A novel CISD2 intragenic deletion, optic neuropathy and platelet aggregation defect in Wolfram syndrome type 2

Wolfram syndrome (WFS; MIM 222300) is a rare neurodegenerative disease with autosomal recessive inheritance and incomplete penetrance, whose diagnosis requires diabetes mellitus (DM) and optic atrophy (OA). WFS includes a wide spectrum of other possible disorders, such as diabetes insipidus, sensorineural deafness, genitourinary tract problems, male hypogonadism, neurological or psychiatric disorders, and less frequently, bowel dysfunction. Clinical wide spectrum is likely sustained by a genetic heterogeneity [1]. To date, 2 types of WFS, type 1 (WFS1; MIM 606201) and type 2 (WFS2; MIM 604928) characterized by different disease genes have been identified. The disease gene for WFS2 is CISD2, encoding for a highly conserved zinc-finger protein of the Endoplasmic Reticulum Intermembrane Small (ERIS), playing a pivotal role in calcium homeostasis (Ca2H) [2]. It was identified in three consanguineous Jordanian families carrying a point mutation in exon 2 [3]. In WFS2, diabetes insipidus and psychiatric disorders are not described, whereas bleeding upper intestinal ulcers (b-UIU), not reported in WFS1 [4], and defective platelet aggregation (PA) may be considered diagnostic criteria. After the first report [3, 5], no further CISD2 mutations have been reported. We describe the first Caucasian patient with WFS2 with a novel homozygous CISD2 intragenic deletion.

Our patient was a Caucasian girl (FS), first child of non-consanguineous parents. Since the age of 5 years old, FS suffered from recurrent b-UIU. When 14 years old, malrotation and partial duodenum intussusception were diagnosed, resection of a duodenal tract and duodenal-jejunal anastomosis were performed. At the age of 16, she suffered from reduced visual acuity and dyschromatopsia, so she underwent eye examination including BCVA (20/40 in the right and 20/50 in the left eye), slit lamp biomicroscopy (normal anterior segment bilaterally), fundus examination (optic disc pallor with preserved pupillary light reflexes bilaterally), and p-VEP according to the ISCEV guidelines [6], (normal latency and normal amplitude responses bilaterally). The b-MRI showed normal optic nerve caliber in both eyes. At 17 years old non-autoimmune diabetes occurred and WFS2 was suspected. She started insulin treatment. Further examinations showed a bilateral sensorineural hearing loss in the high frequencies (SHLHF), and a slow intestinal transit time with entero-gastric bile reflux (EGBR). As visual acuity, pupillary light reflexes, slit lamp biomicroscopy, fundus examination and p-VEP were unchanged after 5 years follow up, additional eye exams were performed in order to extensively define optic nerve involvement. Visual field testing, ERG, MP, and OCT of macula and retinal nerve fiber layer showed a moderate involvement of the optic nerve in both eyes, compatible with a diagnosis of Optic Neuropathy (ON), excluding the OA (Figure 1) and other ocular abnormalities.

Evaluating PA, a reduced and reversible defective PA up to 10 μM of ADP was present, whereas PA in response to other tested agents (collagen, epinephrine, ristocetin) was within normal ranges. Platelet secretion in response to ADP was absent (Figure 2). The bleeding score, according to Tosetto et al. [7], was 3 (normal values: ≤3), the other main coagulative tests were normal.

Ophtalmological abnormalities, DM and deafness were not found in the parents and brother, whereas a reversible PA in response to ADP was found. Currently, FS is 22 years old and presents DM, ON with preserved visual acuity and electrophysiologic tests, no OA, tinnitus due to SHLHF triggered by loud environments, EGBR, slow intestinal transit, reduced and reversible defective PA to ADP, and b-UIU.

We describe the first European Caucasian girl with WFS2, carrying a novel intragenic exon 2 CISD2 homozygous deletion. Only a homozygous mutation of the CISD2, a single nucleotide substitution that leads to a missense change Glu37Gln, was previously associated to WFS2 [3]. This gene, mapping on chromosome 4q24 [1], encodes an extremely small zinc-finger protein, named ERIS, which displays a fairly wide cellular expression profile, including pancreas, brain, blood and platelets [3]. Conlan et al. [10] showed that the domain 2Fe-2S is essential for the activity of this zinc-finger protein, and is located from aminoacid 99 (Cys) to aminoacid 112 (Gly). Genomic data from our subjects show that the triplets coding for aminoacids WRSKT, from the position 102 to 106 of the polypeptide, are within the deleted region, and this may result in a non functional protein because of zinc-finger domain lost. The ERIS protein is highly conserved and has 70% identity and 82% similarity across all the vertebrate species [3]. It plays a pivotal role in Ca2H [2], as well as wolframin, another endoplasmic reticulum (ER) [7] protein encoded by WFS1 gene. Also wolframin is involved in the regulation of ER stress and Ca2H [11, 12], without any interaction with ERIS. The ER function influences some crucial secretory proteins, such as insulin, as well as cell-surface receptors and integral membrane proteins. Imbalance in Ca2H of ER elicits stress in this organelle, leading to the accumulation of misfolded and unfolded protein in the organelle, a state called ‘ER stress’, which plays, in WFS, a key role in β-cells, leaving these cells more prone to oxidative stress and consequently to apoptosis. Activation of the unfolded protein response (UPR), an adaptive response that counteracts the ER stress, serves to restore ER homeostasis. Moreover, UPR maintains pancreatic β-cell function and promotes β-cell survival. Exaggerated ER stress, which normally triggers the UPR, has been hypothesized in the β-cells impairing the cell progression and increasing apoptosis [13]. Neuronal cells have highly developed ER, whose "stress" is related with ganglion cells apoptosis, as demonstrated in a glaucoma chronic injury model [14]. The degeneration of ganglion and glial retinal cells could be related to the ER stress probably accountable for the ocular involvement in our patient that presented moderate ON but not OA. Few and non-specific studies on ERIS localization and expression, in particular in the ganglion and glial cells of the optic nerve, may explain the different optic nerve involvement and a favourable outcome of ocular pathology. In WFS1, early OA causes alterations of the optic nerve caliber due to the loss of a considerable number of ganglion cells [15]. These patients show severe progressive visual acuity reduction and non recordable p-VEP or increased latency or greatly reduced amplitudes [16, 17]. In Jordanian WFS2 patients OA was observed after childhood or was described as “asymptomatic”, but opportune investigations were not reported [1]. Since our patient does not show OA at the age of 22 years, and since “asymptomatic” OA described in Jordanians is not completely supported, we suppose that the ocular pathology in those patients was probably ON and not OA. This is just a case report and thus great caution is needed, even if the absence of OA, one of the main diagnostic criteria of WFS, suggests that the so-called WFS2 could not be a WFS subtype.

As shown in Table 3 the clinical spectrum of WFS1 and WFS2 is only partially coincident, according to the different expression of the two proteins. In fact wolframin is expressed in pancreas, brain, heart, skeletal muscle, placenta, lung, liver and kidney, while ERIS is expressed in pancreas, brain, blood and platelets. Furthermore in WFS2 patients, diabetes insipidus and psychiatric disorders are not described so far, otherwise some additional features are present and may be considered diagnostic criteria: significant bleeding tendency, defective PA with collagen [4], and peptic ulcer disease with b-UIU [5]. The tendency to mucocutaneous bleedings mainly displayed by gastric ulcers is a common feature, observed in 79% and 90% respectively of Jordanian patients [5]. Expression studies using mass spectrometry listed in the Human Protein Reference Database (accession number 17413) show the presence of ZCD2 transcripts in platelets [3]. This may explain, at least in part, the bleeding phenotype [18] described in these patients.

Furthermore, in Table 3 we show the difference in the clinical spectrum of Jordanian cases [1] and our patient. Firstly, our girl presented ON and not OA even though as previously discussed it is possible that the ocular pathology in those patients was probably ON and not OA; secondly, she showed an impaired platelet aggregation to ADP and not to collagen; however collagen- and ADP- induced aggregation are both calcium-mediated, thus it is possible that different experimental conditions or inter-patient variability can explain different results in PA.

The new findings of this report could better characterize the WFS2 phenotype.

This study was approved by institutional ethics committee and was done in accordance with Declaration of Helsinki. Informed consent for genetic analysis was obtained from the study participants. Moreover, as the patient is a girl, written informed consent was obtained from her parents for publication of this Case report. A copy of the written consent is available for review by the Editor of this journal.