Genomic integrity and mitochondrial metabolism defects in Warsaw syndrome cells: a comparison with Fanconi anemia

Abstract

Warsaw breakage syndrome (WABS), is caused by biallelic mutations of DDX11, a gene coding a DNA helicase. We have recently reported two affected sisters, compound heterozygous for a missense (p.Leu836Pro) and a frameshift (p.Lys303Glufs*22) variant. By investigating the pathogenic mechanism, we demonstrate the inability of the DDX11 p.Leu836Pro mutant to unwind forked DNA substrates, while retaining DNA binding activity. We observed the accumulation of patient‐derived cells at the G2/M phase and increased chromosomal fragmentation after mitomycin C treatment. The phenotype partially overlaps with features of the Fanconi anemia cells, which shows not only genomic instability but also defective mitochondria. This prompted us to examine mitochondrial functionality in WABS cells and revealed an altered aerobic metabolism. This opens the door to the further elucidation of the molecular and cellular basis of an impaired mitochondrial phenotype and sheds light on this fundamental process in cell physiology and the pathogenesis of these diseases.

Role of the DDX11 DNA Helicase in Warsaw Breakage Syndrome Etiology

“…WABS cells displays a marked reduction of oxygen consumption and ATP synthesis in the presence of pyruvate/malate, a partial impairment of electron transport between complexes I and III, similarly to what observed in FA cells, and a remarkable decrease of complex IV activity, which is not displayed by FA cells [74,75]. However, the ultrastructure of mitochondria in WABS patient cells appeared to be normal with parallel cristae across the entire body and a dense mitochondrial matrix, in contrast to what observed in FA cell lines [14,72,73]. Therefore, due to the clinical overlap of WABS with other genome instability hereditary syndromes (especially FA), clinicians should analyze metaphase chromosome spreads for detecting possible cohesion defects, which are only observed in WABS (or RBS) patient cell lines, to avoid any possible mistake in diagnosis [71].…”

“…All these symptoms are also common to many WABS probands. Of note, it has been recently reported that another clinical feature is shared by WABS and FA patients: altered mitochondrial metabolism [14,72,73]. WABS cells displays a marked reduction of oxygen consumption and ATP synthesis in the presence of pyruvate/malate, a partial impairment of electron transport between complexes I and III, similarly to what observed in FA cells, and a remarkable decrease of complex IV activity, which is not displayed by FA cells [74,75].…”

“…In addition, Western blot analysis revealed the presence of DDX11 also in the nucleolar fraction of HeLa cell extracts. However, in very recent studies it was found that a great proportion (if not the majority) of the DDX11 protein is present in the cytoplasmic and not in the nuclear fraction of human cell extracts [13,14]. A possible localization of DDX11 at a specific cytoplasmic organelle/compartment needs to be more carefully analyzed and might lead to the discovery of novel cellular functions of this protein.…”

“…Color code is as in Figure 1 for black and grey elements. DDX11 pathogenic mutations described in the same article are highlighted with the same color: light blue [8]; indigo [17]; dark green [76]; light green [77]; pink [78]; orange [14].…”

“…In particular, the following amino acid substitutions were found to compromise the ability of DDX11 to unwind forked duplex DNA substrates: ∆K897 [8], R263Q [76], L836P [14] and C705Y [17]. Of note, the DDX11 mutation R140Q, which was described in a recently discovered WABS affected individual (WABS03 patient) can be considered as a variant of unknown significance (VOUS), as it does not map in any conserved sequence motif of the DDX11 polypeptide chain and, moreover, the corresponding mutated protein displayed a level of helicase activity comparable with the wild type counterpart in assays carried out in vitro with a flayed duplex DNA substrate; in addition, its cellular localization and stability, measured in cells treated with proteasome inhibitors, were found not to be altered compared to wild type DDX11 [17].…”