P756: impaired mitochondrial function and marrow failure in patients carrying a mutation on srsf4 gene

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: An 8-year-old patient was admitted to the Haematology Unit because of leukopenia, lymphopenia, and neutropenia. Two relatives belonging to the maternal branch died due to bone marrow failure and leukaemia, respectively. Trephine biopsy showed reduced cellularity (30%), while the DEB test, telomere length, and marrow colony forming unit assay appeared normal. Leuko-neutropenia and marrow cellularity remained stable over six years of follow-up. WES showed a never reported missense variant of unknown significance on the SRSF4 gene (p.R235W) inherited by the mother who suffered from mild leukopenia. Protein expression resulted lower. Serine/arginine-rich splicing factor (SRSF) proteins are a protein family involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. SRSF4 altered expression has been associated with haematological diseases as its reduced expression has been found in patients with Acute Myeloid Leukemia and mice with a hypomorphic mutation in dyskerin (DKC1). Germline mutations on the SRSF4 gene have never been reported as disease-causing.The role of SRSF proteins on mitochondria function has already been described for SRSF6, which is involved in alternative splicing of mitochondrialfunction-related genes and whose silencing reduced the mitochondrial function and metabolism. Mitochondrial function is crucial for HSC maintenance, and mitochondrial defects have been described in some genetic BMF syndromes as FanconiAnemia. Aims: To validate the pathogenicity of the SRSF4 variant and its role on mitochondria function Methods: Proteins expression were evaluated by Western Blot analysis. Oxygen consumption rate was measured with an amperometric electrode in a closed chamber at 25°C. The FoF1 ATP-synthase activity was evaluated by a luminometric analysis. For both assay pyruvate plus malate were used as respiratory substrates. ATP/AMP value was calculated as the ratio between the ATP and AMP concentration evaluated spectrophotometrically. The malondialdehyde (MDA) concentration was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Results: Energy metabolism showed reduced oxygen consumption and ATP synthesis compared to the control samples, favouring glutamine and fatty acids as energy substrates over glucose. The aerobic metabolism dysfunction caused a decrease in cells’ energetic state and peroxidized lipids accumulation, a sign of increased oxidative stress. WB analyses showed that the altered mitochondrial metabolism did not depend on the expression of proteins involved in OxPhos machinery (i.e., MTCO2, ATP synthase-beta subunit). By contrast, it appeared associated with low mTOR phosphorylation and an imbalance in proteins regulating biogenesis (i.e., CLUH) and mitochondrial dynamics (i.e., DRP1, OPA1). Transfection with the wt-SRSF4 gene restored the mitochondrial function. Interestingly, even though both samples carry the same mutation, all the observed alterations are more evident in the child than in the mother, suggesting a role of epigenetics in energy metabolism regulation Summary/Conclusion: In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to cells’ mitochondrial function impairment and could be responsible for the clinical phenotype observed in our patients.Keywords: Mitochondria, Bone marrow failure, Bone marrow niche, Neutropenia