Article Figures & Data
Figures
- Figure 1. Autosomal recessive inheritance of ADPRHL2 mutations in two hereditary motor neuropathy families.
Pedigrees of families A and B with their respective mutation and the segregation of each by genotype, showing affected (black), unaffected (white). The patient and the partially affected parent (grey) in family B, both carry a known causal EXT1 variant causal for their exostosis phenotype. The father does not present with the hereditary motor neuropathy and neurodevelopmental phenotype.
- Figure 2. In vitro expression and activity of ARH3 (mutant) protein and ribbon representation of ARH3 in complex with ADP ribosylation (yellow) and Mg2+ ions (dark blue).
(A) SDS–PAGE analysis of expression and purification of recombinant ARH3 wild type and mutants in Escherichia coli. ARH3 (theoretical Mw 42.88 kD) was enrich from whole cell lysate by nickel affinity chromatography (for details, see the Materials and Methods section). Both C26F and V335G show similar expression, but lower abundance in the soluble fraction, compared with WT and D77N D778N mutant. (B) Alpha-helix 1, containing Cys26, is highlighted for orientation purposes. Right panels: Van der Waals radii of Cys26 sulphur and Val335 side chain carbon atoms are depicted as transparent spheres. Residue Cys26 is located in the core of a conserved helical bundle (right upper panel). Positioning of this residue within the structure suggest that the increase in Van der Waals volume associated with the C26F mutation incompatible with correct packing. Residue Val335 is located in partial structured surface loop packing against α-helix 1 (right lower panel) and is inserted in a hydrophobic pocket. The structural consequences of the V335G mutation are not immediately appreciable but may weaken the local packing, expose hydrophobic residues and thus affect the overall structural stability of the protein. Note that in the right panels foreground structural elements have been removed to allow representation of the buried residue pockets. Image was created with PyMOL v2.3 (Schrodinger LLC) using human ARH3 in complex with ADP-ribose (PDB 6D36). (C) The (ADP-ribosyl)hydrolase activity of ARH3 WT and mutants was assessed using H3 and poly(ADP-ribose)polymerase (PARP)1 MARylated and PARylated, respectively, in presence of 32P-NAD+ as substrates. After the reaction samples were analyzed by autoradiogram and SDS–PAGE. Both WT and V335G were active under the assay conditions. cntr (control; no ARH3).
- Figure 3. Protein expression in control, C26F and V335G patient fibroblast cells.
(A, B, C) Whole-cell lysates of patient-derived and control (cntr: healthy individual) fibroblast immunoblotted for ARH3 and GAPH (loading control) for the different mutations V335G (family A), C26F (family B) (B) soluble and insoluble whole-cell lysate fractions of patient and control fibroblasts immunoblotted for ARH3 and α-tubulin (loading control) (C) subcellular fractions of patient and control fibroblasts immunoblotted for α-tubulin (cytosolic control), VDAC1 (mitochondrial control) and Histone H3 (H3) (nuclear control) in whole cell lysate, mitochondrial fraction (mito), cytosolic fraction (cyto), and nuclear fraction (nucl). (D, E) Quantification of ARH3 expression of V335G mutant relative to control (cntr: healthy individual) per fraction normalized to the respective subcellular fraction control α-tubulin/H3/α-tubulin/VDAC1 showing (n = 4, mean and SD) (E) quantification of panADPr signal of V335G mutant relative to control (cntr: healthy individual) per fraction normalized to the respective subcellular fraction control α-tubulin/H3 (n = 4, mean and SD).
- Figure S1. ARH3 antibody specificity in U2OS knock-out model.
Western blot of whole cell lysates of WT and ARH3-KO U2OS cell lines showing the specificity of the ARH3 antibody (sc-374162; Santa Cruz) with α-tubulin as a loading control.
- Figure 4. Live cell imaging of U2OS overexpression model.
Localization of ARH3 protein as checked by live cell imaging for GFP-tagged ARH3 (green) with mitotracker (red) and Hoechst (blue) staining in separate and merged images in ARH3(WT)-GFP wild-type and mutants D77N D78N (catalytic null) and V335G in transfected U2OS cells. Size bar indicates 10 µm.
- Figure S2. Live cell imaging of U2OS overexpression model.
(A) Localization of ARH3 protein as checked by live cell imaging for GFP-tagged ARH3 (green) with mitotracker (red) and DAPI (blue) staining in separate and merged images in ARH3(WT)-GFP, ARH3(CAT)-GFP, and ARH3(V335G)-GFP in transfected BE(2)-M17 neuroblastoma cells. Size bar indicates 10 µm. (B) Recruitment of ARH3-GFP to the nucleus and DNA damage sites (white arrows) upon laser-induced DNA damage. The ARH3(V335G)-GFP protein does not relocate upon laser-induced DNA damage. (C) Expression of ARH3(WT)-GFP and ARH3(V335)-GFP protein in transfected U2OS cells. Size bar indicates 10 µm.
- Figure S3. H2O2 induces panADPr signal in control (cntr) and mutant V335G cells.
Western blot of whole cell lysates of wild-type (cntr: healthy individual) and V335G mutant fibroblast cell lines showing the up-regulation of panADPr signal in presence of 2 mM H2O2 stimulation (+) as compared with non-stimulated conditions (−), with HSP60 as a loading control.
- Figure 5. Recruitment of ARH3 to DNA damage site.
(A) Subcellular fractions of patient (V335G) and control (cntr: healthy individual) fibroblasts treated with H2O2 to induce DNA damage, immunoblotted for α-tubulin (cytosolic control), H3 (nuclear control) and VDAC1 (mitochondrial control) in whole cell lysate, nuclear fraction (nucl), cytosolic fraction (cyto) and mitochrondial fraction (mito). (B, C) Quantification of ARH3 expression of V335G mutant relative to control (cntr: healthy individual) per fraction normalized to the respective subcellular fraction control α-tubulin/H3/α-tubulin/VDAC1 (n = 4, mean and SD) (C) quantification of panADPr signal of V335G mutant relative to control (cntr: healthy individual) per fraction normalized to the respective subcellular fraction control α-tubulin/H3 (n = 4, mean and SD).
Tables
- Table 1.
Clinical description of patients carrying homozygous ADPRHL2 missense variants showing variable phenotypes.
Individual A:II:1 (patient 1) A:II:2 (patient 2) B:II:1 (patient 3) Gender M M F Parental consanguinity Reported negative Reported negative + Current age or age at death 34 yr 32 yra 16 yra Circumstances of death — Cardiac arrest/respiratory failure Respiratory failure ADPRHL2 Mutation Genomic position (hg19) Chr1: 36558899T>G Chr1: 36558899T>G Chr1: 36554582G>T cDNA NM_017825: c.1004T>G NM_017825: c.1004T>G NM_017825: c.77G>T Protein p.Val335Gly p.Val335Gly p.Cys26Phe Clinical features Age at onset 13 yr 15 yr 15 mo Symptoms at onset Walking instability and intermittent lateropulsion Fatigue and instability during walking Febrile seizures Psychomotor development Normal Normal Normal speech, moderate intellectual disability (6 yr) General development Normal Normal Growth retardation for which growth hormones were supplied Gait Weakness of foot dorsiflexors, drop foot, and mild spasticity Foot dorsiflexor weakness, drop foot, mild spasticity, and instability; later also affected by fracture Weakness of foot dorsiflexors, drop foot Muscle atrophy Moderate atrophy of distal third of upper and lower limbs Moderate atrophy of distal upper and lower limbs and mild proximal atrophy Moderate atrophy of intrinsic hand muscles (10 yr), mild atrophy of distal lower limbs Proximal strength upper limb 5 5 5 Distal strength upper limb 4 2–4 2/5 to 4-/5 Proximal strength lower limb 5 5 5 Distal strength lower limb 1–2 1–3 4-/5 to 5/5 Reflexes upper limb Normal Diminished Normal Reflexes lower limb Normal Distally diminished Normal Sensory involvement Hypoesthesia in tip toes, deep position, and vibration sense severely diminished in lower legs and hands Hypoesthesia and loss of vibration sense in legs - Seizure type - Myoclonic jerks Febrile seizures Cardiac features Normal Normal Left ventricle hypertrophy and mitral insufficiency Other clinical features Motor tics in childhood, micrognathia, nystagmus, postural tremor, absent trunk hair, pes cavus, mild to moderately restrictive pulmonary function, and scoliosis Nystagmus, postural tremor, mild dysarthria, pes cavus, hyperhidrosis, absent trunk hair, carpal tunnel surgery, and mixed restrictive/obstructive lung function Moderate scoliosis, growth retardation, pes cavus, and exostosis with confirmed causal EXT1 variant Neurological examination EMG Severe axonal motor polyneuropathy and mild sensory involvement Severe axonal motor polyneuropathy and mild sensory involvement Profound axonal motor polyneuropathy, no sensory involvement Brain MRI (age performed) Normal (13 yr) Normal (26 yr) Normal (13 yr) Mild white matter hyperintensity lesions (33 yr) EEG Normal Mild nonspecific changes with intermittent bifrontal theta waves Sporadic epileptiform activity frontocentral localization Other genetic features - - NM_000127.2 (EXT1): c.538_539delAG (p.Leu181Profs) a Individual is deceased
