OC 07.3 - Whole genome expression analysis resolves the correlation between genetic knockout of single genes and the obtained phenotype in relation to FVIII secretion

Saturday, July 9, 2022

1:30 PM – 1:45 PM

Location: ExCel Center, ICC Capital Suite Room 2-4

Presentation Format: In-Person

Presenting Author(s)

Rawya Al Rifai

PhD candidat
Uniklinikum Bonn
bonn, Nordrhein-Westfalen, Germany

Co-Author(s)

MJ

Muhammad Ahmer Jamil

Dr
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
bonn, Nordrhein-Westfalen, Germany
MI

Mira Ibrahim

PhD candidate
Uniklinikum Bonn
Bonn, Nordrhein-Westfalen, Germany
NN

Nicole Nüsgen

Medizinisch-technische Assistentin
Uniklinikum Bonn
Bonn, Nordrhein-Westfalen, Germany
HS

Heike Singer

Dr. rer. nat
University Clinic Bonn, Institue of experimental Haematology and Transfusion medicine
Bonn, Nordrhein-Westfalen, Germany
Johannes Oldenburg

Professor, Chairman and Director
University Clinic Bonn AöR, Institute of Experimental Haematology and Transfusion Medicine, Bonn, Germany
Bonn, Nordrhein-Westfalen, Germany
OE

Osman EL-Maarri

Priv.-Doz. Dr.
Institute of Experimental Haematology and Transfusion Medicine and Center for Rare Diseases, University Hospital Bonn, Bonn, Germany
bonn, Nordrhein-Westfalen, Germany

Background: We have shown that GABARAPs proteins modulate FVIII secretion; however, GABARAP gene KO decreased FVIII secretion by 40%, whereas GABARAPL1 and GABARAPL2 gene KOs increased FVIII secretion by 70 and 100%, respectively.

Aims: To identify the underlying reason, we performed gene rescue experiments and 3’RNAseq to identify DEGs. Our overall aim is to identify the link between the individual gene knockouts and FVIII secretion.

Methods: We rescued each KO by transient transfection of the corresponding gene; FVIII activities in the medium were measured using chromogenic assay. 3’RNA sequencing data results of wild type HEK28 cells and the eight knockouts were analysed using bioinformatics tools to determine DEGs and the affected pathways.

Results: The reintroduction of CANX, CALR, LMAN1, MCFD2 and GABARAP returned the FVIII secretion to direction of wild type values; in contrast, GABARAPL1 and GABARAPL2 did not show a significant rescue. Pairwise comparison between knockouts and HEK28 at 5% FDR showed 199, 68 and 1087 DEGs in CALR, GABARAPL1 and GABARAPL2 knockout respectively; all other KOs showed low DEG counts. IPA analysis of the DEG showed significant enrichment for pathways related to energy metabolism, among which ATP production was predicted high and low for increased and decreased FVIII secretion knockout respectively.

Conclusion(s): The rescue process was successful for transporter molecules (CANX, LMAN1, MCFD2 and GABARAP) returning the FVIII secretion values to normal, while the others (CALR, GABARAPL1 and L2) did not reach normal values. The RNA-seq analyses showed that the latter are involved in the metabolism of the cells requiring longer time and more suitable conditions for rescue. 3'RNA sequencing results underscore the need for high ATP for the release of FVIII from the ER, these knockouts are considerably showing the change in ATP production correlated with change in FVIII secretion.