo
Determinazione attività enzimatica - linfociti
α-glucosidasi acida - spot di sangue
assenza di attività enzimatica attività enzimatica residua
Fenotipo classico: Fenotipo “variante”:
“early-onset” o forma infantile forma giovanile o dell’ adulto
(late-onset)
53. La Malattia di Pompe
(Glicogenosi II)
Popolazioni a rischio
- ipotonia del neonato e del lattante
Fenotipo classico
forma infantile (early-onset) - cardiomiopatia ipetrofica del neonato e del lattante
- cardiomiopatia ipertrofica e ipotonia del lattante
Fenotipo “variante” - miopatia ad esordio clinico variabile (bambino adulto)
forma giovanile/adulta
(late-onset) - iperCPKemia isolata
58. 1) BMT
1) Gaucher : fino al 1990, oggi non c’e indicazione
2) MPS tipo 1: indicazione nel fenotipo Hurler, entro i due anni di età
3) MPS tipo 2 : scarsi risultati
4) M. di Krabbe : tentativi in pazienti di pochi mesi
60. La medicina “genomica” nella medicina moderna
Le biotecnologie
a) Copiare un gene = una informazione
b) Inserire questo gene in un sistema “ospite”:
batterio, lievito, colture cellulare
c) Traduzione dell’informazione in quantità “industriali”:
sintesi in vitro di proteine “ricombinanati)
insulina, GH
vaccini (epatite B)
fattore VIII (emofilia)
enzimi lisosomiali
61. Nell’ambito delle malattie lisosomiali nasce in medicina
il primo modello di terapia enzimatica sostitutiva
1991: Malattia di Gaucher [difetto di β-glucosidasi]
2001: Malattia di Fabry [difetto di α-galattosidasi A]
2002: malattia di Hurler-Scheie (MPS I) [difetto di α-iduronidasi]
2005: malattia di Pompe (glicogenosi II) [difetto di α-glucosidasi acida]
2005: malattia di Hunter (MPS II): [difetto di α-iduronidasi-sulfatasi]
2005: malattia di Maroteaux-Lamy (MPS VI): [difetto di arilsulfatasi B]
63. Gaucher
ERT and bone disease:
British Journal of Radiology, 2002;75:Suppl1
“Gaucher bone disease
can be stabilized
or reversed with ERT” Hemorrhagic
Infarction
Necrosis
“ ERT in pediatrics
patients: Prevention of Osteosclerosis
potential serious
skeletal complications”
(Bembi, Ciana) Severe
Osteoporosis
Loss of
Cortical Bone
64. La malattia di Gaucher: gli effetti delle terapia enzimatica sostitutiva
(alglucerasi imiglucerasi)
- 12 anni di follow-up; > 2500 pazienti in terapia
- Weinrab et al. ERT in 1028 pazienti (Gaucher Registry)
Am J Med, 2002;113
Efficacia della terapia: > 90 % dei pazienti
Dosi/15gg
6 mesi
Anemia
Piastrinopenia 60 U/kg
Epatomegalia 1 anno 30 U/kg
Splenomegalia
20 U/kg
Cardiomiopatia
15 U/kg
Malattia ossea 2-4 anni
65. Shitrit, Zimran, el…
Prediction of severity of bone and lung disease in Gaucher disease
Am J Hematol 2003;73:236
118 pazienti 85 % ERT
15 % wait and see
67 %
very mild
moderate
severe
15 %
18 % Bone disease
Lung disease
66. Efficacy of Enzyme Replacement Therapy in Fabry disease
(Eng et al, N Engl J Med, 2001)
1 mg/kg iv every other week
Primary end p
- oint: clearance of renal capillary endothelium
Baseline (score 3) Week 20 (score 0)
67. ERT in Fabry disease: Indications for Treatment
renal extrarenal
- patients on dialysis: - Cardiomyopathy
to avoid cardiac/cerebrovascular complications
- Arrythmias
- kidney transplanted patients:
to avoid cardiac/cerebrovascular complications
- Previous coronary artery disease
- patients with decreased GFR (CRF):
- Previous strokes/TIAs
to stop the progression of renal failure
- Deafness
- early-stage renal disease patients:
to stop / to reverse progression of nephropathy
- Severe pain syndrome
- childrens/adolescents renal asymptomatic:
to prevent the nephropathy
68. ERT in Fabry disease
a) ERT may prevent or reverse the nephropathy in young patients:
Therapy must be considered in pediatric/adolescent age
b) ERT may arrest the progression to renal failure in patient with
early-stage nephropathy (2 to 3 year follow-up)
c) ERT in chronic renal failure : which is the “point of not return”?
dose-dependent clinical efficacy ?
d) Prevention of CNS events and cardiac disease in ESRD patients:
further follow-up is necessary
e) ERT improves the cardiomyopathy (Circulation, 2003)
f) ERT improves blood flow velocity (Munich, October 2003)
g) ERT may improve cardiac conduction disease
h) ERT may improve hearing loss
69. Mucopolissacaridosi tipo I
(difetto di L-iduronidasi)
- encefalopatia progressiva
- facies grossolana
- macroglossia
- sordità (tipo misto)
- epato-splenomegalia
- opacità corneali
- malattia cardiaca: “ ERT in MPS I ”,
- malattia respiratoria ostruttiva Kakkis et al.
N Engl J Med, 2001
- malattia scheletrica :
a) deformità ossee
b) artropatia cronica progressiva
70. 3) Enzyme Enhancement Therapy (EET)
low-molecular weight pharmacological chaperons
Rescue of misfolded or unstable enzyme proteins
Increase enzyme function
EET in lysosomal disorders
Galactose α-gal A activity (Fabry)
Deoxygalactonojirimycin (DGJ) (specific mutations)
N-deoxynojirimycin (NN-DNJ) β-glucosidasi (Gaucher)
71. 3) Enzyme Enhancement Therapy (EET)
Fabry patient with severe cardiac involvement (cardiac variant)
- heart transplantation list
- NYHA class IV
Galactose (1 g/kg) intravenously Enhancement of α-gal A activity
Dramatic clinical improvement
NYHA class I
No need for heart transplantation
(Frustaci et al, N Engl J Med, 2001)
72. 4) Substrate Reduction Therapy (SRT):
New approach in the treatment of LSDs
New form of treatment, but not a new concept
- In 1970s: first inhibitors of glucosylceramide synthesis
- Aim: to balance the rate of synthesis with the impaired rate of breakdown
- SRT is efficient only if residual enzyme activity is still present
73. Simplified Biochemical Pathways:
ceramide as a cornerstone for sphingolipid byosynthesis
Sphingomyelin Ceramide Galactosyl-ceramide
(Niemann-Pick A,B) (Krabbe, MLD)
GSL
Glucosylceramide
Ganglio-series Globo-series
Lacto-series
(GM1, GM2) (Gaucher) (Fabry)
74. X
iminosugar :
N-butyldeoxynojirimycin
(miglustat)
76. Goal of Therapy
Gaucher disease regular
macrophage macrophage
• Over time, the Gaucher disease cell becomes full of waste material
• These cells store in various parts of body and result in the problems associated
with Gaucher disease
77. Enzyme Replacement
waste material waste material degraded
add Enzyme
Ceramide-Glucose
(Replacement Ceramide
(Glucocerebroside)
Therapy)
Glucose
78. Substrate Deprivation Therapy
less storage of allows the residual enzyme
less waste
waste material in activity to catabolize waste
material
lysosomes material
Own residual
enzyme
84. The extraordinary lesson from PKU
(Bob Guthrie - early 1960s)
Blood spot technology
screening for inborn errors of metabolism
a model in modern preventive medicine
85. Mass or Selective Screening Programms
by blood spot technology
Basic criteria
1) Characteristics of the disease
- treatable
- (frequent ?)
2) Characteristics of the screening method
- simple (easy methodology and technology)
- low-cost (low investment, low cost per analysis)
- sensible
- specific
- efficient (many samples per day)
86. Screening
for Lysosomal Storage Disorders (LSDs)
Treatable LSDs
1991: Gaucher disease
2001: Fabry disease
2002: MPS I
Pompe disease
Results of ERT trials
MPS II
J Inherir Metab Dis 2005;28(suppl 1)
MPS VI
Escolar et al:
Transplantation of umbilical-cord blood
Krabbe
in babies with infantile Krabbe’s disease
N Eng J Med 2005
87. Screening
for Lysosomal Storage Disorders (LSDs)
TWO MAIN AIMS
1) define the real epidemiology (frequency) of the disease
(present knowledge is based on retrospective analysis)
2) make an early diagnosis before the onset of irreversible pathology
- Newborns
- childhood
- young adults
- family members
- selected patient populations
90. POMPE Disease
Giulia, 5 mesi
Glicogenosi tipo II
Malattia cardiaca e muscolare
gravissima
Terapia enzimatica
sperimentale a 13 mesi
91. La prevenzione della Malattia di Pompe:
una frontiera della medicina moderna
Fenotipo “variante” - miopatia ad esordio clinico variabile (bambino adulto)
forma giovanile/adulta
(late-onset) - iperCPKemia isolata
93. Screening for Lysosomal Storage Disorders (LSDs)
strategies
1) Not-specific lysosomal protein markers:
LAMP+saposins by immune-based assay (Meikle, Australia, 1997)
2) Specific lysosomal substrates by tandem-mass spectrometry
(2001-2005)
3) “Protein profiling” : immune-based assay for lysosomal enzymes
(Meikle, Australia, 2004)
4) Direct assay of lysosomal enzymes by tandem mass spectrometry
(Gelb,Usa,2004)
5) Fully-automated fluorescent assay of lysosomal enzymes
(Pagliardini, Spada-2001)
94. Mass and selective screening for LSDs
our strategy
M Spada and S Pagliardini
(Regina Margherita Children’s Hospital, Torino, Italy)
New screening approach for Fabry Disease.
1st European Round Table on Fabry disease
Nice-France 15 June 2001
fully automated fluorescent assay for
Fabry screening α-gal A quantification in blood spots
Hospital:
name or code:
> 1000 samples per die
date of sampling:
Mass and selective screening
in large populations
95. Pagliardini S, Spada M.
A simple and rapid approach for screening lysosomal disorders.
J Inher Metab Dis, 2003;26(Suppl 2):1.
- α-galactosidase A
fully automated
-β-glucosidase enzyme quantification
LSDs screening -chitotriosidase
- α-glucosidase
> 1000 samples per die
-α-iduronidase
-any lysosomal enzyme
Mass and selective screening
in large populations
96. First prospective screening studies for LSDs
Selected populations
Newborns (general population)
Pagliardini S, Spada M.
Spada M. , Pagliardini S First neonatal mass
Screening for Fabry disease in screening program
end-stage nephropathies for lysosomal storages
J Inher Metab Dis, disorders:
2002;28(Suppl 1):148. 18 month experience
J Inher Metab Dis,
2005;28(Suppl 1):148.
97. Selective screening for Fabry disease:
ESRD male population
(Strategies in Regina Margherita Children’s Hospital, Turin, Italy)
α--galactosidase A activity
α galactosidase A activity
25.000 tests on dried blood spot on filter paper Screening test
on dried blood spot on filter paper
(new method)
(new method)
α- galactosidase A activity
α- galactosidase A activity
in plasma
in plasma
(classical method)
(classical method)
Diagnostic tests
a cohort of 8037
ESRD male patients GLA gene sequencing
completed the study GLA gene sequencing
98. Screening for Fabry disease in male patients with ESRD:
five lessons (1)
1) The prevalence of Fabry disease in ESRD male population:
0.25 % = 21:8037 (1:400)
2) Fabry disease is largely undiagnosed amongst ESRD patients
Thadhani et al. ( Kidney International 2002): Spada, Pagliardini ( December 2003):
retrospective study mass screening
USA Europe Europe
73 / 233.552 = 0,030 % 21/ 8037 = 0,25 %
37 / 132.687 = 0,027 %
Clinical strategy Screening strategy
1 10
99. % αGalA activity Mutation Age at diagnosis(yr) Age at ESRD(yr) Cardiac dis. CVD
1.KJ <5% I317T 26 24 - -
2.DE <5% G325S 44 25 - -
3.PS <5% V269M 40 29 - -
4.BM <5% g10214insT 36 31 - -
5.CF <5% R220X 43 39 CMP -
6.BM <5% N228H 52 48 CAD -
7.MC <5% E59K 53 49 CAD -
8.PD <5% A143T 55 53 - Stroke at 50 y
9.CG <5% A143T 71 54 CMP -
10.CC <5% Q279K 58 56 CAD at 50 y Stroke at 51 y
11.SQ <5% A143T 62 59 CMP -
12.SF 25 % G395A 65 59 CAD at 52 y -
13.MK <5% G360E 68 61 nd nd
14.PG <5% A143T 68 66 CMP -
15.OI <5% D313Y 72 71 CAD at 57 y TIA
16.TS <5% A143 T 82 78 CAD at 42 y Stroke at 40 y
101. Le malattie lisosomiali: una storia
Prime descrizioni cliniche
1882: Gaucher (MD Thesis, Faculté de Médicine, Paris)
1898: Fabry (Arch Dermat Syph)/Anderson (Br J Dermatol)
Biochimica dei lisosomi
1955: de Duve (Biochem J): scoperta dei lisosomi (Premio Nobel)
1963: Hers (Biochem J): primo difetto enzimatico lisosomiale descritto
(deficit di maltasi acida nella M. di Pompe)
2006: 40 malattie lisosomiali definite biochimicamente
Genetica LSD (1980-1990)
1985: Sorge et al (Proc Natl Acad Sci USA) : cloning gene glucocerebrosidasi
1986: Bishop et al (Proc Natl Acad Sci USA): cloning gene α-gal-A
Terapia LSD
1984: trapianto di midollo in Gaucher disease-BMT (Rappeport, N Engl J Med)
1991: terapia enzimatica-ERT (Beutler et al, N Engl J Med)
2000: terapia di riduzione del substrato-SRT (Cox et al, Lancet)
1999: terapia di stimolazione enzimatica : EET-chaperon (Fan et al, Nat Med)
Screening-Prevenzione LSD
2006: Screening neonatale LSD (Spada, Pagliardini et al, Am J Hum Gen)
102. Newborn screening for Fabry disease
- Piemonte Region, Italy
- 37.104 consecutive male newborns
Normal mean activity: α--galactosidase A activity
α galactosidase A activity Screening
7.7 ± 3.1 nmol/h/ml blood on dried blood spot
on dried blood spot
Cut-off level: < 1.5 nmol/h/ml
(20% normal mean )
α- galactosidase A activity
α- galactosidase A activity
in plasma
in plasma Definite diagnosis
GLA gene sequencing
GLA gene sequencing
103. RESULTS
12 infants were found posititive at
second blood spot α- gal A determination.
The diagnosis of Fabry disease was then confirmed
Birth prevalence:
12/ 37.104
1/3100
104. Biochemical and molecular data in 12 male newborns with α-gal A deficiency
blood spot α- plasma α-gal α-gal A gene
gal A (> 1.9 A (6-19 mutation
nmol/h/ml) nmol/h/ml)
1 0.0 0.8 N215S
2 0.2 0.7 E66G
3 0.0 0.6 M51I
4 0.0 0.8 A73V
5 0.1 0.7 N215S
6 0.0 0.3 F113L
7 0.1 1.1 A143T
8 0.2 0.6 A143T
9 0.2 0.7 R118C
10 0.0 0.2 IVS5 +1G T
11 0.0 0.2 F113L
12 0.4 0.8 A143T
105. Newborn screening for Fabry disease
1) Definition of the prevalence of the disorder
Newborn screening approach Clinical diagnosis/retrospectve studies
1 : 3000 1: 40.000 - 1: 100.000
chronic nephropathy
A disease largely misunderstood cerebrovascular disease
in medical community hypertrophic cardiomyopathy
2) A simple, low-cost, rapid and efficient tool for prevention strategy
early diagnosis
General population (newborns) Newborn family members
Optimal planning of ERT since pediatric age Start ERT before end-stage organ disease
106. Prevalenza della malattia di Fabry
in popolazioni di pazienti a rischio
Insufficienza renale = 0.25-0.3 %
Spada, 2002. J Inher Metab Dis
Nakao, 2003. Kidney Int
Cardiomiopatia ipertrofica = 3-4 %
Nakao, 1995. N Engl J Med
Sachdev, 2002. Circulation
Stroke = 5 %
Rolfs, 2005. Lancet
107. Fabry disease = a model of X-linked disorder
GLA gene mutation = G 260 E (ex 5)
108. Screening neonatale di massa
Pediatria preventiva Pediatria eugenetica
Sintomi neonatali assenti Sintomi neonatali presenti Sintomi neonatali
Terapia efficace [< 2 gg] [ 2-5 gg] [5-15 gg] assenti/presenti
Terapia efficace
Problemi: Terapia non (poco) efficace
diagnosi clinica/dimissione precoce Prognosi infausta
PKU galattosemia fibrosi cistica
ipotiroidismo SAG
biotinidasi
organico acidurie
Gaucher, Fabry leucinosi
difetti β-ox-AG
Pompe, MPS I
tirosinemia
Wilson
MCAD
109. Screening neonatali: nuove frontiere
1) Malattie cerebrali con danno irreversibile Fenilchetonuria
(entro il primo anno di vita): Ipotiroidismo
ritardo mentale Dif. biotinidasi
severa encefalopatia
2) Malattie acute potenzialmente mortali S. adreno-genitale: disidratazione
ad esordio neonatale Galattosemia: epatopatia acuta
3) Malattie croniche con danno d’organo irreversibile
M. di Gaucher: anemia,piastrinopenia, malattia scheletrica
M. di Fabry: insuff. renale, ictus, infarto
Glicogenosi II: cardiomiopatia, miopatia
MPS1: encefalopatia e malattia scheletrica
