2.8.3. NDUFB8

The NDUFB8 gene is located on chromosome 10. The NDUFB8 subunit is initially synthesized with 186 residues and a mass of about 22 kDa, but the first 28 residues serve as a transit peptide and are cleaved off during processing. NDUFB8 is a single-pass protein with the C-terminus in the IMS and the N-terminus in the matrix (see Figure 17). It is located near the end of complex I, and its single transmembrane span lies near ND5. The helix residues (126–151) are not conserved, but the small domains on each side of the helix are much more conserved. On the matrix side NDUFB8 mainly contacts core subunit ND5 and NDUFB4, but it also contacts core subunit ND4 and NDUFB9. On the IMS side, NDUFB8 contacts NDUFB7 and NDUFB10. In a knockout strain of cultured human cells, the loss of NDUFB8 resulted in no assembly of complex I [11].

‐ ‐ **Figure 17.** Structural features of NDUFB8 from the ND5-module. The sites of three mutations in NDUFB8 are shown in space-filling. NDUFB8 is colored pink, while the sites of Pro76Gln and Tyr62His are shown in blue on the matrix side, and Cys144Trp is shown in yellow. The Cys279 of ND5 is shown in orange, and it appears to form a disulfide with Cys144 of NDUFB8.

‐ In 2018, two unrelated compound heterozygous patients with biallelic NDUFB8 mutations were identified to have Leigh-like encephalomyopathy [86]. At three months of age, Patient 1 showed symptoms of failure to thrive, muscle hypotonia, and elevated lactate levels. Patient 2 began to show these symptoms at six months of age. Similar to patients with Leigh syndrome, both patients showed symmetrical basal ganglia and capsula interna lesions. Hypertrophy of the left cardiac ventricle was observed in Patient 1, and he died at 15 months of age. Patient 2 was still alive at six years of age.

′ Patient 1 harbored a p.Pro76Gln mutation and a p.Cys144Trp mutation. Pro76 is located within a highly conserved region. The residue lies on the matrix side above the lateral helix of core subunit ND5, and it is near core subunit ND4. Cys144 is found within the membrane domain. Though it is weakly conserved, it appears to form a disulfide with Cys279 of chain ND5. Surprisingly, it was discovered that the p.Cys144Trp mutation generated a new exonic splicing silencer (ESS). The Human Splicing Finder algorithm was used to identify the ESS, and Sanger sequencing revealed that exon 4 (Met105–Val156) was skipped in the NDUFB8 transcript, thus confirming the splice defect. Nevertheless, 14% of total cDNA from Patient 1 contained the p.Cys144Trp missense mutation, indicating that while skipping exon 4 is favored, normal splicing also occurs at a lower frequency. Upon the analysis of respiratory chain activity in Patient 1′ s muscle tissue and BN gel electrophoresis in a muscle biopsy sample, isolated complex I deficiency was confirmed.

‐ ‐ Patient 2 carried a p.Tyr62His mutation and a loss-of-function deletion Glu63Aspfs\*35 (c. 189delA). Tyr62 is completely surrounded by mostly conserved residues of NDUFB8 and is located in the same conserved region as Pro76; the two residues are only 4–5 Å distant from each other. Furthermore, the side chain hydroxyl group of Tyr62 forms a hydrogen bond with the side chain and backbone oxygen atoms of Asp74 in NDUFB8. Thus, although His is a fairly conserved substitution for Tyr, His substitution would likely be disruptive at this residue. Complex I deficiency in Patient 2 was observed through the BN gel electrophoresis of muscle homogenate. The complementation of both mutant cell lines was tested using the lentiviral expression of wild-type NDUFB8. Cells from Patient 1

showed normal levels of complex I activity by in-gel assays after BN gel electrophoresis, and normal levels of the NDUFB8 subunit by immunoblotting. Cells from Patient 2 did not grow well enough for such analysis, but both cell lines showed complementation in assays using microrespirometry and flow cytometry.
