SCARB2 mutations as modifiers in Gaucher disease: the wrong enzyme at the wrong place?

(1)

SCARB2

MUTATIONS AS

MODIFIERS

IN

GAUCHER DISEASE:

the wrong enzyme at the wrong place?

C O U T I N H O M F

1 _{, L A C E R D A L}

2 _{, G A S PA R A}

3 _{, P I N T O E}

2 _{, R I B E I R O I}

2 _{, L A R A N J E I R A}

F

2 _{, R I B E I R O H}

2 _{, S I LV A E}

2 _{, F E R R E I R A C}

2 _{, P R ATA M J}

4 , 5

_{, A LV E S S}

1

15th

_{International Symposium}

(2)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)

Original illustration by Marcos Bernardino for Cristiana Petriz´s “Gigi e a Doença de Gaucher”, 2010

(3)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



Atypical form:



Deficient enzyme:

Saposin C



Gene: PSAP

(10q21-q22)

(4)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



Large spectrum of severity

& symptoms

(5)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)

(6)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…

(7)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants

based on the presence

of neuronopathic disease

abscence

(8)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants

based on the presence

of

neuronopathic disease

abscence

(9)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants

GD type

1

2

3

(10)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants

Non-Neurological

GD type

1

2

3

(11)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants

Non-Neurological

Neurological

GD type

1

2

3

(12)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants

(13)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



At the clinical level…



3 variants



Genotyope-Phenotype correlations

(14)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



One of the

most frequent LSD

(15)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



One of the

most frequent LSD



Available ERT

(16)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)

(17)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)



1 st

described

(18)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)



1 st

described



1 st

drug approved

(19)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)



1 st

described



1 st

drug approved



> nr therapeutic approaches

(20)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)



Still…

it does have some

significant differences

(21)



At the populational level…



Surprising link to common disorders

(22)



At the populational level…

GBA mutation carriers

(23)



At the populational level…

GBA mutation carriers

Parkinson’s disease

(24)

Gaucher Disease

(GD)



GCase

(25)

Gaucher Disease

(GD)



GCase

(26)

Gaucher Disease

(GD)



GCase

(27)

Gaucher Disease

(GD)



GCase

(28)

Gaucher Disease

(GD)



GCase

(29)

Gaucher Disease

(GD)



GCase



At the molecular level…

(30)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2

(31)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2



Gene:

SCARB2

Action myoclonus-renal failure

(32)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2



Gene:

SCARB2

Variation in this gene may account

(33)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2



Gene:

SCARB2

Variation in this gene may account

(34)

Objective

Understand the role of

variations

in

SCARB2

in the

(35)

Objective

Understand the role of

variations

in

SCARB2

in the

broad

phenotype spectrum

observed for GD patients

(36)

Sample Collection



Portuguese

GD population

(37)

Sample Collection



Portuguese

GD population



91 samples



Biochemically



molecularly

characterized GD patients,

diagnosed at CGMJM

(38)

Sample Collection



Portuguese

GD population



91 samples



Biochemically



molecularly

characterized GD patients,

diagnosed at CGMJM

until 2013

+



Controls

(39)

Sample Collection



Portuguese

GD population



91 samples



Biochemically



molecularly

characterized GD patients,

diagnosed at CGMJM

until 2013

+



Controls



50 samples

(40)

Genetic analysis



DNA



_{Peripheral blood}

(41)

Genetic analysis



Sanger sequencing



12 SCARB2

exons +

intronic bounderies

Intron 1 Intron 2 Intron 3

Exon 1 Exon 2 Exon 3 Exon 4

Exon 18 Exon 18 Exon 18 Exon 18 Exon 19 Exon 20 Exon 20 Exon 20 Exon 21 Exon 21 Exon 21 Exon 21 Normal transcript Mutant transcripts Alu-Sz element 62 bp of intron 18 (a) (b) (c1) (3) (2) Exon 18 Exon 20

C

A

Gg

c

a

c

g

t

(c2) 5’ss GC Alu-Sz element

C

A

GG

C

T

G

A C

T

C 5’ss GC 5’ss flanking sequence fitting the parameters described by Sorek et al., 2004 for Alu exonization

Alu element

5’ UTR Intron 4 Intron 5 Intron 6 Intron 7

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

C5’ss GC 5’ss flanking sequence fitting the parameters described by Sorek et al., 2004 for Alu exonization

Alu element

Intron 8 Intron 9 Intron 10 Intron 11

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

Intron 12



DNA



_{Peripheral blood}

(42)

Genetic analysis



Sanger sequencing



12 SCARB2

exons +

intronic bounderies

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

Intron 12



In silico analyses



Evaluation of the

deleterious potential

of the novel variant(s)



DNA



_{Peripheral blood}

(43)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element Intron 12

N

o

n

-co

d

in

g

vari

an

ts

rs3733259

rs894248

_rs2289512

rs2289513

rs35369082

rs77814624

rs35583533

(44)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

N

o

n

-co

d

in

g

vari

an

ts

rs3733259

rs894248

_rs2289512

rs2289513

rs35369082

rs77814624

rs35583533

Registered polymorphisms

(45)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

p.M159V

p.V396I

Co

d

in

g

vari

an

ts

(46)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

p.M159V

p.V396I

Co

d

in

g

vari

an

ts

known polymorphic variations

(47)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

p.T398M

Co

d

in

g

variant

s

(48)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

p.T398M

Co

d

in

g

variant

s

Novel coding variant

(49)

Results

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

C

A

GG

C

T

G

A C

T

p.T398M

Co

d

in

g

variant

s

Novel coding variant

not detected in 50 controls

(50)

In silico analyses

(51)

In silico analyses

(52)

Reassessment

of the

clinical case



2 nd

child of young parents

healthy

unrelated

(53)

Reassessment

of the

clinical case



Symptoms’ onset:

7 months



2 nd

child of young parents

healthy

unrelated

(54)

Reassessment

of the

clinical case



Symptoms’ onset:

7 months



Multi-symptomatic clinical presentation



_{abdominal distension;}



diarrhea;



anorexia;



feeding difficulties;



_{rough coughing;}



_{progressive weight loss.}



2 nd

child of young parents

healthy

unrelated

(55)

Reassessment

of the

clinical case



2 nd

child of young parents

healthy

unrelated

Cape Verdean origin



Symptoms’ onset:

7 months



Disease progression:



_{severe anemia;}



poor facial mimic;



stridor;



thrombocytopenia;



_{cardiomegaly;}



_{marked splenomegaly;}

(56)

Reassessment

of the

clinical case



Symptoms’ onset:

7 months



Disease progression:



_{severe anemia;}



poor facial mimic;



stridor;



thrombocytopenia;



_{cardiomegaly;}



_{marked splenomegaly;}



interstitial lung disease with multiple recurrent infections;



_{bilateral convergent strabismus;}



marked axial hypotonia



2 nd

child of young parents

healthy

unrelated

(57)

Reassessment

of the

clinical case



Symptoms’ onset:

7 months



Disease progression:



_{severe anemia;}



poor facial mimic;



stridor;



thrombocytopenia;



_{cardiomegaly;}



_{marked splenomegaly;}



interstitial lung disease with multiple recurrent infections;



_{bilateral convergent strabismus;}



marked axial hypotonia

global psychomotor developmental delay



2 nd

child of young parents

healthy

unrelated

(58)



Symptoms’ onset:

7 months



Disease progression:



_{severe anemia;}



poor facial mimic;



stridor;



thrombocytopenia;



_{cardiomegaly;}



_{marked splenomegaly;}



interstitial lung disease with multiple recurrent infections;



_{bilateral convergent strabismus;}



marked axial hypotonia

global psychomotor developmental delay



2 nd

child of young parents

healthy

unrelated

Cape Verdean origin

Neurological GD

(59)

Reassessment

of the

clinical case



Symptoms’ onset:

7 months



Disease progression:



_{severe anemia;}



poor facial mimic;



stridor;



thrombocytopenia;



_{cardiomegaly;}



_{marked splenomegaly;}



interstitial lung disease with multiple recurrent infections;



_{bilateral convergent strabismus;}



marked axial hypotonia

global psychomotor developmental delay



2 nd

child of young parents

healthy

unrelated

Cape Verdean origin

(60)

Reassessment

of the

GBA genotype



Genotype:

L444P/L444P



2 nd

child of young parents

healthy

unrelated

(61)

Reassessment

of the

GBA genotype



Genotype:

L444P/L444P



_{Common mutation}



Known genotype-phenotype correlation



2 nd

child of young parents

healthy

unrelated

(62)

Reassessment

of the

GBA genotype



Genotype:

L444P/L444P



_{Common mutation}



Known genotype-phenotype correlation





2 nd

child of young parents

healthy

unrelated

Cape Verdean origin

(63)

Reassessment

of the

GBA genotype



Genotype:

L444P/L444P



_{Common mutation}



Known genotype-phenotype correlation





2 nd

child of young parents

healthy

unrelated

Cape Verdean origin

(64)

p.T398M: GD modulator?



No conclusions

can be drawn by the analysis of the

phenotype

alone

(65)

A LOOK FORWARD…



Functional studies

(66)

A LOOK FORWARD…



Functional studies



Western Blot



Real time PCR



Immunofluorescence



GCase activity assay

(67)

A LOOK FORWARD…



Functional studies



Western Blot



Real time PCR



Immunofluorescence



GCase activity assay

p.T398M

dysfunction

or

(68)

A LOOK FORWARD…



Functional studies



Western Blot



Real time PCR



Immunofluorescence



GCase activity assay

p.T398M

dysfunction

or

r

ed

uc

tion

of

LIMP-2

levels

Lower receptor density



key factor for

recombinant GCase uptake

(69)

A LOOK FORWARD…

Parkinson’s disease

(70)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese population

(71)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese population

(72)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese population

(73)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese population

(74)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese population



(75)

SUMMARY



1 st

_{time a}

_{whole GD population}

_{is screened for}

_SCARB2

_mutations;

(76)

SUMMARY



1 st

_{time a}

_{whole GD population}

_{is screened for}

_SCARB2

_mutations;



SCARB2 variability does

not account much

to the

Portuguese GD phenotypic spectrum

(77)

SUMMARY



1 st

_{time a}

_{whole GD population}

_{is screened for}

_SCARB2

_mutations;



SCARB2 variability does

not account much

to the

Portuguese GD phenotypic spectrum



Still,

one novel variant here identified

(p.T398M)

, deserves further attention and

extra studies

(78)

SUMMARY



1 st

_{time a}

_{whole GD population}

_{is screened for}

_SCARB2

_mutations;



SCARB2 variability does

not account much

to the

Portuguese GD phenotypic spectrum



Still,

one novel variant here identified

(p.T398M)

, deserves further attention and

extra studies



Plenty of questions remain unanswered…

(79)

Acknowledgments

Dr. Sandra Alves

Prof. Mª João Prata

UID-SA, DHG, INSA

Biochemical Genetics Unit, CGMJM, CHP

PTDC/SAU-GMG/102889/2008

SFRH/BD/124372/2016

(80)