Novel compound heterozygous pathogenic variants in nucleotide-binding protein like protein (NUBPL) cause leukoencephalopathy with multi-systemic involvement.

TLDR: Exome sequencing of a patient presenting with infantile-onset hepatopathy, renal tubular acidosis, developmental delay, short stature, leukoencephalopathy with minimal cerebellar involvement and multiple OXPHOS deficiencies revealed the presence of two novel pathogenic compound heterozygous variants in NUBPL.

About: This article is published in Molecular Genetics and Metabolism.The article was published on 2020-01-01 and is currently open access. It has received 9 citations till now. The article focuses on the topics: Compound heterozygosity.

Figures

Figure 4 – Stable lentiviral transfection of wild-type NUBPL restored the defective Complex I as demonstrated by analysing the A) Steady state levels of NUBPL and CI subunits (NDUFS1, NDUFS2, NDUFA9, NDUFB8) by SDS page analysis; B) 1D and C) 2D BN PAGE WB of samples treated with 1% DDM and D) 1D BN PAGE of Rieske protein in 1%DDM treated samples; E) spectrophotometer analysis of mitochondrial respiratory chain activities; F) 35S-methionine labelling for mitochondrial proteins translation. Data in figures are given as mean +/- SEM based on at least 3 biological replicates. CI= NADH:ubiquinone oxidoreductase; CII= succinate:ubiquinone oxidoreductase ; SDH= Succinate dehydrogenase; CIII= ubiquinol-cytochrome c reductase; CIV= cytochrome c oxidase.

Figure 3 - Functional characterisation of NUBPL defective immortalised fibroblasts vs three independent controls: the analyses of A) steady-state level of CI (NDUFV1, NDUFS1, NDUFS2, NDUFS3, NDUFA9, NDUFB11, NDUFB8), CII (SDHA), CIII (UQCRC2, UQCRFS1), CIV (MTCO1, COXIV), CV (ATPA5) and loading control (GAPDH) by WB analysis of SDS page from fibroblast lysates; B) 1D BN-PAGE and C) 2D BN- PAGE WB study with 1%DDM treated proteins; D) 1D BN-PAGE and E) 2D BN- PAGE study with 1% Digitonin treated proteins; F) 35S-methionine labelling for mitochondrial protein translation were performed in patient’s and controls immortalised fibroblasts; and, G) Spectrophotometer analysis of mitochondrial respiratory chain complex activities in fibroblast lysates. Data in figures are given as mean +/- SEM based on at least 3 biological replicates.

Table II: mitochondrial respiratory chain activities (μmole/min/g) in muscle homogenate

Figure 2. Brain MRI and MR Spectroscopy findings of the patient obtained at 2.5 years (A-E, K, L) and 7 years of age (F-J, M). Axial T2-weighted (A, F), fluid-attenuated inversion recovery (FLAIR) (B, G), diffusion-weighted images (DWI) (C, H), sagittal T2-weighted (D, I) and coronal T2-weighted (E, J) images are shown. The initial MRI shows diffuse cerebral white matter abnormalities, mainly in the frontal and right parietal lobes (A, B), involving both the periventricular and subcortical regions, with central rarefaction (asterisks, B). The abnormal cerebral white matter is surrounded by a rim of abnormal solid tissue, with linear areas of restricted diffusion (arrows, C). There is swelling and T2-hyperintensities at the level of the anterior portions of the corpus callosum and splenium, with preservation of the fibres connecting the pericentral cortex (arrow, D). There is a very small area of signal abnormality of the cerebellar cortex in the right hemisphere (empty arrow, E). The late MRI reveals improvement of the deep white matter changes with absence of restricted diffusion (G, H), and thinning of the affected white matter with mild enlargement of the lateral ventricles (asterisks, F). There is atrophy of the corpus callosum (arrowheads, I). The cerebellar abnormalities slightly worsen (empty arrow, J). MR spectroscopy at 2.5 years of age demonstrate very high lactate peaks in the affected white matter (arrow, K) and smaller lactate peaks in the spared, normal-appearing white matter (arrow, L). Follow-up MR spectroscopy performed at 7 years of age reveals no significant lactate peaks in the affected cerebral white matter (M).

Table I – Clinical, molecular genetics and biochemical features of NUBPL patients

Frequently asked questions

Q1. What are the contributions in "Novel compound heterozygous pathogenic variants in nucleotide-binding protein like protein (nubpl) cause leukoencephalopathy with multi- systemic involvement" ?

The authors investigated patient ’ s and control immortalized fibroblasts and demonstrated that both the peripheral and the membrane arms of complex I are undetectable in mutant NUBPL cells, resulting in virtually absent CI holocomplex and loss of enzyme activity. In conclusion, this is the first report describing a complex multisystemic disorder due to NUBPL defect. In addition, the authors confirmed the role of NUBPL in Complex I assembly associated with secondary effect on Complex III stability and they demonstrated a defect of mtDNA-related translation which suggests a potential additional role of NUBPL in mtDNA expression. 

Q2. What future works have the authors mentioned in the paper "Novel compound heterozygous pathogenic variants in nucleotide-binding protein like protein (nubpl) cause leukoencephalopathy with multi- systemic involvement" ?

Further studies with additional identified patients are needed to confirmed their hypothesize. This observation suggests that NUBPL is involved in the formation of both the peripheral and membrane arms of the enzyme. A current standard model suggests that partially assembled CIII2 is incorporated in CI+CIII2+CIV SC, before the insertion of the Rieske protein ( UQCRFS1 ), which is preferentially localized in SC rather than in free CIII2 [ 30 ]. In conclusion, their results confirm the role of NUBPL in the assembly of CI and suggest an involvement of the protein in the stability of the whole enzyme. 

Q3. What is the common cause of CI deficiency?

Isolated mitochondrial CI deficiency (MIM# 252010), due to pathogenic variants in CI subunits or assembly factors, causes severe decrease of energy production and is the most common cause of OXPHOS disorders (Nouws, Nijtmans, Smeitink, & Vogel, 2012). 

Q4. What is the CI function of the two pathogenic variants?

While the first pathogenic variant is localized in a Walker B motif essential for nucleotide bind, the second pathogenic variant is localized in proximity to the CxxC binding residues to the Fe-S clusters. 

Q5. How many patients have been reported with variable disease course?

10 patients have been reported with variable disease course: four patients presented episodes of regression, two patients a progressive disease course; three had a stable disease course with acquisition of some milestones. 

Q6. What is the role of the N module in the assembly of CI?

whilst the Q module is added to the membrane arm at an early stage, the insertion of the N module is one of the final steps of the complete assembly of CI (Guerrero-Castillo et al., 2017; SanchezCaballero et al., 2016). 

Q7. What did the authors do to confirm the pathogenicity of the variants?

To further confirm the pathogenicity of the pathogenic variants, the authors performed complementation experiments by overexpressing NUBPL wild-type cDNA in patient’s and control fibroblasts by stable lentivirus transduction. 

Q8. What is the effect of CI on the CI assembly pathway?

As expected, in the absence of CI there is a redistribution of the remaining complexes, which leads to an increase in CIII2 and CIII2+CIV species, while monomer CIV does not seem to be affected. 

Q9. What is the role of NUBPL in the CI?

NUBPL has been identified as a CI assembly factor, involved in the insertion of 4Fe-4S clusters in the CI N module (Sheftel et al., 2009). and in two 4Fe-S metallated subunits of the Q module, NDUFS2 and NDUFS3 (Sanchez-Caballero, Guerrero-Castillo, & Nijtmans, 2016; Sheftel et al., 2009). 

Q10. What was the reaction used to transfer the protein to the membrane?

Proteins were transferred to a PVDF membrane (Immobilon-P #IPVH00010) at 100 V for 1 hour at 4°C in Tris-Glycine transfer buffer, 20% methanol, 0.25% SDS. 

Q11. What is the role of the CI assembly pathway?

Previous structural analysis of CI by blue native gel electrophoresis in patients’ cells with pathogenic variants in N and Q module subunits has shown the accumulation of stable CI intermediates corresponding to the unconnected N module and to the incomplete membrane arm, which can contain also Q module subunits, such as NDUFA9 and NDUFS2 (Lazarou, McKenzie, Ohtake, Thorburn, & Ryan, 2007). 

Q12. What is the effect of the two pathogenic variants on the CI?

SDS-PAGE based Western blot (WB) immunodetection showed strong reduction of the steady-state level of CI subunits localized in both the peripheral and membrane arms, suggesting a deleterious effect of the NUBPL pathogenic variants on thewhole enzyme (Figure 3A-S2). 

Q13. What is the CI function of the respiratory chain complexes?

Spectrophotometric analysis of the respiratory chain complexes confirmed severe CI deficiency with 22% residual activity of patient’s vs controls’ fibroblasts (Figure 3G), whereas the activities of the other complexes, including those containing mtDNA encoded subunits, were normal. 

Q14. What was the result of the MRI?

Follow-up brain MRI showed improvement of the cerebral white matter abnormalities, with decrease in both white matter swelling and extent of the signal abnormalities, whereas the cerebellar abnormalities had slightly worsened (Figure 2F-J). 

Q15. What is the effect of digitonin on CI?

The analysis of mitochondrial complexes extracted with digitonin in 1D and 2D blue native gels allowed us to investigate the formation and the stability of supercomplexes in their patient cell line. 

Q16. What is the effect of the two variants on the CI?

1st and 2nd dimension (1D and 2D) BN-PAGE analysis of DDMtreated samples demonstrated a dramatic reduction of the fully assembled CI in patient’s fibroblasts, whereas both CIII and CII, as well as CIV, holocomplexes were normal (Figures 3B and 3C).