A homozygous missense mutation of WFS1 gene causes Wolfram's syndrome without hearing loss in an Iranian family (a report of clinical heterogeneity)

Abstract Background Wolfram's syndrome (WFS) is a hereditary (autosomal recessive) neurodegenerative disorder. The clinical features are related to diabetes insipidus, diabetes mellitus, optic atrophy, and deafness (DIDMOAD) with other variable clinical manifestations. Pathogenic variants in the WFS1 gene, encoding wolframin, are known to be the main cause of Wolfram's syndrome. In this study, we present the clinical and genetic characteristics of two WFS patients from an Iranian family. Methods The mutation screening was performed by polymerase chain reaction (PCR) followed by direct Sanger sequencing of all exons from two affected WFS. Results The complete Sanger sequencing of the WFS1 gene detected a homozygous missense variant, c.2207G>A (p.Gly736Asp), in the eighth exon of the WFS1 gene. Both cases developed all the major symptoms of the disease, interestingly, except hearing loss. Conclusions Because of the rarity and clinical heterogeneity of WFS, the molecular genetic assay is essential to confirm the diagnosis and management of the WFS patients.

called wolframin. 8 The wolframin protein is expressed in several tissues (pancreas, lung, liver, heart, bone, muscle, brain, and kidney). 9 Its function in the endoplasmic reticulum (ER) that is involved in protein production, processing, and transport, maintaining homeostasis of the amount of intracellular calcium. 8 Perturbations in ER function result in the accumulation of misfolded proteins, a state entitled ER stress, 10 and result in WFS. Several mutations have been reported in WFS1 gene, mostly located in the exon 8, including missense, insertion, deletion, and splice site mutations in the form homozygote and compound heterozygote. 11 The purpose of the present study was mutational analysis of the WFS1 gene and investigation of the genotype-phenotype correlation in a consanguineous Iranian family with Wolfram's syndrome.

| MATERIAL S AND ME THODS
The current study was approved by the Ethics Committee of the Arak University of Medical Sciences (IR.ARAKMU.REC.1398.115).
After obtaining written informed consent, two affected and four unaffected subjects from the family members of consanguineous Iranian with WFS were enrolled in this present study. The research related to human use has been complied with all the relevant national regulations, institutional policies, and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors' institutional review board or equivalent committee. The pedigree of the WFS family with an autosomal recessive pattern of inheritance is shown in Figure 1A.
F I G U R E 1 A, The family pedigree of Wolfram's syndrome. B and C, The mutation status of WFS1 (c.2207G>A) was identified by the Sanger sequencing (marked with a red box), the patients (IV-1 and IV-5) were homozygous for c.2207G>A and her parents (III-1 and III-2), and brothers (IV-4) were in the heterozygous state. D, Multiple sequence alignment demonstrates high conservation of the p.G736 residue (marked with a black box) Genomic DNA was extracted from peripheral blood samples obtained from all subjects using the salting-out method. Forward and reverse primers (Table 1)   We performed a full three-dimensional (3D) structural modeling of the WFS1 protein with the based on 5IJO protein configuration in the Protein Data Bank (PDB) using I-TASSER online software ( Figure 2B). Then computational visualization of the protein residues was done by PyMOL script (https://pymol.org/2/). Finally, we used MetaDome software to determine the tolerance of the mutated residue for better interpret of the variant with unknown significance.
MetaDome mapped population variations from the gnomAD and pathogenic variants from ClinVar, and allowed to create genetic intolerance regions at the amino acid resolution for human protein domains.

| Clinical description
The probands, two male (IV-1 and IV-5), were born to healthy Iranian consanguineous parents originating from a Bakhtiari ethnicity

| Mutational analysis
One homozygous missense variant, Figure 1C, was found in the exon According to the HOPE program, the wild-type and mutant residue charge were neutral versus negative, respectively. Furthermore, the wild-type residue is more hydrophobic than the mutant residue, and this mutation decreases the stability of the protein structure with Confidence Score: −0.602 (probably damaging). The new amino acid affects the α-helix structure by creating different hydrogen bonds ( Figure 2C,D). The p.G736D was predicted to be highly deleterious using the SIFT and PolyPhen programs. Also, MutationTaster predicted this mutation to be "disease-causing" variant, and with 29.5 CADD Phred score, which is over the deleterious threshold. Multiple sequence alignment exhibited that p.Gly736 residue in WFS1 is evolutionarily conserved across different species ( Figure 1D), representing its importance in the function of WFS1 protein. MetaDome software also showed that this residue is an intolerance region for any mutation ( Figure 2E). Furthermore, according to the HOPE, the mutation is located within a section of residues that is repeated in the protein named as lumenal. Under this conservation information, this alteration is most likely damaging to the wolframin protein. In our survey, we found a probably pathogenic missense mutation (c.2207G>A) in an Iranian family with significant consequences on WFS1 configuration and function. Previous studies have reported that the residue G736 is a hot spot mutation point (G736R, G736D, G736S). 13 Our description of the c.2207G>A (p.G736D) mutation in WFS1 is the second description of a mutation related with WFS in an Iranian family and is the third report worldwide. 14,15 However, two previous studies did not investigate parental segregation of the mutation and carrier situation. 14,15 Furthermore, in our study, the patients revealed no signs of hearing loss, while in the previous survey, loss of auditory acuity in both ears was reported at the age of 16. 14 Moreover, the hearing status was not determined in a Japanese patient. 15 The clinical heterogeneity between the patients with the same mutation may be partly due to the effects of modifier genes and the natural variants in the WFS1 gene. Consistently, the effects of modifier genes and alleles on the clinical heterogeneity have been reported in previous studies. 16,17 Interestingly, similar to our research, it was recently reported that c.376G>A mutation in the exon 4 did not result in the hearing loss in an Iranian patient with WFS. 18 In one study, subjects with WFS were subdivided into three groups consistent with the predicted functional consequences of each mutation. 15  According to the mentioned classification, the c.2207G>A mutation falls into the group 2, and the age of onset of diabetes mellitus and optic atrophy in our subjects was similar to this category. So, these differences in the age of onset may be correlated with special effects of such alterations on WFS1 protein function.

| D ISCUSS I ON
Management of Wolfram's syndrome is symptomatic and supportive. It involves a multidisciplinary work to manage the different features of this disorder such as insulin therapy (for diabetes mellitus), vasopressin (for diabetes insipidus), and hearing aids or cochlear implants (for hearing loss). 5 Early diagnosis through genetic testing can help to dismiss the symptoms, avoiding future complications and improving the quality of life for WFS subjects.
In conclusion, we found a missense mutation which was shared between the considered patients. Altogether, according to previous