Whole-exome sequencing of the affected individual identified a likely pathogenic variant in homozygous state, c.358C > T in exon 4 of MICU1 gene. Pathogenic variants in the MICU1 gene which is located on 10q22.1 chromosome region have been shown to be associated with proximal muscle weakness and learning disabilities due to deregulated mitochondrial Ca2+ uptake, resulting in mitochondrial Ca2+ overload, excessive production of ROS, and increased cell death.
Mitochondrial function is crucial for energy provision especially in excitable cells including skeletal and cardiac muscle cells, neurons, and glia cells and protects them from damage via fluctuation of Ca2+ (18).
Ca2+, a versatile and ubiquitous intracellular messenger (28), plays a central role in a remarkably wide range of cellular processes especially in nervous system and muscle. Calcium ions have been implicated to mediate neuronal gene expression, neuronal development and plasticity, synaptic transmission, neurotransmitter release, neuronal excitability, data processing, cognition, learning and memory in brain; excitation-contraction coupling, energy metabolism, adaptation to exercise and sarcolemmal repair in muscles (19, 29–31).
Ca2+ uptake regulation in the mitochondria is vital to control crucial functions like ATP production or cell death. This regulation in the outer membrane is mediated through the voltage-dependent anion selective channel (VDACs) and in the inner membrane occurs through channels such as Mitochondrial Calcium Uniporter (MCU) which mediates mitochondrial Ca2+ uptake through its regulators, MICU1 and MICU2 (2).
In the inner mitochondrial membrane (IMM), there is a mitochondrial calcium uniporter complex, containing four subunits: MCU, MICU1, MICU2, and EMRE. Ca2+ selectivity contributed by the MCU. MICU1–MICU2 are essential for Ca2+ uptake and EMRE is required for Ca2+ infiltration. Loss of MICU1–MICU2 results in Ca2+ leakage and interrupts respiration dependent to ATP synthesis which causes brain and muscle disorders in humans (14).
The MCU has been shown to mediate mitochondrial Ca2+ uptake which needs the mitochondrial calcium uptake 1 (MICU1) protein as a crucial element for its regulation. MCU is a channel that does not have much affinity for Ca2+, thus it uses different channels and proteins such as MICU1 which bind to Ca2+, to be activated. It has been shown that down-regulation of MICU1 suppresses mitochondrial Ca2+ significantly.
MICU1, which is localized to the mitochondrial inner membrane, is a ~ 54-kDa protein consists of 476 amino acids. It contains two parts including a transmembrane helix (aa ~ 33–52) and a cytosolic C-terminus (aa ~ 53–476) which contains two EF-hand Ca2+-binding domains (EF1 and EF4) which help activating MCU.
The structure of Ca2+-free MICU1-xtal is shown to contain four regions, the N-domain, including three a-helices and three antiparallel b-strands, the N-lobe and the C-lobe including six and seven a-helices respectively and the C-helix which plays an important role in calcium uptake in the mitochondria by mediating binding of MICU1 to MCU and formation of a ~ 480 kDa complex and oligomerization in the presence of Ca2+ (32). (Fig. 3b)
As MICU1 has an essential role in mitochondrial Ca2+ uptake, defects in this protein results in increasing Ca2+ level in mitochondria and reduction of cytosolic Ca2+ which facilitates apoptosis process, learning anomalies, muscle weakness due to difficulties in myofiber regeneration, and extra-pyramidal movement disorder. Studies showed that MICU1 plays a gatekeeper role in interaction with MCU (33).
Also it has been shown that different neuromuscular diseases are caused due to mutations in MICU1 which lead to the deregulation of cytoplasmic and mitochondrial Ca2+ levels and interrupting Ca2+-dependent processes such as muscle contraction and synaptic transmission (17).
MICU1 consists of 6 chains. The nonsense variant found in this study affects the exon 4 of this gene and therefore affects 5 of these chains. The structural analysis revealed that EF-hand motif, which has an important role in transferring Ca2+ through mitochondrial membrane (34), begins from amino acid 218, thus the R129* mutation disrupts this motif as well as the structure and function of MICU1 protein and leads to a complete loss of function of MICU1 protein. Figure 3b shows the topological structure of wild type and mutant form of MICU1 protein.
According to the ACMG-CAP guideline (35): 1) Nonsense variant in MICU1 gene, which leads to loss-of-function, is associated with myopathy and is a known mechanism of disease. (PVS1). 2) Pattern of inheritance is found to be autosomal recessive (PM3). 3) Co-segregation with the disease as heterozygous carriers are not affected while the homozygous individual shows myopathy phenotype. In addition it was not found in ethnically matched healthy controls, Iranome (PS4). 4) This variant was not found in Human Gene Mutation Database (HGMD), ClinVar, 1000 genome project, Exome Aggregation Consortium (ExAC) Exome Sequencing Project (ESP), and gnomAD database (PM2). 5) Pathogenic computational verdict based on 5 pathogenic predictions from BayesDel_addAF, DANN, EIGEN, FATHMM-MKL and MutationTaster vs no benign predictions (PP3). According to ACMG-CAP rules for combining criteria to classify sequence variants (PVS1 + PM3 + PS4 + PM2 + PP3), this variant is classified as pathogenic.
In this study, we reported a novel nonsense variant in the MICU1 gene in an Iranian patient with extrapyramidal signs. This is the second report of MPXPS from Iran.
Accurate identification of causative mutations in families play a key role to provide them with appropriate genetic counseling, Pre-implantation Genetic Diagnosis (PGD), Prenatal Diagnosis (PND), management and/or further therapy strategies.