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)



Atypical form:



Deficient enzyme:

Saposin C



Gene: PSAP

(10q21-q22)

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)



Large spectrum of severity

& symptoms

(4)

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

(5)

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

(6)

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

(7)

Gaucher Disease

(GD)



Autosomal recessive



Lysosomal Storage Disorder



Deficient enzyme:

β-glucocerebrosidase

(Gcase)



Gene:

GBA

(1q21)



One of the

most frequent LSD



Available ERT

(8)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)



1 st

described



1 st

drug approved



> nr therapeutic approaches

(9)

Gaucher Disease

(GD)





“The prototype lysosomal disease…”

(

Zhao and Grabowski, 2002

)



Still…

it does have some

significant differences

(10)



At the populational level…

GBA mutation carriers

Parkinson’s disease

(11)

Gaucher Disease

(GD)



GCase

(12)

Gaucher Disease

(GD)



GCase

(13)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2



Gene:

SCARB2

Action myoclonus-renal failure

(14)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2



Gene:

SCARB2

Variation in this gene may account

for

GD phenotype

(15)

Gaucher Disease

(GD)



GCase



At the molecular level…



LIMP2



Gene:

SCARB2

Variation in this gene may account

for

GD phenotype

(16)

Objective

Understand the role of

variations

in

SCARB2

in the

broad

phenotype spectrum

observed for GD patients

(17)

Sample Collection



Portuguese

GD cohort



91 samples



Biochemically



molecularly

characterized GD patients,

diagnosed at CGMJM

from ? to 2013

+



Controls



50 samples

(18)

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

CA

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

CA

GG

C

T

G

A C

T

Alu element

Intron 8 Intron 9 Intron 10 Intron 11

C

A

Gg

c

a

c

g

t

CA

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}

(19)

Results

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

CA

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

Registered polymorphisms

(20)

Results

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

p.M159V

p.V396I

Co

d

in

g

vari

an

ts

known polymorphic variations

(21)

Results

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

Alu element

C

A

Gg

c

a

c

g

t

CA

GG

C

T

G

A C

T

p.T398M

Co

d

in

g

vari

an

ts

Novel coding variant

not detected in 50 controls

(22)

In silico analyses

(23)

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

(24)



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

(25)

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

(26)

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

(27)

p.T398M: GD modulator?



Still…

therapeutic follow-up with assessment of

chitotriosidase

levels



enzyme levels disparate from the ones

expected for a GD patient under ERT

low response to treatment?



No conclusions

_{can be drawn by the analysis of the}

_phenotype

_alone

(28)

A LOOK FORWARD…



Functional studies



Western Blot



Real time PCR



Immunofluorescence



GCase activity assay

(29)

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

(30)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese cohort

(31)

A LOOK FORWARD…

Parkinson’s disease

Dementia with Lewy Bodies



SCARB2

screening



Portuguese cohort

(32)

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…

(33)

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

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