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Case Reports
. 2024 Feb 8;25(4):2083.
doi: 10.3390/ijms25042083.

Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene

Maurizio Miano  1 , Nadia Bertola  2 , Alice Grossi  3 , Gianluca Dell'Orso  1 , Stefano Regis  4 , Marta Rusmini  3 , Paolo Uva  5 , Diego Vozzi  6 , Francesca Fioredda  1 , Elena Palmisani  1 , Michela Lupia  1 , Marina Lanciotti  1 , Federica Grilli  1 , Fabio Corsolini  7 , Luca Arcuri  1 , Maria Carla Giarratana  1 , Isabella Ceccherini  3 , Carlo Dufour  1 , Enrico Cappelli  1 , Silvia Ravera  8
Affiliations
Case Reports

Impaired Mitochondrial Function and Marrow Failure in Patients Carrying a Variant of the SRSF4 Gene

Maurizio Miano et al. Int J Mol Sci. .

Abstract

Serine/arginine-rich splicing factors (SRSFs) are a family of proteins involved in RNA metabolism, including pre-mRNA constitutive and alternative splicing. The role of SRSF proteins in regulating mitochondrial activity has already been shown for SRSF6, but SRSF4 altered expression has never been reported as a cause of bone marrow failure. An 8-year-old patient admitted to the hematology unit because of leukopenia, lymphopenia, and neutropenia showed a missense variant of unknown significance of the SRSF4 gene (p.R235W) found via whole genome sequencing analysis and inherited from the mother who suffered from mild leuko-neutropenia. Both patients showed lower SRSF4 protein expression and altered mitochondrial function and energetic metabolism in primary lymphocytes and Epstein-Barr-virus (EBV)-immortalized lymphoblasts compared to healthy donor (HD) cells, which appeared associated with low mTOR phosphorylation and an imbalance in the proteins regulating mitochondrial biogenesis (i.e., CLUH) and dynamics (i.e., DRP1 and OPA1). Transfection with the wtSRSF4 gene restored mitochondrial function. In conclusion, this study shows that the described variant of the SRSF4 gene is pathogenetic and causes reduced SRSF4 protein expression, which leads to mitochondrial dysfunction. Since mitochondrial function is crucial for hematopoietic stem cell maintenance and some genetic bone marrow failure syndromes display mitochondrial defects, the SRSF4 mutation could have substantially contributed to the clinical phenotype of our patient.

Keywords: CLUH; DRP1; OPA1; SRSF4; mTOR; marrow failure; mitochondria.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Cellular energy status in SRSF4 cell lines. All data were evaluated in healthy donor (HD) cell lines, cell lines mutated for SRSF4 (Pt1 and Pt2), mutated cell lines corrected with empty vector (Pt1scr and Pt2scr), and mutated cell lines corrected with wild-type SRSF4 (Pt1corr and Pt2corr). (A) Intracellular ATP content; (B) intracellular AMP content; (C) ATP/AMP ratio as a cell energy status marker. Data are reported as mean ± standard deviation (SD) and are representative of three independent experiments. *** and **** indicate a p < 0.001 or 0.0001, respectively, between Pt1 or Pt2 and HD. #### indicates a p < 0.0001 between the mutated sample and the corrected cells within the same patient. §§ and §§§§ indicate a p < 0.01 or 0.0001, respectively, between Pt1 and Pt2.
Figure 2
Figure 2
Expression of SRSF4 in patients and healthy donors. All data were evaluated in healthy donor (HD) cell lines, cell lines mutated for SRSF4 (Pt1 and Pt2), mutated cell lines corrected with empty vector (Pt1scr and Pt2scr), and mutated cell lines corrected with wild-type SRSF4 (Pt1corr and Pt2corr). (A) SRSF4 Western blot (WB) signal; (B) densitometric analysis of the SRSF4 signal normalized to the actin signal. Data are reported as mean ± SD and are representative of four independent experiments. **** indicates a p < 0.0001 between Pt1 or Pt2 and HD. #### indicates a p < 0.0001 between the mutated sample and the corrected cells within the same patient. §§§§ indicates a p < 0.0001 between Pt1 and Pt2.
Figure 3
Figure 3
OxPhos function in SRSF4 cell lines. All data were evaluated in healthy donor (HD) cell lines, cell lines mutated for SRSF4 (Pt1 and Pt2), mutated cell lines corrected with empty vector (Pt1scr and Pt2scr), and mutated cell lines corrected with wild-type SRSF4 (Pt1corr and Pt2corr). (A) Pyruvate/malate (P/M)-induced ATP synthesis; (B) P/M-induced oxygen consumption rate (OCR); (C) P/M-induced P/O ratio as an OxPhos efficiency marker; (D) succinate (Succ)-induced ATP synthesis; (E) Succ-induced OCR; (F) Succ-induced P/O ratio as an OxPhos efficiency marker. Data are reported as mean ± SD and are representative of three independent experiments. **** indicates a p < 0.0001 between Pt1 or Pt2 and HD. #### indicates a p < 0.0001 between the mutated sample and the corrected cell within the same patient. §§ and §§§§ indicate a p < 0.01 or 0.0001, respectively, between Pt1 and Pt2.
Figure 4
Figure 4
Lipid peroxidation accumulation in SRSF4 cell lines. All data were evaluated in healthy donor (HD) cell lines, cell lines mutated for SRSF4 (Pt1 and Pt2), mutated cell lines corrected with empty vector (Pt1scr and Pt2scr), and mutated cell lines corrected with wild-type SRSF4 (Pt1corr and Pt2corr). The graph represents the intercellular malondialdehyde (MDA) content as a lipid peroxidation accumulation marker. Data are reported as mean ± SD and are representative of four independent experiments. **** indicates a p < 0.0001 between Pt1 or Pt2 and HD. #### indicates a p < 0.0001 between the mutated sample and the corrected cell within the same patient. §§§§ indicates a p < 0.0001 between Pt1 and Pt2.
Figure 5
Figure 5
Respiratory complex activity in SRSF4 cell lines. All data were evaluated in healthy donor (HD) cell lines, cell lines mutated for SRSF4 (Pt1 and Pt2), mutated cell lines corrected with empty vector (Pt1scr and Pt2scr), and mutated cell lines corrected with wild-type SRSF4 (Pt1corr and Pt2corr). (A) NADH-ubiquinone oxidoreductase (Complex I) activity; (B) succinate-coenzyme Q reductase (Complex II) activity; (C) coenzyme Q-cytochrome c reductase (Complex III) activity; (D) cytochrome oxidase (Complex IV) activity. Data are reported as mean ± SD and are representative of four independent experiments. ** and **** indicate a p < 0.01 or 0.0001, respectively, between Pt1 or Pt2 and HD. ### and #### indicate a p < 0.001 or 0.0001, respectively, between the mutated sample and the corrected cell within the same patient. §§§§ indicates a p < 0.0001 between Pt1 and Pt2.
Figure 6
Figure 6
Expression of mTOR and proteins involved in the mitochondrial network dynamic in SRSF4 cell lines. All data were evaluated in healthy donor (HD) cell lines, cell lines mutated for SRSF4 (Pt1 and Pt2), mutated cell lines corrected with empty vector (Pt1scr and Pt2scr), and mutated cell lines corrected with wild-type SRSF4 (Pt1corr and Pt2corr). (A) WB signals and relative densiometric analysis of phosphorylated and total mTOR ratio (B), CLUH (C), OPA1 (D), and DRP1 (E). Each signal was normalized to the actin signal. Data are reported as mean ± SD and are representative of four independent experiments. *** and **** indicate a p < 0.001 or 0.0001, respectively, between Pt1 or Pt2 and HD. # indicate a p < 0.05 and #### indicate a p < 0.0001, respectively, between the mutated sample and the corrected cell within the same patient. §, §§ and §§§§ indicate a p < 0.05, 0.01 or 0.0001, respectively, between Pt1 and Pt2.
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