CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes
Nunziata Maio, … , Tracey A. Rouault, Carsten G. Bönnemann
Nunziata Maio, … , Tracey A. Rouault, Carsten G. Bönnemann
Published June 17, 2024
Citation Information: J Clin Invest. 2024;134(12):e179559. https://doi.org/10.1172/JCI179559.
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Research Article Metabolism Muscle biology

CIAO1 loss of function causes a neuromuscular disorder with compromise of nucleocytoplasmic Fe-S enzymes

Abstract

Cytoplasmic and nuclear iron-sulfur (Fe-S) enzymes that are essential for genome maintenance and replication depend on the cytoplasmic Fe-S assembly (CIA) machinery for cluster acquisition. The core of the CIA machinery consists of a complex of CIAO1, MMS19 and FAM96B. The physiological consequences of loss of function in the components of the CIA pathway have thus far remained uncharacterized. Our study revealed that patients with biallelic loss of function in CIAO1 developed proximal and axial muscle weakness, fluctuating creatine kinase elevation, and respiratory insufficiency. In addition, they presented with CNS symptoms including learning difficulties and neurobehavioral comorbidities, along with iron deposition in deep brain nuclei, mild normocytic to macrocytic anemia, and gastrointestinal symptoms. Mutational analysis revealed reduced stability of the variants compared with WT CIAO1. Functional assays demonstrated failure of the variants identified in patients to recruit Fe-S recipient proteins, resulting in compromised activities of DNA helicases, polymerases, and repair enzymes that rely on the CIA complex to acquire their Fe-S cofactors. Lentivirus-mediated restoration of CIAO1 expression reversed all patient-derived cellular abnormalities. Our study identifies CIAO1 as a human disease gene and provides insights into the broader implications of the cytosolic Fe-S assembly pathway in human health and disease.

Authors

Nunziata Maio, Rotem Orbach, Irina T. Zaharieva, Ana Töpf, Sandra Donkervoort, Pinki Munot, Juliane Mueller, Tracey Willis, Sumit Verma, Stojan Peric, Deepa Krishnakumar, Sniya Sudhakar, A. Reghan Foley, Sarah Silverstein, Ganka Douglas, Lynn Pais, Stephanie DiTroia, Christopher Grunseich, Ying Hu, Caroline Sewry, Anna Sarkozy, Volker Straub, Francesco Muntoni, Tracey A. Rouault, Carsten G. Bönnemann

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Figure 6

A second cell line derived from P2 demonstrates abnormal characteristics similar to those seen in P1 cells, and these defects in P2-derived cells are entirely reversed when the WT CIAO1 gene is reintroduced.

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A second cell line derived from P2 demonstrates abnormal characteristics...
(A)SDS IBs were used to detect CIA components, FAM96A, and Fe-S recipient proteins (ERCC2, ELP3, POLD1, DPYD, and RTEL1) in fibroblasts from P1, his parents, a control cell line with 2 wild-type CIAO1 copies (CTRL), and P2. α-Tubulin was used as a loading control (n = 3 biological replicates). (B) SDS IBs also detected CIA components, FAM96A, and Fe-S recipient proteins (POLD1, DPYD) in the same cell lines as in (A). Lysates from P1 and P2 cell lines transduced with V5-tagged wild-type CIAO1 showed full restoration of CIA components and Fe-S recipient levels (n = 3 biological replicates). (C) SDS IBs to detect subunits of the mitochondrial respiratory chain complexes I (NDUFS1, NDUFS8), II (SDHA, SDHB), III (UQCRC1), and IV (MTCO1) in lysates from the cell lines presented in B. Levels of the mitochondrial marker TOM20 are shown as a reference for the loading control (n = 3 biological replicates). (D) SDS IBs to detect the mitochondrial respiratory chain subunits of complex V (CV) (ATP5A) and complex III (CIII) (UQCRFS1) in lysates from the cell lines presented in B and C (n = 3 biological replicates). (E) Representative 55Fe incorporation into POLD1-FLAG/MYC expressed in cell lines as presented the same cell lines presented in panels B and C (n = 4 biological replicates). (F) Quantification of radioactive iron incorporated into POLD1-F/M as assessed by scintillation counter. [55Fe]-POLD1-F/M levels in control cells (father of P1) were quantified and set to 100%. Values are expressed as a percentage of the control and shown as the mean ± SEM. ****P < 0.0001, by 1-way ANOVA Šidák’s multiple-comparison test for P1 versus the father, P1 versus the mother; P1 versus P1_CIAO1-V5 and P2 versus P2_CIAO1-V5. P1 versus P2 was not statistically significant (NS, P = 0.9991). n = 4 biological replicates.