- Open Access
Novel mutations and the ophthalmologic characters in Chinese patients with Wolfram Syndrome
Orphanet Journal of Rare Diseases volume 14, Article number: 190 (2019)
Wolfram Syndrome (WFS) is a rare autosomal recessive neurodegenerative disease which has a wide spectrum of manifestations including diabetes insipidus, diabetes mellitus, optic atrophy and deafness. WFS1 and CISD2 are two main causing genes of WFS. The aim of this study was to illustrate the ophthalmologic manifestations and determine the genotype of Chinese WFS patients.
Completed ophthalmic examinations and family investigations were performed on 4 clinically diagnosed WFS patients from 4 unrelated families. Genetic testing was done by the next generation sequencing of candidate genes. One patient carried a homozygous mutation (c.272_273del) in CISD2, two patients carried compound heterozygous mutations (c.1618 T > G + c.2020G > A and c.1048 T > A + c.2020G > A) in WFS1, and one patient carried a heterozygous mutation (c.937C > T) in WFS1. Three of them were novel mutations.
Our study indicated WFS in Chinese is a neurodegenerative disease with both wide spectrum of clinical features and genetic heterogeneity. We found three novel mutations in WFS patients, and to our best knowledge, this is the first report of Chinese WFS patient with mutation in CISD2.
Wolfram syndrome (WFS; MIM #222300), first described in 1938 by Wolfram and Wagener, is a rare hereditary autosomal recessive disease. The prevalence of WFS was estimated to be 1 in 770,000 in UK  and 1 in 710,000 in the Japanese population . As a progressive neurodegenerative disorder, WFS has a wide spectrum of clinical manifestations. The main phenotypes of WFS are diabetes insipidus (DI), diabetes mellitus (DM), optic atrophy (OA) and deafness (D) [1, 3]. Around 50% patients harbor all these manifestations, so WFS was also referred to as the acronym DIDMOAD syndrome [1, 3, 4]. Other common manifestations include neurologic and psychiatric disorders, renal tract abnormalities, endocrine disorders, as well as many others . The major diagnosis criterion of WFS is the coincidence of early onset type 1 DM and bilateral OA before the second decade [1, 5,6,7]. Because of the multi-system neurodegeneration, the prognosis of WFS is very poor and the patients’ median life expectancy is about 30 years (range 25–49 years) .
WFS1 on chromosome 4 is the causative gene of Wolfram Syndrome type 1 (WFS1) , and the loss-of-function mutations of WFS1 have been identified in most of patients with WFS [5, 9]. WFS1 encodes wolframin, an endoplasmic reticulum (ER) transmembrane protein . Wolframin is widely expressed in neurons, pancreas, heart, muscle, liver, spleen and kidney . It has also been detected in optic nerve glial cells and retinal ganglion cells [12, 13]. The main function of wolframin are reducing the ER stress, maintaining the Ca2+ homeostasis and regulating the biosynthesis and secretion of insulin [14,15,16].
In addition, mutations of CISD2 are responsible for Wolfram Syndrome type 2 (WFS2; MIM #604928), which has variant features including gastrointestinal ulceration and bleeding tendency without diabetes insipidus [17,18,19]. CISD2, CDGSH iron-sulfur domain-containing protein 2, located on chromosome 4q22–24, encodes endoplasmic reticulum intermembrane small protein (ERISP) . Although the biological functions of CISD2 still remain incompletely defined, some studies show that it has a similar role with WFS1 in maintaining the homeostasis of Ca2+ and ER and the cross-talk between ER and mitochondria [20, 21].
In this study, we performed a clinical and genetic investigation on 4 unrelated Chinese patients with WFS. We systematically reviewed their clinical ophthalmologic features and identified 3 novel mutations in WFS1 and CISD2 gene. And we reported the first Chinese patient with WFS2 carried a homozygous mutation in CISD2.
Patients and methods
We retrospectively reviewed 4 consecutive patients diagnosed with WFS at Ophthalmology Department of Eye Ear Nose and Throat Hospital of Fudan University from 2013 to 2018. This study was approved by the Eye Ear Nose and Throat Hospital of Fudan University Institutional Review Board, and written formal consent was obtained from all enrolled patients or their legal guardians. Patients were enrolled in our study when meeting one of the following two criteria: 1) the early onset DM and progressive OA, not explained by any other diseases; 2) the identification of 2 pathological WFS1/CISD2 mutations. DM was diagnosed by WHO criteria . OA was confirmed by funduscopic examination of the optic nerve head with pallid appearance and by the evidence of atrophy of the peripapillary nerve fiber layer on optical coherence tomography (OCT). Magnetic resonance imaging (MRI) or computed tomography (CT) scan was also utilized to exclude compressive optic neuropathies. 110 healthy Chinese people, without diagnosis of DM, OA or any other serious ocular or systematic diseases, were also included in this study.
All patients underwent a complete ophthalmologic examination, including visual acuity (VA) examination, intraocular pressure measurement, slit-lamp biomicroscopy, ophthalmoscope, visual fields assessment (Carl Zeiss Meditec, Inc., Dublin, CA, United States), electroretinography (ERG) and visual evoked potentials (VEP) (LKC UTAS E3000 LKC Technologies, Inc., United States). The OCT (Cirrus OCT 5000, Carl Zeiss Meditec, Inc., Dublin, CA, United States) was performed for each patient to evaluate retinal nerve fiber layer (RNFL) thickness. The MRI was performed in 2 patients and CT scan was completed in the other 2 patients. The audiological, urological, neurological and psychiatric examinations results were recorded from the medical records.
Genomic DNA samples were extracted from whole blood samples of the patients, their relatives, and 110 healthy Chinese people. Genetic testing was performed in all four patients by next generation sequence (NGS). A panel including 790 ophthalmology associated genes were sequenced by Illumina HiSeq 2000 (Illumina, Inc., San Diego, CA, United States) sequencing system. The average depth was 200x. Family members of the probands were validated by Sanger sequence.
The detected mutations were checked in 110 Chinese normal controls by Sanger Sequence. Conservation of the mutation sites was evaluated by Clustal Omega . Polymorphism Phenotyping 2 (PolyPhen2)  and Sorting Intolerant from Tolerant (SIFT)  were applied for the assessment of pathogenicity of detected mutations.
General clinical manifestations
Four Chinese WFS patients from 4 different families were enrolled in our study. The demographic and clinical features of the 4 patients are shown in Table 1. All patients were male. The median age of patients was 25 years (range 11–42 years). Patient 1 came from consanguineous family and has an elder brother diagnosed of DM at age of 10 years and died from ketosis encephalopathy at age of 17 years. Patient 2 has an elder sister diagnosed of WFS with same symptoms.
All patients presented to our ophthalmology clinic because of progressive loss of vision (Table 1). They all had the coincidence of DM and OA. The median age at DM onset was 10 years (range 9–28 years). Three of them had various degree of hearing impairment: two patients had bilateral high-frequency hearing impairment and one had bilateral sensorineural deafness. Patient 1 was diagnosed as central diabetes insipidus by the water deprivation and desmopressin challenge test, and he also showed absence of the physiological high signal of the posterior pituitary gland on T1-weighted images of cranial MRI and an abnormal electroencephalography (EEG). No patient presented renal tract abnormalities, psychiatric diseases or behavioral disorders.
In general, optic atrophy was observed in all four patients, demonstrated by the examination of fundus, MRI and OCT (Figs. 1, 2 and 3). The median age of presenting impaired vision and OA diagnosis was 12 years (range 7–39 years) and 17 years (range 7–42 years), respectively (Table 1). All patients presented severe vision loss and most of them had best corrected vision acuity (BCVA) less than 20/400 (Table 1). All of them had normal pupillary responses. All patients presented color vision loss, especially patient 1 and patient 2 had all color vision defect. Perimetry examination demonstrated various types of vision field loss, presenting as central scotomas, constriction of peripheral visual filed, segmental arcuate defect or diffuse decreased sensitivity. OCT were abnormal in all patients, showing diffused thinning of peripapillary RNFL and macular ganglion cell lay complex (GCC) (Fig. 3). ERG were normal in all patients, VEP showed latency increase and amplitude reduction in P100 waves. No one had cataract and diabetic retinopathy.
Mutations in WFS1 or CISD2 gene were detected in all these patients, including one homozygous mutation on CISD2 and four missense mutations on WFS1 (Table 2). No other gene mutations or mitochondrial genome mutations were detected. Pedigrees with WFS in our study are shown in Fig. 4. For patient 1, we detected one novel frameshift mutation (p.Leu91fs) in exon 2 of CISD2 caused by the deletion of two nucleotides (c.272_273del). The homozygous mutations were inherited from his parents, who were first-cousin (Fig. 5). For patient 2, compound heterozygotic mutations (c.2020G > A+ c.1618 T > G) in WFS1 were identified and inherited from his father and mother respectively. The same mutations were also found in his sister, who had DM and OA as well (Additional file 1: Figure S1). Compound heterozygotic mutations in WFS1 were also detected in patient 3 (c.2020G > A+ c.1048 T > A) (Additional file 2: Figure S2). Patient 4 carried a de novo heterozygotic mutation (c.937C > T) in WFS1, which was absent in his parents (Additional file 3: Figure S3). Four variants, including c.1618 T > G, c.1048 T > A, and c.937C > T in WFS1 and c.272_273del in CISD2, were sequenced in 110 normal Chinese controls and none of mutations were detected.
Three of them were reported for the first time, including a frameshift mutation c.272_273del in CISD2 and two missense mutations c.1618 T > G, c.1048 T > A in WFS1. These mutations all locate in evolutionary conserved positions of CISD2 and wolframin by multiple sequence alignment across species (Additional file 4: Figure S4). Two novel missense mutations of WFS1 both located in exon 8 which encodes wolframin, a protein with nine predicted transmembrane domains and extracellular loops (Fig. 6). The novel variations of p.Trp540Gly (c.1618 T > G) and p.Phe350Ile (c.1048 T > A) in WFS1 and p.Leu91fs (c.272_273del) in CISD2 are all predicted to be highly deleterious by SIFT or PolyPhen2 (Table 2).
In this study, we evaluated four Chinese WFS patients and descried their ophthalmologic characteristics, as well as reported three novel WFS1 and CISD2 mutations. Most patients presented at least three clinical manifestations and developed at least one in their first decade, which was consistent with the systematic review of WFS . A wide range of ophthalmological findings were detected including severe vision acuity lost, declined color vision, constriction of visual fields and abnormal VEP, which were consistent with previous studies [31,32,33]. Notably, the presenting ages of impaired vision of some patients were early than OA diagnosis age, which suggested the insidiousness of vision loss in WFS. This indicates that ophthalmologist should be aware of the possibility of WFS in young patients with severe bilateral optic atrophy. Detailed medical history inquiry and appropriate genetic testing are highly recommended for these patients.
There are two genes, WFS1 and CISD2, were proven to cause WFS. CISD2 is a rare causative gene and autosomal-recessive mutations in CISD2 is the pathogeny of WFS2. So far, very limited mutations have been reported in this gene (Table 3) [17,18,19,20]. In our study, patient 1 was homozygous for the frame-shift mutation c.272_273del in CISD2, due to the parental consanguinity. This mutation was not detected in our Chinese control population. Patient 1 presented the most severe phenotype with rapid progression of disease and multisystem manifestations. The mutant CISD2 protein exerts a deleterious influence on ER-mitochondrial structure and function and ultimately participate in multisystem neurodegeneration . WFS2 firstly was regarded as a subtype which has various unique features such as peptic ulcer and bleeding tendency [17,18,19]. In contrast, our patient presented classical features of WFS1, including early-onset DM, progressive OA, DI and neurodegenerative features. Haematological abnormalities and peptic ulcer has not been detected so far. Our study may support the point of view that WFS1 and WFS2, caused by different genes, has a continuous clinical spectrum . Since this patient was still young, with the progression of WFS2, he may develop other signs of WFS2 in the future, so long term follow-up is needed.
Mutations in WFS1 gene are responsible for most WFS patients. Since the discovery of WFS1 in 1998, more than 300 different mutations have been identified in this gene  and majority of them located in the exon 8 encoding the nine transmembrane segments and the C-terminal tail of wolframin . In this study, we found four missense mutations located in exon 8 of WFS1, two of them were first reported including c.1618 T > G (p.Trp540Gly) and c.1048 T > A (p.Phe350Ile). The Sanger Sequence results in control population showed that these mutations are less likely to be polymorphisms. These two novel missense mutations are located in transmembrane domain. Multiple sequence alignment showed that they were positioned within evolutionary conserved regions of wolframin. And they were predicted to be deleterious by different tools (Table 2). Notably, the mutation c.2020G > A was found in two unrelated patients in our study. This mutation was previously reported in 4 patients with DM and OA without DI and deafness [26, 27]. The allele frequency of A is < 0.0001 in Han Chinese by the 1000 Genomes Project . Our result indicates that this mutation is probably a hotspot in Chinese WFS patients, which needs to be verified by more cases. Only one heterozygous mutation (c.937C > T, p.His313Tyr) was found in patient 4, which was previously detected in three patients with OA, very early DM diagnosis and profound hearing loss [28,29,30]. Coincidentally, patient 4 was diagnosed hearing loss much earlier than OA, which might provide an evidence that this mutation cause more hearing impairment than visual disability.
Our study showed a group of Chinese patients with WFS who had various clinical features. Genetic analysis detected three novel mutations in WFS1 and CISD2. This is the first report of Chinese patient with WFS2. Our study also illustrates the complexity and heterogeneity of WFS. So genetic testing is recommended for clinical optic nerve atrophy patients with highly suspected WFS, especially when diabetes mellitus is concomitant.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Best corrected vision acuity
- CISD2 :
CDGSH iron-sulfur domain-containing protein 2
Encodes endoplasmic reticulum intermembrane small protein
Ganglion cell lay complex
Magnetic resonance imaging
Next generation sequence
Optic coherence tomography
Polymorphism Phenotyping 2
Retinal nerve fiber layer
Sorting Intolerant from Tolerant
Visual evoked potentials
Wolfram Syndrome type 1
Wolfram Syndrome type 2
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We are grateful to the biobank of the Eye Ear Nose and Throat Hospital of Fudan University. We would also like to thank all the patients and their families.
This work was supported by funds from the National Science Foundation of China [No. 81570887].
Ethics approval and consent to participate
This study was approved by the Eye Ear Nose and Throat Hospital of Fudan University Institutional Review Board.
Consent for publication
Written informed consent was obtained from the patients or their legal guardians.
The authors declare that they have no competing interests.
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Figure S1. Electropherograms of identified mutations in patient 2 and his families. (JPG 139 kb)
Figure S2. Electropherograms of identified mutations in patient 3 and his families (his father has passed away). (JPG 133 kb)
Figure S3. Electropherograms of identified mutations in patient 4 and his families. (JPG 56 kb)
Figure S4. Multiple alignment of amino acid sequences of WFS1 and CISD2 across species. (JPG 273 kb)