A homozygous mutation in a novel zinc-finger protein, ERIS, is responsible for Wolfram syndrome 2

Abstract

A single missense mutation was identified in a novel, highly conserved zinc-finger gene, ZCD2, in three consanguineous families of Jordanian descent with Wolfram syndrome (WFS). It had been shown that these families did not have mutations in the WFS1 gene (WFS1) but were mapped to the WFS2 locus at 4q22-25. A G-->C transversion at nucleotide 109 predicts an amino acid change from glutamic acid to glutamine (E37Q). Although the amino acid is conserved and the mutation is nonsynonymous, the pathogenesis for the disorder is because the mutation also causes aberrant splicing. The mutation was found to disrupt messenger RNA splicing by eliminating exon 2, and it results in the introduction of a premature stop codon. Mutations in WFS1 have also been found to cause low-frequency nonsyndromic hearing loss, progressive hearing loss, and isolated optic atrophy associated with hearing loss. Screening of 377 probands with hearing loss did not identify mutations in the WFS2 gene. The WFS1-encoded protein, Wolframin, is known to localize to the endoplasmic reticulum and plays a role in calcium homeostasis. The ZCD2-encoded protein, ERIS (endoplasmic reticulum intermembrane small protein), is also shown to localize to the endoplasmic reticulum but does not interact directly with Wolframin. Lymphoblastoid cells from affected individuals show a significantly greater rise in intracellular calcium when stimulated with thapsigargin, compared with controls, although no difference was observed in resting concentrations of intracellular calcium.

Figure 1.

Identification of a single base-pair change causative of WFS2. A, Sequencing results from a control individual (left panel) with a G at nucleotide 109 in exon 2 of the ZCD2 gene, which codes for glutamic acid, and sequence from an affected individual (right panel) with a homozygous C at the same position, which changes the amino acid to glutamine. B, Truncated pedigrees of WFS2-affected families. Genotyping of the mutation is shown below the pedigrees. All pedigrees represent consanguineous matings. Only children with DNA samples are shown. Children are numbered using designations from the original published pedigree. The mutation disrupts an XmnI restriction-enzyme site (GAANNNNTTC), and homozygous affected individuals show a 249-bp fragment, whereas the wild-type allele is cut into a 210-bp fragment and a 39-bp fragment (not shown).

Figure 2.

Comparison of amino acid sequence of ERIS in nine species. The human sequence is shown, and identical amino acids in other species are represented with a dash. An asterisk (*) represents a missing amino acid in that species. The bold amino acids signify predicted conserved phosphorylation sites (β-adrenergic receptor kinase substrate at 25–29; PKA/PKC kinase substrate motif at 32–34 and 67–69; and CK2 substrate motif at 34–37). The glutamic acid that is mutated in ERIS in the Jordanian families is indicated by an arrow. The underlined amino acids represent a putative transmembrane domain. The double-underlined amino acids highlight the zinc-finger domain of the protein. The unblackened arrowheads indicate the region corresponding to splice junctions in the cDNA.

Figure 3.

ZCD2 expression and WFS2 disease pathogenesis. A, RT-PCR of the WFS2 gene, ZCD2, in a variety of tissues (top row) and the GADPH control (bottom row). B, RT-PCR of lymphoblastoid RNA from two control individuals (C1 and C2) and an affected individual (A), by use of human ZCD2 cDNA primers. The primers amplify the full-length cDNA; the large band is 551 bp, and the smaller band is 336 bp. Lane M is the molecular marker. C, Genomic sequence of the intron-exon boundary shows that the mutation (bold and underlined) is within 6 bp of the 3′ splice-acceptor site. Below, cDNA and amino acid sequences are shown at the boundary between exon 1 and exon 2, compared with the premature stop codon that occurs when exon 1 and exon 3 are spliced together in an affected individual.

Figure 4.

Localization and immunoprecipitation of ERIS. A, P19 cells transfected with pN-FLAG ZCD2. In panel 1, the transfected cells were stained with DAPI. Panel 2 shows the ER marker calnexin (red). Panel 3 shows pN-FLAG ZCD2 (green). Only a proportion of the cells that were transfected showed pN-FLAG ZCD2 expression. Panel 4 shows the merged picture, with pN-FLAG ZCD2 colocalizing with calnexin in the ER. B, Cell lysates from HEK293 cells transfected with pC-FLAG ZCD2, immunoprecipitated using anti-WFS1 antibodies (lane 1) and anti-FLAG antibodies (lane 2). The top half of the blot was probed with anti-WFS1 antibodies, and the bottom half with anti-FLAG antibodies. Lane 1 shows a strong Wolframin band at 100 kDa but does not show pC-FLAG ZCD2 (at 17.5 kDa). pC-FLAG ZCD2 is observed in lane 2, with no Wolframin. The 19-kDa band represents immunoglobulin G, whereas other bands represent nonspecific binding of the antibodies. The same results were observed when the experiment was performed with pN-FLAG ZCD2 (data not shown).

Figure 5.

Intracellular Ca²⁺ measurements. A, No difference in the mean basal [Ca²⁺]i levels observed between wild-type and mutant cells (n=30–37; P=.49). B, The mean [Ca²⁺]i increase, significantly greater in mutant cells than in wild-type cells when stimulated with 2 mM TG (n=8; the asterisk [*] indicates significance [P=.03]). Also shown are representative single-cell measurements of [Ca²⁺]i changes in mutant (C) and wild-type (D) cells when stimulated by TG.