Representative fusions of individual patient cell lines and of patient cell lines and a ρ0 (r0) cell line (lacking mtDNA) and a control cell line. Results are shown as complex I activity relative to the mitochondrial matrix marker enzyme CS, as percentage of the mean of fusions between control cell lines (observed range for complex I activity relative to CS activity: 164_169, n = 2 fusions). Error bars represent the observed range for all values for each cell line. N, transformed control fibroblasts; ρ0, transformed 143BTK_ρ0 osteosarcoma cell line; A, B, C, D, E, and G, complex I_deficient transformed fibroblasts from patients A_G; others, hybrids between the parental cell lines as indicated. For the parental cell lines, the mean and observed ranges for the G418-resistant and hygromycin B_resistant lines combined are shown. Number of estimations for each cell line were: N, n = 23; ρ0, A, and C, n = 8; B, n = 11; D, n = 14; E, n = 9; G, n = 7; G2, n = 1; A × B, B × ρ0, A × N, C × ρ0, D × ρ0, E × N, G × G2, G × ρ0, and G × N, n = 2; A × ρ0, n = 3; B × C, C × N, D × N, and E × ρ0, n = 4; D × E, n = 6; B × N, n = 8. (A) Affected brothers G and G2 are in the same (nuclear) complementation group. (B) Patients A and B represent 2 different (nuclear) complementation groups. (C) Patients B and C represent the same (nuclear) complementation group. (D) Patients D and E represent the same (mtDNA) complementation group.

Pedigrees and haplotyping of families B and C. The symbols denote the following: open squares, unaffected males; open circles, unaffected females; open diamonds, an additional number of unaffected siblings; slashed symbols, unexplained infant deaths; triangle, miscarriages; black symbols, affected children. The homozygous regions in the probands are indicated by rectangles. The polymorphic marker shown in italics is not part of the original 5-cM genome-wide scan marker set. (A) Family B pedigree and the chromosome 5 haplotype of the affected child. (B) Family C pedigree and the chromosome 5 haplotype shared by the affected siblings.

Analysis of NDUFS6 cDNA and genomic DNA in families B and C. (A) Two percent agarose gel of NDUFS6 cDNA fragments generated by standard PCR. Lanes show control (Co); patient B (B); patient C (C); patient H (H); dH₂O blank (Bl). PCR of NDUFS6 cDNA gave no product in patient B and a larger fragment than the control in patient C. (B) A representation depicting the frameshift and creation of a premature stop codon caused by the 26-bp insertion of intronic sequence in the proband from family C (bottom). Translation of the control sequence is also shown for comparison (top). The diagram shows the exon 2/exon 3 boundary, with the inserted sequence shown in bold and underlined and a stop codon represented by an asterisk. (C) 0.8% agarose gel of NDUFS6 fragments generated by long-range PCR using forward and reverse primers located 6.2 kb apart. Lanes 2_5, unrelated complex I_deficient patients. The PCR product in patient B is approximately 4 kb smaller than that of other samples. The approximately 2-kb fragment from lane 6 was sequenced and the deletion breakpoints elucidated. (D) Schematic representation of the 4.175-kb deletion, which encompasses exons 3 and 4 (shaded box) of the 14.5-kb NDUFS6 gene in the proband from family B. The exact homozygous deletion breakpoints are also shown with the immediate sequence before and after each breakpoint included.

Immunoblot analysis and quantitation of complexes I and II from fibroblasts of patients A_J and controls (N) following separation by BN-PAGE. (A) Fully assembled complex I detected by an antibody to the 39-kDa subunit is at approximately 900 kDa. Partially assembled complex I detected by the same antibody is at approximately 750 kDa, and fully assembled complex II detected by an antibody to the 70-kDa subunit is at approximately 130 kDa. (B) Histogram showing percentage fully (white bars) and partially (gray bars) assembled complex I relative to fully assembled complex II in primary fibroblasts. The error bar represents the observed range for 2 different control cell lines.

A chromosome map showing the location of markers that were homozygous in all 3 affected children (as horizontal bars) in the initial 5-cM genome-wide scan. Consecutive homozygous markers on chromosomes 2p (D2S2369, D2S337, and D2S2368), 2q (D2S2188 and D2S364), and 21p (D21S1904 and D21S1911) are highlighted by boxes. The approximate locations of all complex I subunit genes are shown in black text, and those subunit genes located adjacent to homozygous regions from the genome-wide scan are shown in bold italicized text. The prefix “NDU” is omitted from subunit gene names for clarity. The locations of the 7 most downregulated genes in both patients compared with control as revealed by microarray analysis are shown by gray underlined text. NDUFS6 is highlighted by an ellipse, as it was the only subunit gene located adjacent to a homozygous region that showed significant underexpression on microarray analysis.