2.3.1. NDUFA1

The NDUFA1 gene is located on the X chromosome. The NDUFA1 subunit, also known as MWFE, contains 70 amino acids and has a mass of 8.0 kDa. It is a single-pass transmembrane protein that lies at the junction of the membrane and matrix arms of complex I. The N-terminus lies at the matrix surface. The protein consists of two separate domains: residues 1–31 form an alpha-helix that is situated in a groove between the first and seventh transmembrane helices of core subunit ND1. The C-terminal domain lies in the IMS and contains an alpha-helix (residues 42–56) and a short 3–10 helix (residues 65–70). This domain primarily contacts NDUFA8 (see Figure 7). A Ser residue is phosphorylated at position 55, but the significance of this modification remains unknown [42]. In a knockout strain of cultured human cells, the loss of NDUFA1 resulted in a reduced expression of complex I, and it migrated as a smaller-than-normal-sized complex in BN gel electrophoresis [11]. NDUFA1 mainly contacts core subunit ND1 and supernumerary subunit NDUFA8, but it also weakly contacts core subunit ND6 and supernumerary subunits NDUFS5, NDUFS8, and NDUFA13. ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

‐ ‐ ‐ **Figure 7.** Structural features of ND1-module subunits. The proteins are portrayed in ribbons. ND1 is in the background and colored light gray. NDUFA1 is colored blue with the sites of 4 mutations, Gly8Arg, Pro19Ser, Gly32Arg, and Arg37Ser, shown in space-filling and colored yellow. NDUFA3 is shown in orange. No point mutations have been discovered yet. NDUFA13 is colored pink. The site of one mutation, Arg57His, is shown in space-filling and colored black.

‐ Four clinical mutations have been identified in this subunit: p.Gly8Arg, p.Arg37Ser, p.Gly32Arg, and p.Pro19Ser, and so would be hemizygous in males. The p.Gly8Arg mutation was identified in two half-brothers diagnosed with Leigh syndrome who both died in infancy [43]. The brothers had different biological fathers but shared a mother who carried the p.Gly8Arg mutation. The older brother developed psychomotor retardation at nine months and generalized hypotonia and choreoathetosis. At 19 months of age, brainstem lesions were observed, and the patient died from cardiorespiratory failure. The younger brother presented with axial hypotonia, vertical rolling nystagmus, choreoathetosis, and bilateral lesions. He developed respiratory insufficiency at 13 months of age and died at 14 months of age from cardiorespiratory arrest. The family pedigree shows that three maternal uncles had died of an unknown disease. The severity of the disease is reflected in the structural importance of the Gly8 residue on NDUFA1. Gly8 lies in the conserved hydrophobic N-terminal region of NDUFA1. Gly8 is in loose contact with residues Thr23, Lys26, and Leu43 in core subunit ND1. The substitution of the glycine for an arginine would be disruptive to interactions in this region.

The p.Arg37Ser mutation was identified in a boy who was diagnosed with generalized hypotonia, myoclonic epilepsy, and cerebellar atrophy [43]. His clinical evolution stabilized, and he was still living at 10 years old. His mother was a heterozygous carrier for the p.Arg37Ser mutation. BN gel

electrophoresis demonstrated a low level of complex I formation, and muscle cells showed 15–30% of the normal level of complex I activity. The loss of a positive charge may disrupt the local structure or affect the phosphorylation of the subunit at Ser55. Arg37 contacts two residues from supernumerary subunit NDUFA8: it forms a hydrogen bond with Ser22 and is in contact with Gln92. It is also located near Asp90 and Gly93 from NDUFA8. Furthermore, according to mouse active (PDB id-6g2j) and deactive (PDB id = 6g72) structures [37], the residue may engage in different intermolecular interactions depending on the state of complex I, although this was not seen when comparing open and closed structures from the ovine enzyme [38].

The p.Gly32Arg mutation has been well-studied. Three separate clinical studies have been performed on this mutation. The first study [44] focused on two male patients who were maternal cousins from the same healthy non-consanguineous family. One patient experienced deterioration of motor and verbal skills at age four and an unsteady gait, retinitis pigmentosa, and cerebellar atrophy at age seven. His cousin developed an ataxia and proximal muscle weakness at age five and myoclonic seizures and bilateral sensorineural hearing loss at age 10. Both probands had mothers who were heterozygous carriers. Both men had lived into their thirties when the study was conducted. A six-year-old boy was also found to carry the p.Gly32Arg mutation [45]. Though the age of onset was not recorded, he experienced episodic neuroregression and encephalopathy but was seizure-free. His family pedigree was unknown. This finding is in concordance with the fact that the p.Gly32Arg mutation is not as deleterious as the p.Gly8Arg mutation. In 2011, a female patient with a heterozygous p.Gly32Arg mutation was identified [46]. She did not show signs of deficiency until 11 months of age, when she developed an X-linked respiratory chain deficiency in skeletal muscle tissue. She developed frequent upper airway infections and experienced somnolence and muscle hypotonia during these illnesses. However, at age five, she showed nearly normal psychomotor development. Though there was only a 25% expression of the normal allele in her skeletal muscle tissue, she showed a relatively mild clinical phenotype. It is hypothesized that X-inactivation due to selection advantage may have favored the expression of the normal allele. Neither her mother nor her father had the p.Gly32Arg mutation, suggesting that the mutation arose spontaneously. The fact that all four patients carrying the p.Gly32Arg mutation survived into adulthood may be attributed to the fact that Gly32 in NDUFA1 lies in the membrane near the cytoplasmic side and is not located at the interfaces of NDUFA1 and other subunits.

In 2014, a patient diagnosed with Leigh syndrome and mitochondrial respiratory chain disorder (MRCD) was discovered to have the p.Pro19Ser mutation [47]. The age of onset was five years old, but before 10 months of age, the boy had already experienced hypotonia, nystagmus, generalized epilepsy, and high blood lactate and pyruvate levels. Pro19 in NDUFA1 is a highly conserved in vertebrates and lies in the N-terminal membrane spanning helix (Pro7–Arg28). The residue contacts Leu9, Pro12, and Met91 of core subunit ND1 in the membrane region of the bilayer, and the substitution of the polar serine for proline could be deleterious.
