Diphthamide-deficiency syndrome: a novel human developmental disorder and ribosomopathy

Abstract

We describe a novel type of ribosomopathy that is defined by deficiency in diphthamidylation of translation elongation factor 2. The ribosomopathy was identified by correlating phenotypes and biochemical properties of previously described patients with diphthamide biosynthesis gene 1 (DPH1) deficiencies with a new patient that carried inactivating mutations in both alleles of the human diphthamide biosynthesis gene 2 (DPH2). The human DPH1 syndrome is an autosomal recessive disorder associated with developmental delay, abnormal head circumference (microcephaly or macrocephaly), short stature, and congenital heart disease. It is defined by variants with reduced functionality of the DPH1 gene observed so far predominantly in consanguineous homozygous patients carrying identical mutant alleles of DPH1. Here we report a child with a very similar phenotype carrying biallelic variants of the human DPH2. The gene products DPH1 and DPH2 are components of a heterodimeric enzyme complex that mediates the first step of the posttranslational diphthamide modification on the nonredundant eukaryotic translation elongation factor 2 (eEF2). Diphthamide deficiency was shown to reduce the accuracy of ribosomal protein biosynthesis. Both DPH2 variants described here severely impair diphthamide biosynthesis as demonstrated in human and yeast cells. This is the first report of a patient carrying compound heterozygous DPH2 loss-of-function variants with a DPH1 syndrome-like phenotype and implicates diphthamide deficiency as the root cause of this patient's clinical phenotype as well as of DPH1-syndrome. These findings define "diphthamide-deficiency syndrome" as a special ribosomopathy due to reduced functionality of components of the cellular machinery for eEF2-diphthamide synthesis.

Fig. 1. Clinical presentation of the patient.

a Image at 5 months and b at 8 months, note the prominent forehead, high hairline, sparse scalp hair, and single palmar crease on the right hand. c Notched primary lower incisor at 14 months. Images were partially obscured to maximize privacy for the patient while highlighting pertinent clinical findings.

Fig. 2. Structural comparison of Pyrococcus Dph2 and human DPH2.

a Structure of the Pyrococcus horikoshii. Dph2 is described as a homodimer. To facilitate the structural comparison and localization of the variants, only one monomer of that structure is shown. Cys residues (C59, C163, C287) that carry a [4Fe–4S] cluster essential for catalytic activity (PDB:3LZD [19]) are shown in orange. b Model of human DPH2. Structure alignments identify Cys residues (orange) (C88/89 and C341) homologous to cysteines (C59 and C287) that bind the [4Fe–4S] cluster in archaeal Dph2. An additional cysteine is located at position 110. Mutated residues found in the patient (c.922 C > T (p.(Gln308*)) and c.601 C > T (p.(Arg201Cys))) are highlighted in yellow and wheat, respectively. The c.601 C > T (p.(Arg201Cys)) variant is located close to the catalytical center. The region predicted to be absent in the c.922 C > T (p.(Gln308*)) variant is shown in gray. This truncation leads to the absence of a large portion of the catalytical center including C341 and may additionally destabilize or misfold the protein.

Fig. 3. Toxin-mediated ADP-ribosylation (ADPR) assay.

Cell extracts from MCF7 DPH2ko cells transfected with plasmids expressing DPH2 wt, c.922 C > T (p.(Gln308*) (Q308*)) or c.601 C > T (p.(Arg201Cys) (R201C)) variants were subjected to the ADPR activity of DT (+). Reactions without DT (−) and extracts of mock-transfected cells served as negative controls. Capability of cells to synthesize diphthamide is revealed by the generation of ADPR-eEF2 as a prominent band at ~100 kDA (indicated by red arrowheads). For this, DPH2ko cells transfected with wild-type DPH2 serve as positive control, left panel: short exposure; right panel: long exposure.

Fig. 4. Analysis of yeast Dph2 variants.

a Western blot to quantify total cellular eEF2 and unmodified eEF2 in several dph2 mutants. Top panel: Anti-eEF2(pan) was used to detect eEF2 regardless of its modification status. Middle panel: Anti-eEF2(no diphthamide) specifically detects unmodified eEF2. Lower panel: to ensure equal loading, anti-Cdc19 was used to detect pyruvate kinase Cdc19 used as loading control for protein extracts from yeast. b Resistance toward growth inhibition by the diphthamide-dependent diphtheria toxin (DT). Cells carrying the glucose-repressible and galactose-inducible DT expression vector pSU9 [5] were serially diluted and cultivated on medium containing glucose (DT expression: off) or galactose (DT expression: on) at 30 °C for 3 days. c Resistance to sordarin was determined by yeast cultivation in the presence of 10 and 12.5 µg/ml sordarin [29]. d Sensitivity to diphthamide-responsive translation inhibitor hygromycin B was assessed by cultivation on medium containing the indicated doses of hygromycin B.