Molecular detection of novel WFS1 mutations in patients with Wolfram syndrome by a DHPLC-based assay

Wolfram syndrome (WS) is a recessively inherited mendelian form of diabetes and neurodegeneration also known by the acronym DIDMOAD from the major clinical features, including diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Affected individuals may also show renal tract abnormalities as well as multiple neurological and psychiatric symptoms. The causative gene for WS (WFS1) encoding wolframin maps to chromosome 4p16.1 and consists of eight exons, spanning 33.44 Kb of genomic DNA. In this study we report on the mutational analysis of the WFS1 coding region in 19 Italian WS patients and 25 relatives, using a DHPLC-based protocol. A total of 19 different mutations in WFS1 were found in 18 of 19 patients (95%). All these mutations, except one, are novel, preferentially located in WFS1 exon 8, and include deletions, insertions, duplications, and nonsense and missense changes. In particular, a 16 base-pair deletion in WFS1 codon 454 was detected in five different unrelated nuclear families, being the most prevalent alteration in this Italian group. Nine neutral changes and polymorphisms were also identified. Overall, this study represents the molecular characterization of the largest cohort of Italian WS patients and carriers studied so far, and increases the number of identified WFS1 allelic variants worldwide.     

Study of the WFS1 gene and mitochondrial DNA in Spanish Wolfram syndrome families

Wolfram syndrome (WS) is an autosomal recessive neurodegenerative disorder characterized by early onset diabetes mellitus and progressive optic atrophy. Patients with WS frequently develop deafness, diabetes insipidus, renal tract abnormalities, and diverse psychiatric illnesses, among others. A gene responsible for WS was identified on 4p16.1 (WFS1). It encodes a putative 890 amino acid transmembrane protein present in a wide spectrum of tissues. A new locus for WS has been located on 4q22-24, providing evidence for the genetic heterogeneity of this syndrome. Six Spanish families with a total of seven WS patients were screened for mutations in the WFS1-coding region by direct sequencing. We found three previously undescribed mutations c.873C > A, c.1949_50delAT, and c.2206G > C, as well as the duplication c.409_424dup16, formerly published as 425ins16. Several groups had detected deletions in the mitochondrial DNA (mtDNA) of WS patients. For this reason, we also studied the presence of mtDNA rearrangements as well as Leber's hereditary optic neuropathy, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes, and A1555G point mutations in the WS families. No mtDNA abnormalities were detected.     

Autoimmune disease in a DFNA6/14/38 family carrying a novel missense mutation in WFS1

Most familial cases of autosomal dominant low frequency sensorineural hearing loss (LFSNHL) are attributable to mutations in the wolframin syndrome 1 (WFS1) gene at the DFNA6/14/38 locus. WFS1 mutations at this locus were first described in 2001 in six families segregating LFSNHL that was non-progressive below 2,000 Hz; the causative mutations all clustered in the C-terminal domain of the wolframin protein. Mutations in WFS1 also cause Wolfram syndrome (WS), an autosomal recessive neurodegenerative disorder defined by diabetes mellitus, optic atrophy and often deafness, while numerous single nucleotide polymorphisms (SNPs) in WFS1 have been associated with increased risk for diabetes mellitus, psychiatric illnesses and Parkinson disease. This study was conducted in an American family segregating autosomal dominant LFSNHL. Two hearing impaired family members also had autoimmune diseases-Graves disease (GD) and Crohn disease (CD). Based on the low frequency audioprofile, mutation screening of WFS1 was completed and a novel missense mutation (c.2576G --> A) that results in an arginine-to-glutamine substitution (p.R859Q) was identified in the C-terminal domain of the wolframin protein where most LFSNHL-causing mutations cluster. The family member with GD also carried polymorphisms in WFS1 that have been associated with other autoimmune diseases.     

Presence of a major WFS1 mutation in Spanish Wolfram syndrome pedigrees

Wolfram syndrome (WS) is an autosomal recessive neurodegenerative disease mainly characterized by familial diabetes mellitus and optic atrophy. WS patients frequently present with other clinical features such as diabetes insipidus, renal abnormalities, psychiatric disorders, and a variety of neurologic symptoms: deafness, ataxia, peripheral neuropathy. A gene responsible for Wolfram Syndrome (WFS1) has been recently identified on chromosome 4p16.1. Twenty-two Wolfram patients from 16 Spanish families were screened for mutations in the WFS1 coding region by SSCP analysis and direct sequencing. Since WS has been considered a mitochondrial disorder for some time, mitochondrial DNA (mtDNA) in these families was also examined. WFS1 mutations were detected in 75% of families (12 of 16). One of these mutations, an insertion of 16 base pairs in exon 4, turned out to be notably frequent in Spanish pedigrees. As many as 50% of pedigrees with WFS1 mutations harbored this insertion, either in one (33% of cases) or in two chromosomes (67%). Ten other mutations were identified: 7 missense changes, 2 deletions, and 1 nonsense mutation. Only 3 of these changes had been previously described in non-Spanish pedigrees. Large mtDNA rearrangements and LHON point mutations were detected in four and six families, respectively. No correlation could be established between WFS1 gene mutations and specific point mutations or rearrangements in mtDNA. We would suggest first screening for the 16-bp insertion in exon 4 when a new Spanish WS case is reported.     

Wolfram syndrome and WFS1 gene

Rigoli L, Lombardo F, Di Bella C. Wolfram syndrome and WFS1 gene. Wolfram syndrome (WS) (MIM 222300) is a rare multisystem neurodegenerative disorder of autosomal recessive inheritance, also known as DIDMOAD (diabetes insipidus, insulin‐deficient diabetes mellitus, optic atrophy and deafness). A Wolfram gene (WFS1) has been mapped to chromosome 4p16.1 which encodes an endoplasmic reticulum (ER) membrane‐embedded protein. ER localization suggests that WFS1 protein has physiological functions in membrane trafficking, secretion, processing and/or regulation of ER calcium omeostasis. Disturbances or overloading of these functions induce ER stress responses, including apoptosis. Most WS patients carry mutations in this gene, but some studies provided evidence for genetic heterogeneity, and the genotype–phenotype relationships are not clear. Here we review the data regarding the mechanisms and the mutations of WFS1 gene that relate to WS.     

WFS1/wolframin mutations, Wolfram syndrome, and associated diseases

Wolfram syndrome (WS) is the inherited association of juvenile-onset insulin-dependant diabetes mellitus and progressive bilateral optic atrophy. A nuclear gene, WFS1/wolframin, was identified that segregated with disease status and demonstrated an autosomal recessive mode of inheritance. Mutation analysis of the WFS1 gene in WS patients has identified mutations in 90% of patients. Most were compound heterozygotes with private mutations distributed throughout the gene with no obvious hotspots. The private nature of the mutations in WS patients and the low frequencies make it difficult to determine the biological or clinical relevance of these mutations. Mutation screening in patients with psychiatric disorders or diabetes mellitus has also been performed to test the hypothesis that heterozygous carriers of WFS1 gene mutations are at an increased risk following the observation that WS first-degree relatives have a higher frequency of these disorders. Most studies showed no association, however two missense mutations were identified that demonstrated significant association with psychiatric disorders and diabetes mellitus. Population association studies and functional studies of these variants will need to be performed to confirm these preliminary results. The elucidation of functions and functional pathways for the WFS1 gene product and variants will shed light on the effect of such disparate mutations on gene function and their role in the resulting clinical phenotype in WS and associated disorders.     

Wolfram syndrome in French population: characterization of novel mutations and polymorphisms in the WFS1 gene

Wolfram syndrome (WS), a rare autosomal recessive neurodegenerative disorder, results in most cases from mutations in the WFS1 gene. In this study, a total of 19 patients with Wolfram syndrome and 36 relatives from 17 families were screened for mutations in the WFS1 gene. WFS1 mutations were identified on both alleles in 16 of 19 patients and on 1 allele of 3 patients, showing that WFS1 is the major gene involved in WS in the french population. We identified 25 different mutations, twelve of which were novel. We found 6 frameshift mutations, 6 nonsense mutations, 6 missense mutations, 6 in-frame deletions, and one new homozygous mutation in the splice donor site of exon 7 (c.861+1G>A) resulting in a frameshift. Most patients were compound heterozygotes. No common founder mutation or mutational hot spot were found in the WFS1 gene. Although most mutations occurred in exon 8, in some cases molecular screening requires analysis of all exons, including the non-coding exon 1. We also identified 3 new polymorphisms. Furthermore, genotype-phenotype correlation suggests that the presence of inactivating mutations on both alleles may be associated with an early onset of diabetes mellitus.     

Rapidly progressive renal disease as part of Wolfram syndrome in a large inbred Turkish family due to a novel WFS1 mutation (p.Leu511Pro)

Wolfram syndrome, also named "DIDMOAD" (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), is an inherited association of juvenile-onset diabetes mellitus and optic atrophy as key diagnostic criteria. Renal tract abnormalities and neurodegenerative disorder may occur in the third and fourth decade. The wolframin gene, WFS1, associated with this syndrome, is located on chromosome 4p16.1. Many mutations have been described since the identification of WFS1 as the cause of Wolfram syndrome. We identified a new homozygous WFS1 mutation (c.1532T>C; p.Leu511Pro) causing Wolfram syndrome in a large inbred Turkish family. The patients showed early onset of IDDM, diabetes insipidus, optic atrophy, sensorineural hearing impairment and very rapid progression to renal failure before age 12 in three females. Ectopic expression of the wolframin mutant in HEK cells results in greatly reduced levels of protein expression compared to wild-type wolframin, strongly supporting that this mutation is disease-causing. The mutation showed perfect segregation with disease in the family, characterized by early and severe clinical manifestations.     

Positional cloning of the gene(WFS1) for Wolfram syndrome

Wolfram syndrome(DIDMOAD syndrome) is an autosomal recessive neurodegenerative disorder characterized by juvenile-onset, insulin-requiring diabetes mellitus and optic atrophy. Other symptoms including diabetes insipidus, neurosensory deafness, urinary tract and neurological abnormalities are often accompanied. In patients, beta-cells are selectively lost from their pancreatic islets of Langerhans. The gene was previously mapped to 4p16.1. By haplotype analysis and recombination mapping in 5 families, we localized the gene within a region less than 250 kb on chromosome 6p. In the region, we identified a novel gene(WFS1) encoding a putative transmembrane protein. Mutations were identified in all affected members of the families and these mutations were associated with disease phenotype. This finding was further confirmed by other investigators and to date, more than 50 mutations were identified in the WFS1 gene from the patients with Wolfram syndrome. The WFS1 gene encodes a protein of 100.3 kDa with 9 to 10 putative transmembrane domains. The protein appears to be important in the survival and maintenance of normal pancreatic beta-cells and neurons. Physiological function of the WFS1 protein and mechanisms by which defective WFS1 lead to the development of Wolfram syndrome need to be clarified.     

Genetic diagnosis of diabetes mellitus: Wolfram syndrome--from positional cloning to DNA diagnosis

Wolfram syndrome(WFS, DIDMOAD syndrome) is an autosomal recessive neurodegenerative disorder characterized by juvenile-onset, insulin-dependent diabetes mellitus and optic atrophy, often accompanied by other symptoms including diabetes insipidus, neurosensory deafness, urinary tract and neurological abnormalities. In patients, beta-cells are selectively lost from their pancreatic islets of Langerhans. By positional cloning, a novel gene named WFS1 was identified and many mutations were subsequently identified in patients with WFS. Most of the mutations were identified in the largest 8th exon, however, they also existed in other exons. No common mutations were found and founder effect was not observed except in Spanish families. The WFS1 gene encodes a putative transmembrane protein of 100.3 kDa localized in endoplasmic reticulum. More recently, WFS1 mutations were also identified in patients with dominantly inherited low-frequency sensorineural hearing loss. Clarification of the functions of the WFS1 protein, as well as the phenotype-genotype relationship, will help improve understanding of the pathophysiology of diseases caused by the WFS1 gene.     

Mutation update and uncommon phenotypes in a French cohort of 96 patients with WFS1‐related disorders

WFS1 mutations are responsible for Wolfram syndrome (WS) characterized by juvenile‐onset diabetes mellitus and optic atrophy, and for low‐frequency sensorineural hearing loss (LFSNHL). Our aim was to analyze the French cohort of 96 patients with WFS1‐related disorders in order (i) to update clinical and molecular data with 37 novel affected individuals, (ii) to describe uncommon phenotypes and, (iii) to precise the frequency of large‐scale rearrangements in WFS1. We performed quantitative polymerase chain reaction (PCR) in 13 patients, carrying only one heterozygous variant, to identify large‐scale rearrangements in WFS1. Among the 37 novel patients, 15 carried 15 novel deleterious putative mutations, including one large deletion of 17,444 base pairs. The analysis of the cohort revealed unexpected phenotypes including (i) late‐onset symptoms in 13.8% of patients with a probable autosomal recessive transmission; (ii) two siblings with recessive optic atrophy without diabetes mellitus and, (iii) six patients from four families with dominantly‐inherited deafness and optic atrophy. We highlight the expanding spectrum of WFS1‐related disorders and we show that, even if large deletions are rare events, they have to be searched in patients with classical WS carrying only one WFS1 mutation after sequencing.     

Non-syndromic progressive hearing loss DFNA38 is caused by heterozygous missense mutation in the Wolfram syndrome gene WFS1.

Dominantly inherited progressive hearing loss DFNA38 is caused by heterozygosity for a novel mutation in WFS1, the gene for recessively inherited Wolfram syndrome. Wolfram syndrome is defined by juvenile diabetes mellitus and optic atrophy and may include progressive hearing loss and other neurological symptoms. Heterozygotes for other Wolfram syndrome mutations generally have normal hearing. Dominant deafness defined by DFNA38 is more severe than deafness of Wolfram syndrome patients and lacks any syndromic features. In a six-generation kindred from Newfoundland, Canada, WFS1 Ala716Thr (2146 G-->A) was shared by all deaf members of the family and was specific to deaf individuals. The causal relationship between this missense mutation and deafness was supported by two observations based on haplotype and mutation analysis of the kindred. First, a relative homozygous for the mutation was diagnosed at age 3 years with insulin-dependent diabetes mellitus, the central feature of Wolfram syndrome. Second, two relatives with normal hearing had an identical haplotype to that defining DFNA38, with the exception of the base pair at position 2146. Other rare variants of WFS1 co-inherited with deafness in the family could be excluded as disease-causing mutations on the basis of this hearing-associated haplotype. The possibility that 'mild' mutations in WFS1 might be a cause of non-syndromic deafness in the general population should be explored.     

Mutation analysis of the WFS1 gene in seven Danish Wolfram syndrome families; four new mutations identified

Wolfram syndrome (WS) is a neuro-degenerative autosomal recessive (AR) disorder (OMIM #222300) caused by mutations in the WFS1 gene on 4p16.1. More than 120 mutations have been identified in WFS1 associated with AR WS, as well as autosomal dominant nonsyndromic low-frequency sensorineural hearing loss (LFSNHL). WFS1 variants were identified in eight subjects from seven families with WS, leading to the identification of four novel mutations, Q194X (nonsense), H313Y (missense), L313fsX360 (duplication frame shift) and F883fsX951 (deletion frame shift), and four previously reported mutations, A133T and L543R (missense), V415del (in frame triple deletion) and F883fsX950 (deletion frame shift). A mutation was found in 11/14 disease chromosomes, two subjects were homozygous for one mutation, one subject was compound heterozygous for two nucleotide substitutions (missense), one subject was compound heterozygous for a duplication and a deletion (frame shift), and in three families only one mutation was detected (Q194X and H313Y). All affected individuals shared clinically early-onset diabetes mellitus and progressive optic atrophy with onset in the first and second decades, respectively. In contrast, diabetes insipidus was present in two subjects only. Various degrees and types of hearing impairment were diagnosed in six individuals and cataract was observed in five subjects.     

Mutational spectrum of the WFS1 gene in Wolfram syndrome,nonsyndromic hearing impairment,diabetes mellitus,and psychiatric disease

WFS1 is a novel gene and encodes an 890 amino-acid glycoprotein (wolframin), predominantly localized in the endoplasmic reticulum. Mutations in WFS1 underlie autosomal recessive Wolfram syndrome and autosomal dominant low frequency sensorineural hearing impairment (LFSNHI) DFNA6/14. In addition, several WFS1 sequence variants have been shown to be significantly associated with diabetes mellitus and this gene has also been implicated in psychiatric diseases. Wolfram syndrome is highly variable in its clinical manifestations, which include diabetes insipidus, diabetes mellitus, optic atrophy, and deafness. Wolfram syndrome mutations are spread over the entire coding region, and are typically inactivating, suggesting that a loss of function causes the disease phenotype. In contrast, only non-inactivating mutations have been found in DFNA6/14 families, and these mutations are mainly located in the C-terminal protein domain. In this paper, we provide an overview of the currently known disease-causing and benign allele variants of WFS1 and propose a potential genotype-phenotype correlation for Wolfram syndrome and LFSNHI.     

Identification of novel mutations in WFS1 and genotype-phenotype correlation in Wolfram syndrome

Mutations in the WFS1 gene have been reported in Wolfram syndrome (WS), an autosomal recessive disorder defined by early onset of diabetes mellitus (DM) and progressive optic atrophy. Because of the low prevalence of this syndrome and the recent identification of the WFS1 gene, few data are available concerning the relationships between clinical and molecular aspects of the disease. Here, we describe 12 patients from 11 families with WS. We report on eight novel (A214fsX285, L293fsX303, P346L, I427S, V503fsX517, R558C, S605fsX711, P838L) and seven previously reported mutations. We also looked for genotype-phenotype correlation both in patients included in this study and 19 additional WS patients that were previously reported. Subsequently, we performed a systematic review and meta-analysis of five published clinical and molecular studies of WFS1 for genotype-phenotype correlation, combined with our current French patient group for a total of 96 patients. The presence of two inactivating mutations was shown to predispose to an earlier age of onset of both DM and optic atrophy. Moreover, the clinical expression of WS was more complete and occurred earlier in patients harboring no missense mutation.     

Genetic variations in the WFS1 gene in Japanese with type 2 diabetes and bipolar disorder

Diabetic and psychiatric symptoms often appear in patients with Wolfram syndrome, and obligate carriers of WFS1 have increased prevalence of type 2 diabetes and are more likely to require hospitalization for psychiatric illness including bipolar disorder. To identify the polymorphisms in Japanese, we examined a region of approximately 50 kb covering the entire WFS1 gene, and evaluated the patterns of linkage disequilibrium. We found a total of 42 variations including 8 novel coding single nucleotide polymorphisms (A6T, A134A, N159N, T170T, E237K, R383C, V412L, and V503G), 14 novel non-coding polymorphisms, and 2 linkage disequilibrium blocks. We also performed association studies in patients with type 2 diabetes mellitus and patients with bipolar disorder. The haplotype comprising R456 and H611 was most associated with type 2 diabetes (p = 0.013) and the haplotype comprising g. -15503C/T and g. 16226G/A was most associated with bipolar disorder (p = 0.006), but neither reached significant difference after multiple adjustment. These genetic variations and linkage disequilibrium patterns in WFS1 in Japanese should be useful in further investigation of genetic diversities of WFS1 and various related disorders.     

Mutations in the Wolfram syndrome 1 gene (WFS1) are a common cause of low frequency sensorineural hearing loss.

Non-syndromic low frequency sensorineural hearing loss (LFSNHL) affecting only 2000 Hz and below is an unusual type of hearing loss that worsens over time without progressing to profound deafness. This type of LFSNHL may be associated with mild tinnitus but is not associated with vertigo. We have previously reported two families with autosomal dominant LFSNHL linked to adjacent but non-overlapping loci on 4p16, DFNA6 and DFNA14. However, further study revealed that an individual with LFSNHL in the DFNA6 family who had a recombination event that excluded the DFNA14 candidate region was actually a phenocopy, and consequently, DFNA6 and DFNA14 are allelic. LFSNHL appears to be genetically nearly homogeneous, as only one LFSNHL family is known to map to a different chromosome (DFNA1). The DFNA6/14 critical region includes WFS1, the gene responsible for Wolfram syndrome, an autosomal recessive disorder characterized by diabetes mellitus and optic atrophy, and often, deafness. Herein we report five different heterozygous missense mutations (T699M, A716T, V779M, L829P, G831D) in the WFS1 gene found in six LFSNHL families. Mutations in WFS1 were identified in all LFSNHL families tested, with A716T arising independently in two families. None of the mutations was found in at least 220 control chromosomes with the exception of V779M, which was identified in 1/336 controls. This frequency is consistent with the prevalence of heterozygous carriers for Wolfram syndrome estimated at 0.3-1%. An increased risk of sensorineural hearing loss has been reported in such carriers. Therefore, we conclude that mutations in WFS1 are a common cause of LFSNHL.     

Identification of novel WFS1 mutations in Italian children with Wolfram syndrome

Six unrelated Italian children with Wolfram syndrome (WS) were analyzed for mutations in the WFS1. Four novel mutations (1387delCTCT, S443I, 1519del16, and IVS6+16g->a) were identified. In addition, we found two new, probably neutral changes (A684V and R708C). Other previously described variants were a heterozygous I333V in three alleles and the H611R in two. The 1519del16 mutation was carried by two patients whereas the CTCT deletion occurred in three subjects from two apparently unrelated families with WS. The current study expands the spectrum of mutations in WFS1 and represents the first molecular characterization of Italian WS patients.     

The wolframin His611Arg polymorphism influences medication overuse headache

Homozygosis for wolframin (WFS1) mutations determines Wolfram syndrome (WS), and common polymorphisms of WFS1 are associated with psychiatric illnesses and dependence behaviour. To test the influence of WFS1 polymorphisms on medication overuse headache (MOH), a chronic headache condition related to symptomatic drugs overuse, we analyzed 82 MOH patients for the WFS1 His611Arg polymorphism, and performed a comparison between clinical features of Arg/Arg (R/R) and non-R/R individuals. Individuals harbouring the R/R genotype showed significantly higher monthly drug consumption (t=-3.504; p=0.00075) and more severe depressive symptoms on the BDI questionnaire (t=-3.048; p=0.003) than non-R/R. WFS1 polymorphism emerged as the only significant predictor of drug consumption, at the multivariate regression analysis (F=12.277; d.f.=1,80; p=0.00075, adjusted R2=0.122). These results implicate WFS1 in the clinical picture of MOH, may be through an influence on need for drugs as in other conditions of abuse behaviour.     

Association of aggression with a novel microRNA binding site polymorphism in the wolframin gene

Rare mutations in the WFS1 gene lead to Wolfram syndrome, a severe multisystem disorder with progressive neurodegeneration and diabetes mellitus causing life‐threatening complications and premature death. Only a few association studies using small clinical samples tested the possible effects of common WFS1 gene variants on mood disorders and suicide, the non‐clinical spectrum has not been studied yet. Self‐report data on Aggression, Impulsiveness, Anxiety, and Depression were collected from a large (N = 801) non‐psychiatric sample. Single nucleotide polymorphisms (SNPs) were selected to provide an adequate coverage of the entire WFS1 gene, as well as to include putative microRNA binding site polymorphisms. Molecular analysis of the assumed microRNA binding site variant was performed by an in vitro reporter‐gene assay of the cloned 3′ untranslated region with coexpression of miR‐668. Among the 17 WFS1 SNPs, only the rs1046322, a putative microRNA (miR‐668) binding site polymorphism showed significant association with psychological dimensions after correction for multiple testing: those with the homozygous form of the minor allele reported higher aggression on the Buss–Perry Aggression Questionnaire (P = 0.0005). Functional effect of the same SNP was also demonstrated in a luciferase reporter system: the minor A allele showed lower repression compared to the major G allele, if co‐expressed with miR‐668. To our knowledge, this is the first report describing a microRNA binding site polymorphism of the WFS1 gene and its association with human aggression based on a large, non‐clinical sample. © 2013 Wiley Periodicals, Inc.