Titel: A homozygous hypomorphic BNIP1 variant causes an increase in autophagosomes and reduced autophagic flux and results in a spondylo-epiphyseal dysplasia
ID: W9-005
Art: Invited talk
Redezeit: 15 min
Session: Workshop 9
Monogenic Syndromes

Referent: Tess Holling (Hamburg/DE)

Abstract - Text


BNIP1 (BCL2 interacting protein 1) is a soluble N-ethylmaleimide-sensitive factor-attachment protein receptor involved in membrane fusion at the ER. Exome sequencing identified the homozygous BNIP1 intronic variant c.84+3A>T in the apparently unrelated patients 1 and 2 with disproportionate short stature and normal intelligence. Radiographs showed abnormalities affecting both the axial and appendicular skeleton with signs of a spondylo-epiphyseal dysplasia in both probands. Transcript analysis revealed ~80% aberrantly spliced BNIP1 pre-mRNAs and a reduced BNIP1 mRNA level to ~80% that collectively caused a BNIP1 protein level reduction by ~50% in patient 1 compared to control fibroblasts. Cell viability and proliferation was normal in patient 1 cells. The BNIP1 ortholog in drosophila, Sec20, is an important regulator of endocytosis, autophagy, and lysosomal degradation. We qualitatively and quantitatively assessed lysosome positioning and identified a decrease in lysosomes in the perinuclear region and an increase in the cell periphery in patient 1 cells. However, lysosomal function appeared to be unaffected in patient-derived fibroblasts as centripetal and centrifugal lysosomal movement, lysosomal enzyme activity, and membrane-bound LAMP2 level were all normal, while slightly increased levels of cathepsin D and Z were observed. LC3B turnover assays by qualitative and quantitative immunofluorescence microscopy and immunoblotting demonstrated an increase in LC3B-positive puncta per cell and in LC3B-II levels, respectively, in patient 1 fibroblasts under steady-state condition, suggesting an accumulation of autophagosomes. By treating serum-starved fibroblasts with or without bafilomycin A1, we identified a significant increase in LC3B-positive puncta and in LC3B-II levels in serum-starved patient 1 cells compared to control cells, while autophagic flux was decreased. Together, our data suggest a block at the terminal stage of autolysosome formation and/or clearance in patient cells with a homozygous hypomorphic BNIP1 variant. BNIP1 together with RAB33B and VPS16, in which biallelic variants cause the Smith-McCort dysplasia type 2 and a multisystem disorder with short stature, respectively, highlight the importance of membrane trafficking, autophagosome-lysosome fusion, and autophagy in skeletal development.