Large-scale deletion and point mutations of the nuclear NDUFV1 and NDUFS1 genes in mitochondrial complex I deficiency

Abstract

Reduced nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest complex of the mitochondrial respiratory chain and complex I deficiency accounts for approximately 30% cases of respiratory-chain deficiency in humans. Only seven mitochondrial DNA genes, but >35 nuclear genes encode complex I subunits. In an attempt to elucidate the molecular bases of complex I deficiency, we studied the six most-conserved complex I nuclear genes (NDUFV1, NDUFS8, NDUFS7, NDUFS1, NDUFA8, and NDUFB6) in a series of 36 patients with isolated complex I deficiency by denaturing high-performance liquid chromatography and by direct sequencing of the corresponding cDNA from cultured skin fibroblasts. In 3/36 patients, we identified, for the first time, five point mutations (del222, D252G, M707V, R241W, and R557X) and one large-scale deletion in the NDUFS1 gene. In addition, we found six novel NDUFV1 mutations (Y204C, C206G, E214K, IVS 8+41, A432P, and del nt 989-990) in three other patients. The six unrelated patients presented with hypotonia, ataxia, psychomotor retardation, or Leigh syndrome. These results suggest that screening for complex I nuclear gene mutations is of particular interest in patients with complex I deficiency, even when normal respiratory-chain-enzyme activities in cultured fibroblasts are observed.

Figure 1

Abnormal D-HPLC patterns of NDUFV1 and NDUFS1 RT-PCR products. “F2,” “F3,” and “F5” refer to the different PCR fragments (table 2). C = control; 1–5 = patients 1–5.

Figure 2

Molecular analysis of the NDUFV1 gene. Sequence analysis was performed on genomic DNA for patients 1 and 2, and on cDNA derived from skin fibroblast for patient 3. Sequence alignment of the NDUFV1 proteins from various species is presented. Boxes indicate the FMN binding site. Arrows show the mutated amino acids (patient 1: E214K; patient 2: A432P; patient 3: Y204C and C206G).

Figure 3

Molecular analysis of the NDUFS1 gene. Sequence analysis was performed on cDNA for patient 4 and on genomic DNA for patients 5 and 6. Sequence alignment of the NDUFS1 proteins from various species is presented. Arrows show the mutated amino acids (patient 4: D252G and del 222; patient 5: R241W; patient 6: M707V).

Figure 4

Haplotype analysis of family 6. Haplotypes are given (top to bottom) for loci D2S113, D2S368, D2S156, D2S138, D2S389, D2S311, D2S369, D2S155, D3S164, D2S159, and D2S125. The box indicates the deleted region between 2q32.1 and q34.