Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency

Abstract

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease.

Figure 1

Mitochondrial respiratory chain expression profile linking complex I, complex IV and porin levels in patients with isolated Complex I deficiency caused by defects in nuclear-encoded Complex I subunits. Graphs show complex I and complex IV expression profile from (A) Normal adult control and patients with (B–D) homozygous c.64T>C, p.(Trp22Arg) NDUFB3 variant, P1–n = 4372 fibres analysed, P2–n = 559, P3–n = 13422 (E) compound heterozygous NDUFS4 variant, P4, n = 5964 (F) Homozygous exon 3 and 4 deletion in NDUFS4, P5, n = 4683 (G) homozygous NDUFS6 variant, P6, n = 880 (H) Homozygous NDUFS2 variant, P7, n = 5337 (I) Homozygous NDUFS3 variant, P8, n = 7154. Each dot represents a single muscle fibre, colour co-ordinated according to its mitochondrial mass: very low – blue, low – light blue, normal – beige, high – orange, very high - red. Black dashed lines represent the SD limits for the classification of the fibres. Lines adjacent to X and Y axis represent the levels of NDUFB8 and COX-1: beige: normal (<−3), light beige: intermediate (+) (−3 to −4.5), light blue: intermediate (−) (−4.5 to −6) and blue: deficient (>−6). Bold dashed lines indicate the mean expression level of normal fibres.

Figure 2

Mitochondrial respiratory chain expression profile linking complex I, complex IV and porin levels in patients with isolated Complex I deficiency caused by defects in nuclear-encoded Complex I assembly factors. Graphs show complex I and complex IV expression profile from patients with (A) Compound heterozygous NDUFAF6 variant, P9, n = 9504 fibres analysed (B) Homozygous NDUFAF6 variant, P10, n = 5355 (C) Compound heterozygous NDUFAF5 variant, P11, n = 7352 (D) Compound heterozygous FOXRED1 variant, P12, n = 1708 (E–F) Compound heterozygous ACAD9 variant, (E = P13, n = 2684, F = P14, n = 239) (G) Homozygous TMEM126B variant, P15, n = 131. Each dot represents a single muscle fibre, colour co-ordinated according to its mitochondrial mass: very low – blue, low - light blue, normal – beige, high – orange, very high - red. Black dashed lines represent the SD limits for the classification of the fibres. Lines adjacent to X and Y axis represent the levels of NDUFB8 and COX-1: beige: normal (<−3), light beige: intermediate (+) (−3 to -4.5), light blue: intermediate (−) (−4.5 to −6) and blue: deficient (>−6). Bold dashed lines indicate the mean expression level of normal fibres.

Figure 3

Mitochondrial respiratory chain expression profile linking complex I (NDUFB8), complex IV and porin levels in patients with isolated Complex I deficiency caused by defects in mtDNA-encoded Complex I subunits. Graphs show complex I and complex IV expression profile from patients with (A) m.3356T>C MTND1 variant, P16, n = 696 fibres analysed (B) m.10158T>C MTND3 variant, P17, n = 5842 (C) m.10197G>A MTND3 variant, P18, n = 7302 (D) m.13514A>G MTND5 variant, P19, n = 2427 (E) m.12425delA MTND5 variant, P20, n = 3795 (F) m.13094T>C MTND5 variant, P21, n = 1311 (G) m.13513G>A MTND5 variant, P22, n = 3341 (H) m.13513G>A MTND5 variant, P23, n = 2730 (I) m.13513G>A MTND5 variant, P24, n = 675 (J) m.13513G>A MTND5 variant, P25, n = 785. Each dot represents a single muscle fibre, colour co-ordinated according to its mitochondrial mass: very low – blue, low - light blue, normal – beige, high – orange, very high - red. Black dashed lines represent the SD limits for the classification of the fibres. Lines adjacent to X and Y axis represent the levels of NDUFB8 and COX-1: beige: normal (<−3), light beige: intermediate (+) (−3 to −4.5), light blue: intermediate (−) (−4.5 to −6) and blue: deficient (>−6). Bold dashed lines indicate the mean expression level of normal fibres.

Figure 4

Mitochondrial respiratory chain expression profile linking complex I (NDUFS3), complex IV and porin levels in patients with Isolated complex I deficiency caused by defects in mtDNA-encoded Complex I subunits. Graphs show complex I and complex IV expression profile from patients with (A) m.12425delA MTND5 variant, P20, n = 5536 fibres analysed (B) m.10197G>A MTND3 variant, P18, n = 6645 (C) m.13514A>G MTND5 variant, P19, n = 2730 (D) m.13094T>C MTND5 variant, P21, n = 3979 (E) m.13513G>A MTND5 variant, P23, n = 10009 (F) m.13513G>A MTND5 variant, P24, n = 575 (G) m.13513G>A MTND5 variant, P25, n = 1168. Each dot represents a single muscle fibre, colour co-ordinated according to its mitochondrial mass: very low – blue, low - light blue, normal – beige, high – orange, very high - red. Black dashed lines represent the SD limits for the classification of the fibres. Lines adjacent to X and Y axis represent the levels of NDUFB8 and COX-1: beige: normal (<−3), light beige: intermediate (+) (−3 to −4.5), light blue: intermediate (−) (−4.5 to −6) and blue: deficient (>−6). Bold dashed lines indicate the mean expression level of normal fibres.

Figure 5

Analysis of Complex I assembly by BN-PAGE. Complex I assembly profiles were analysed using one dimensional blue native polyacrylamide gel electrophoresis (BN-PAGE) (4-16% gradient). Analysis showed a decrease in fully-assembled CI in patients P17, P18, P23 and P24, whilst normal assembly is seen in patients P19, P21, P22 and P25. Complex II was used a loading control. Both OXPHOS complexes were detected by immunoblotting using subunit specific antibodies – NDUFB8 (Complex I) and SDHA (complex II). The original, full length blots are included in the Supplementary Information File (Supplementary Fig. S6).