TY  - CONF
AU  - Parezanović, Marina
AU  - Anđelković, Marina
AU  - Stevanović, Nina
AU  - Klaassen, Kristel
AU  - Spasovski, Vesna
AU  - Ugrin, Milena
AU  - Komazec, Jovana
AU  - Stanković, Sara
AU  - Jocić, Nikola
AU  - Pavlović, Sonja
AU  - Stojiljković, Maja
AU  - Skakić, Anita
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2121
AB  - Introduction: Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by fasting hypoglycemia and the accumulation of glycogen in the liver, kidneys and intestinal mucosa. Recent studies revealed that chronic endoplasmic reticulum (ER) stress and increased apoptosis
play a role in the progression of disease manifestations. Although dietary control is commonly utilized
to manage hypoglycemia, there is still a lack of effective pharmacological therapy. Therefore, the establishment of proper model system is essential for testing novel treatment approaches.
Methods: To create GSD-Ib in vitro model system, CRISPR/Cas9-knockout (KO) method was used to introduce a deletion in SLC37A4 gene in the FlpInHEK293 cells. Characterization of CRISPR/Cas9-KO model
system was performed using Sanger sequencing, RT-qPCR and Western Blot. Additionally, the expression analysis of ER stress and apoptotic markers was performed.
Results: Sanger sequencing confirmed the presence of c.14_100del in SLC37A4 gene. The expression
level of SLC37A4 was decreased to 26.8% in the SLC37A4-/- cell line compared to the SLC37A4 wild-type
along with Western blot analysis, which confirmed reduced target protein level in SLC37A4-/- clones. Furthermore, ER stress (ATF4, DDIT3, HSPA5, XBP1s) and apoptotic (BCL2, BAX, CASP3, CASP7) markers expression levels were significantly altered in SLC37A4-/- clones compared to wild-type, which proved that
we created a suitable GSD-Ib in vitro model system.
Conclusion: Utilizing CRISPR/Cas9 technology, we established cellular GSD-Ib modelsystem that mirrors
increased ER stress and apoptosis. This model system could be used to facilitate a comprehensive understanding of disease mechanisms and enable testing of potential treatment effectiveness.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB
EP  - 65
SP  - 65
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2121
ER  - 

@conference{
author = "Parezanović, Marina and Anđelković, Marina and Stevanović, Nina and Klaassen, Kristel and Spasovski, Vesna and Ugrin, Milena and Komazec, Jovana and Stanković, Sara and Jocić, Nikola and Pavlović, Sonja and Stojiljković, Maja and Skakić, Anita",
year = "2023",
abstract = "Introduction: Glycogen storage disease type Ib (GSD-Ib) is an autosomal recessive disorder characterized by fasting hypoglycemia and the accumulation of glycogen in the liver, kidneys and intestinal mucosa. Recent studies revealed that chronic endoplasmic reticulum (ER) stress and increased apoptosis
play a role in the progression of disease manifestations. Although dietary control is commonly utilized
to manage hypoglycemia, there is still a lack of effective pharmacological therapy. Therefore, the establishment of proper model system is essential for testing novel treatment approaches.
Methods: To create GSD-Ib in vitro model system, CRISPR/Cas9-knockout (KO) method was used to introduce a deletion in SLC37A4 gene in the FlpInHEK293 cells. Characterization of CRISPR/Cas9-KO model
system was performed using Sanger sequencing, RT-qPCR and Western Blot. Additionally, the expression analysis of ER stress and apoptotic markers was performed.
Results: Sanger sequencing confirmed the presence of c.14_100del in SLC37A4 gene. The expression
level of SLC37A4 was decreased to 26.8% in the SLC37A4-/- cell line compared to the SLC37A4 wild-type
along with Western blot analysis, which confirmed reduced target protein level in SLC37A4-/- clones. Furthermore, ER stress (ATF4, DDIT3, HSPA5, XBP1s) and apoptotic (BCL2, BAX, CASP3, CASP7) markers expression levels were significantly altered in SLC37A4-/- clones compared to wild-type, which proved that
we created a suitable GSD-Ib in vitro model system.
Conclusion: Utilizing CRISPR/Cas9 technology, we established cellular GSD-Ib modelsystem that mirrors
increased ER stress and apoptosis. This model system could be used to facilitate a comprehensive understanding of disease mechanisms and enable testing of potential treatment effectiveness.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB",
pages = "65-65",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2121"
}

Parezanović, M., Anđelković, M., Stevanović, N., Klaassen, K., Spasovski, V., Ugrin, M., Komazec, J., Stanković, S., Jocić, N., Pavlović, S., Stojiljković, M.,& Skakić, A.. (2023). Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 65-65.
https://hdl.handle.net/21.15107/rcub_imagine_2121

Parezanović M, Anđelković M, Stevanović N, Klaassen K, Spasovski V, Ugrin M, Komazec J, Stanković S, Jocić N, Pavlović S, Stojiljković M, Skakić A. Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:65-65.
https://hdl.handle.net/21.15107/rcub_imagine_2121 .

Parezanović, Marina, Anđelković, Marina, Stevanović, Nina, Klaassen, Kristel, Spasovski, Vesna, Ugrin, Milena, Komazec, Jovana, Stanković, Sara, Jocić, Nikola, Pavlović, Sonja, Stojiljković, Maja, Skakić, Anita, "Application of CRISPR/cas9 technology for in vitro disease modelling in glycogen storage disease type IB" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):65-65,
https://hdl.handle.net/21.15107/rcub_imagine_2121 .

TY  - CONF
AU  - Jocić, Nikola
AU  - Parezanović, Marina
AU  - Anđelković, Marina
AU  - Stevanović, Nina
AU  - Ugrin, Milena
AU  - Spasovski, Vesna
AU  - Klaassen, Kristel
AU  - Stanković, Sara
AU  - Komazec, Jovana
AU  - Pavlović, Sonja
AU  - Stojiljković, Maja
AU  - Skakić, Anita
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2141
AB  - Introduction: Glycogen storage disease type Ib (GSD-Ib) is characterized by a deficiency of glucose-6-
phosphate translocase (G6PT) encoded by the SLC37A4 gene, affecting glucose homeostasis and disrupting autophagy. Recent findings suggest that G6PT may also play a role in autophagy and
glycogen-selective autophagy (glycophagy) activation independent of its transport function. To investigate this hypothesis, two groups of GSD-Ib patients carrying variants with different effects on G6PT
transport activity and stability (p.Asn27Lys and p.Leu348Valfs*53), were compared to the control group
of subjects.
Methods: The relative expression levels of SLC37A4 gene, autophagy (mTOR, ULK1, PRKAG1), and glycophagy markers(GABARAPL1, GAA, STBD1) were assessed in mononuclear cells of GSD Ib patients(four
carrying p.Asn27Lys and four carrying p.Leu348Valfs*53 variant) compared to control group using RTqPCR. Statistical analysis was performed using one-way ANOVA followed by a post-hoc t-test.
Results: The p.Asn27Lys group exhibited 1.5-2.5 times higher expression of SLC37A4 and autophagy
markers, while the p.Leu348Valfs*53 group showed downregulation by approximately 50% compared to
the control group. Glycophagy markers were increased twofold in both patient groups, except for GAA,
which had similar expression levels as the control group.
Conclusion: Individuals carrying the p.Asn27Lys variant display the presence of SLC37A4 transcript in
their cells, which correlates with autophagy activation. Conversely, in patients with the p.Leu348Valfs*53
variant SLC37A4 is downregulated, indicating compromised autophagy activation. These findings support the role of G6PT in autophagy activation, independent of itstransport activity. Furthermore, the elevated expression of glycophagy markers observed in both patient groups can be attributed to the
accumulated glycogen.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - Investigation of the role of the glucose-6-phosphate translocase in the activation of autophagy and glycogen-selective autophagy in glycogen storage disease type IB patients
EP  - 92
SP  - 92
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2141
ER  - 

@conference{
author = "Jocić, Nikola and Parezanović, Marina and Anđelković, Marina and Stevanović, Nina and Ugrin, Milena and Spasovski, Vesna and Klaassen, Kristel and Stanković, Sara and Komazec, Jovana and Pavlović, Sonja and Stojiljković, Maja and Skakić, Anita",
year = "2023",
abstract = "Introduction: Glycogen storage disease type Ib (GSD-Ib) is characterized by a deficiency of glucose-6-
phosphate translocase (G6PT) encoded by the SLC37A4 gene, affecting glucose homeostasis and disrupting autophagy. Recent findings suggest that G6PT may also play a role in autophagy and
glycogen-selective autophagy (glycophagy) activation independent of its transport function. To investigate this hypothesis, two groups of GSD-Ib patients carrying variants with different effects on G6PT
transport activity and stability (p.Asn27Lys and p.Leu348Valfs*53), were compared to the control group
of subjects.
Methods: The relative expression levels of SLC37A4 gene, autophagy (mTOR, ULK1, PRKAG1), and glycophagy markers(GABARAPL1, GAA, STBD1) were assessed in mononuclear cells of GSD Ib patients(four
carrying p.Asn27Lys and four carrying p.Leu348Valfs*53 variant) compared to control group using RTqPCR. Statistical analysis was performed using one-way ANOVA followed by a post-hoc t-test.
Results: The p.Asn27Lys group exhibited 1.5-2.5 times higher expression of SLC37A4 and autophagy
markers, while the p.Leu348Valfs*53 group showed downregulation by approximately 50% compared to
the control group. Glycophagy markers were increased twofold in both patient groups, except for GAA,
which had similar expression levels as the control group.
Conclusion: Individuals carrying the p.Asn27Lys variant display the presence of SLC37A4 transcript in
their cells, which correlates with autophagy activation. Conversely, in patients with the p.Leu348Valfs*53
variant SLC37A4 is downregulated, indicating compromised autophagy activation. These findings support the role of G6PT in autophagy activation, independent of itstransport activity. Furthermore, the elevated expression of glycophagy markers observed in both patient groups can be attributed to the
accumulated glycogen.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "Investigation of the role of the glucose-6-phosphate translocase in the activation of autophagy and glycogen-selective autophagy in glycogen storage disease type IB patients",
pages = "92-92",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2141"
}

Jocić, N., Parezanović, M., Anđelković, M., Stevanović, N., Ugrin, M., Spasovski, V., Klaassen, K., Stanković, S., Komazec, J., Pavlović, S., Stojiljković, M.,& Skakić, A.. (2023). Investigation of the role of the glucose-6-phosphate translocase in the activation of autophagy and glycogen-selective autophagy in glycogen storage disease type IB patients. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 92-92.
https://hdl.handle.net/21.15107/rcub_imagine_2141

Jocić N, Parezanović M, Anđelković M, Stevanović N, Ugrin M, Spasovski V, Klaassen K, Stanković S, Komazec J, Pavlović S, Stojiljković M, Skakić A. Investigation of the role of the glucose-6-phosphate translocase in the activation of autophagy and glycogen-selective autophagy in glycogen storage disease type IB patients. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:92-92.
https://hdl.handle.net/21.15107/rcub_imagine_2141 .

Jocić, Nikola, Parezanović, Marina, Anđelković, Marina, Stevanović, Nina, Ugrin, Milena, Spasovski, Vesna, Klaassen, Kristel, Stanković, Sara, Komazec, Jovana, Pavlović, Sonja, Stojiljković, Maja, Skakić, Anita, "Investigation of the role of the glucose-6-phosphate translocase in the activation of autophagy and glycogen-selective autophagy in glycogen storage disease type IB patients" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):92-92,
https://hdl.handle.net/21.15107/rcub_imagine_2141 .