Unlike most lysosomal proteins, β-glucocerebrosidase (GCase), the hydrolase defective in Gaucher disease (GD), is delivered to lysosomes through its interaction with the transmembrane protein LIMP2. A few years ago, mutations in its coding gene, SCARB2, were reported to modify the severity of GD phenotype.  
The existence of a great variety of GD phenotypes is well-known, with numerous patients who carry identical genotypes presenting remarkable phenotypic variability. Over the years, that variability has been attributed to other genetic, epigenetic and/or environmental factors. Still, there is still much to learn on this subject. Recently, an association between Parkinson's disease (PD) and the presence of mutations in the GBA gene has been demonstrated. Moreover, there are also studies suggesting that genetic variants in the SCARB2 gene may also be risk factors for PD.
We analysed the SCARB2 gene in the Portuguese cohort of 91 GD patients, having identified 3 different SCARB2 coding variants. Of those, 2 were known polymorphisms with high prevalence in the normal population (p.M159V and p.V396I) and the third was a novel coding variant, p.T398M, present in heterozigousity in a single patient.  
Our study demonstrated that, at least for the Portuguese population, genetic variability at SCARB2 does not account much to the GD phenotypic spectrum. Nevertheless, in vitro analyses of the novel p.T398M are envisaged, in order to further characterize the effect of this variant on the levels and sub-cellular location of GCase. The clinical presentation of the patient harbouring this coding variant will also be discussed.FCT PTDC/SAU-GMG/102889/2008; SFRH/BD/124372/2016N/

Alves, S.

Coutinho, Maria Francisca

Ferreira, C.

Gaspar, A.

Lacerda, L.

Laranjeira, F.

Pinto, E.

Prata, M.J.

Ribeiro, H.

Ribeiro, I.

Silva, E.

English

Unlike most lysosomal proteins, β-glucocerebrosidase (GCase), the hydrolase defective in Gaucher disease (GD), is delivered to lysosomes through its interaction with the transmembrane protein LIMP2. A few years ago, mutations in its coding gene, SCARB2, were reported to modify the severity of GD phenotype.  
The existence of a great variety of GD phenotypes is well-known, with numerous patients who carry identical genotypes presenting remarkable phenotypic variability. Over the years, that variability has been attributed to other genetic, epigenetic and/or environmental factors. Still, there is still much to learn on this subject. Recently, an association between Parkinson's disease (PD) and the presence of mutations in the GBA gene has been demonstrated. Moreover, there are also studies suggesting that genetic variants in the SCARB2 gene may also be risk factors for PD.
We analysed the SCARB2 gene in the Portuguese cohort of 91 GD patients, having identified 3 different SCARB2 coding variants. Of those, 2 were known polymorphisms with high prevalence in the normal population (p.M159V and p.V396I) and the third was a novel coding variant, p.T398M, present in heterozigousity in a single patient.  
Our study demonstrated that, at least for the Portuguese population, genetic variability at SCARB2 does not account much to the GD phenotypic spectrum. Nevertheless, in vitro analyses of the novel p.T398M are envisaged, in order to further characterize the effect of this variant on the levels and sub-cellular location of GCase. The clinical presentation of the patient harbouring this coding variant will also be discussed.N/

Coutinho, M.F.

Repositório Científico do Instituto Nacional de Saúde

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

SCARB2 MUTATIONS AS MODIFIERS INGAUCHER 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 TO E 2 , R I B E I R O I 2 , L A R A N J E I R AF 2 , R I B E I R O H 2 , S I LVA E 2 , F E R R E I R A C 2 , P R ATA M J 4 , 5 , A LV E S S 115th International Symposium14th-15th March, 2019Gaucher 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”, 2010Gaucher Disease (GD)  Autosomal recessive  Lysosomal Storage Disorder Deficient enzyme: β-glucocerebrosidase (Gcase) Gene: GBA (1q21) Large spectrum of severity & symptoms Original illustration by Marcos Bernardino for  Cristiana Petriz´s “Gigi e a Doença de Gaucher”, 2010Gaucher Disease (GD)  Autosomal recessive  Lysosomal Storage Disorder Deficient enzyme: β-glucocerebrosidase (Gcase) Gene: GBA (1q21)At the clinical level… 3 variantsbased on the presence of neuronopathic diseaseabscence& progressivityGaucher Disease (GD)  Autosomal recessive  Lysosomal Storage Disorder Deficient enzyme: β-glucocerebrosidase (Gcase) Gene: GBA (1q21)At the clinical level… 3 variantsNon-NeurologicalNeurologicalGD type 123Gaucher Disease (GD)  Autosomal recessive  Lysosomal Storage Disorder Deficient enzyme: β-glucocerebrosidase (Gcase) Gene: GBA (1q21)At the clinical level… 3 variants Genotyope-Phenotype correlations only possible for a few mutationsGaucher Disease (GD)  Autosomal recessive  Lysosomal Storage Disorder Deficient enzyme: β-glucocerebrosidase (Gcase) Gene: GBA (1q21) One of the most frequent LSD Available ERTOriginal illustration by Marcos Bernardino for  Cristiana Petriz´s “Gigi e a Doença de Gaucher”, 2010Gaucher Disease (GD)  “The prototype lysosomal disease…”(Zhao and Grabowski, 2002) 1st  described 1st drug approved > nr therapeutic approachesOriginal illustration by Marcos Bernardino for  Cristiana Petriz´s “Gigi e a Doença de Gaucher”, 2010Gaucher Disease (GD)  “The prototype lysosomal disease…”(Zhao and Grabowski, 2002) Still…it does have some significant differencesOriginal illustration by Marcos Bernardino for  Cristiana Petriz´s “Gigi e a Doença de Gaucher”, 2010At the populational level…GBA mutation carriers Parkinson’s diseaseGaucher Disease (GD) Gaucher Disease (GD)  GCaseAt the molecular level…Gaucher Disease (GD)  GCaseAt the molecular level…Gaucher Disease (GD)  GCaseAt the molecular level… LIMP2 Gene: SCARB2Action myoclonus-renal failure (AMRF)Gaucher Disease (GD)  GCaseAt the molecular level… LIMP2 Gene: SCARB2Variation in this gene may account for GD phenotypeGaucher Disease (GD)  GCaseAt the molecular level… LIMP2 Gene: SCARB2Variation in this gene may account for GD phenotypeObjectiveUnderstand the role of variations in SCARB2 in thebroad phenotype spectrum observed for GD patientsin the Portuguese populationSample Collection Portuguese GD cohort 91 samples Biochemically molecularly characterized GD patients, diagnosed at CGMJM from ? to 2013+ Controls 50 samples  screening of the novel variantsGenetic 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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 Exon 5 Exon 6 Exon 7 Exon 8 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 8  Intron 9 Intron 10 Intron 11 Exon 9 Exon 10 Exon 11 Exon 12 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 12  In silico nalyses Evaluation of the deleterious potential of the novel variant(s) DNA Peripheral blood Fibroblasts from patients’ skin biopsies Results 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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 Exon 5 Exon 6 Exon 7 Exon 8 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 8  Intron 9 Intron 10 Intron 11 Exon 9 Exon 10 Exon 11 Exon 12 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 12 Non-codingvariantsrs3733259 rs894248 rs2289512rs2289513rs35369082rs77814624rs35583533Registered polymorphismsAlso present in the controlsResults 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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 Exon 5 Exon 6 Exon 7 Exon 8 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 8  Intron 9 Intron 10 Intron 11 Exon 9 Exon 10 Exon 11 Exon 12 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 12 p.M159V p.V396ICodingvariantsknown polymorphic variationshigh prevalence in the normal populationResults 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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 Exon 5 Exon 6 Exon 7 Exon 8 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 8  Intron 9 Intron 10 Intron 11 Exon 9 Exon 10 Exon 11 Exon 12 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 CAGgcacgt (c2) 5’ss GC Alu-Sz element CAGGC  G T 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  Intron 12 p.T398MCodingvariantsNovel coding variantnot detected in 50 controlsN= 1In silico analysesp.T398MReassessment 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 hypotoniaglobal psychomotor developmental delay 2nd child of young         parents healthy unrelatedCape Verdean origin  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 hypotoniaglobal psychomotor developmental delay 2nd child of young         parents healthy unrelatedCape Verdean origin Neurological GDReassessment of the clinical caseReassessment 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 hypotoniaglobal psychomotor developmental delay 2nd child of young         parents healthy unrelatedCape Verdean origin Neurological GDReassessment of the GBA genotype Genotype: L444P/L444P Common mutation  Known genotype-phenotype correlation 2nd child of young         parents healthy unrelatedCape Verdean origin Neurological GDp.T398M: GD modulator? Still…therapeutic follow-up with assessment of chitotriosidase levels enzyme levels disparate from the ones expected for a GD patient under ERTlow response to treatment? No conclusions can be drawn by the analysis of the phenotype aloneGBA genotype A LOOK FORWARD… Functional studies Western Blot  Real time PCR  Immunofluorescence  GCase activity assayp.T398MA LOOK FORWARD… Functional studies  Western Blot  Real time PCR  Immunofluorescence  GCase activity assayp.T398Mdysfunction orreduction of LIMP-2 levelsLower receptor densitykey factor for recombinant GCase uptake(Desnick and Schuchman, 2012)A LOOK FORWARD…Parkinson’s diseaseDementia with Lewy Bodies SCARB2 screening Portuguese cohortis there an association?A LOOK FORWARD…Parkinson’s diseaseDementia with Lewy Bodies SCARB2 screening Portuguese cohortSUMMARY 1st 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 andextra studiesPlenty of questions remain unanswered…SCARB2 MUTATIONS IN GAUCHER DISEASE AcknowledgmentsDr. Sandra AlvesProf. Mª João PrataUID-SA, DHG, INSABiochemical Genetics Unit, CGMJM, CHPPTDC/SAU-GMG/102889/2008SFRH/BD/124372/2016António Reis Thank You!

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

http://repositorio.insa.pt/bitstream/10400.18/6991/1/2019.03.15.%20Coutinho%20MF%20-%20SPDM%202019.pdf

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

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Repositório Científico do Instituto Nacional de Saúde

Repositório Científico do Instituto Nacional de Saúde

Scientific Repository of the National Health Institute Doutor Ricardo Jorge

Scientific Repository of the National Health Institute Doutor Ricardo Jorge