MUTATION IN BRIEF
HUMAN MUTATION Mutation in Brief #403 (2001) Online
© 2001 WILEY-LISS, INC.
Received 27 October 2000; accepted 31 January 2001.
Identification of Novel WFS1 Mutations in Italian
Children With Wolfram Syndrome
A.Tessa1, I. Carbone2,3, M.C. Matteoli1, C. Bruno2,3, C. Patrono1, I.P. Patera1, F. De Luca4,
R. Lorini3, and F.M. Santorelli1*
1Molecular Medicine and Diabetology, IRCCS-Bambino Gesù, Rome; 2Neuromuscular Service and 3Dept. of
Pediatric, University of Genova, IRCCS-G.Gaslini, Genova; 4University of Messina, Italy
*Correspondence to: Dr. F.M. Santorelli, Molecular Medicine, IRCCS-Children’s Hospital Bambino Gesù, Piazza
S. Onofrio, 4 – 00165 Rome, Italy; Tel: +390668592105; Fax: +390668592024; E-mail: firstname.lastname@example.org
Communicated by Mark H. Paalman
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. © 2001 Wiley-Liss, Inc.
KEY WORDS: Wolfram syndrome; WS; WFS1; Italian; diabetes; DIDMOAD
The Wolfram syndrome (WS, MIM# 222300) is an autosomal rececessive disorder characterized by juvenile-
onset diabetes mellitus and progressive optic atrophy. It is also known by the acronym “DIDMOAD” (diabetes
insipidus, diabetes mellitus, optic atrophy, and deafness). It is a progressive neurodegenerative disorder, and
many patients also develop urinary-tract atony, ataxia, peripheral neuropathy, and psychiatric illness. Some
mtDNA alterations have been inconsistently described (Barrientos, 1996). Recently, different mutations of
WFS1 on chromosome 4p16 have been reported in WS patients (Inoue, 1998). Here, we describe the first
molecular characterization of Italian WS patients.
PATIENTS AND METHODS
Subjects were six Italian patients from five unrelated families in whom the clinical diagnosis of WS was
sustained by the occurrence of minimal ascertainment criteria: juvenile-onset (age <20 years) diabetes mellitus
and optic atrophy (Barrett, 1995). Clinical history and disease status was ascertained using clinical
2 Tessa et al.
and all eight exons (with flanking sequences) were examined in all subjects. Abnormal SSCP fragments were
confirmed in a second run and then analyzed by direct sequencing on an ABI 377. Mutations were assessed in
patients, relatives, and control subjects using SSCP when appropriate.
Table 1 summaries clinical features, mutation screening, parent of origin of the mutation (when available) and
effects on translation. SSCP analysis of WFS1 revealed mutations in 10 of 12 alleles. Direct sequencing of PCR
products led to the identification of the remaining mutant alleles. A total of eight distinct variants were found,
including five missense mutations (I333V, S443I, A684V, R708C, and H611R), one frameshift mutation
(1387delCTCT), a 16-bp (base pairs) deletion at nucleotide position 1519, and an intronic mutation (IVS6+16g-
>a). Six changes are new and four (S433I, 1387delCTCT, 1519del16, and IVS6+16g->a) have presumably
pathogenic consequences. The 1387delCTCT mutation was shared by two apparently unrelated families (three
patients) in association with a similar age at onset, clinical features and disease progression. At this time, we
cannot rule out a common ancestry, though parents originate from different areas of our Country. The 16-bp
deletion mutation at nucleotide position 1520-1536 was found in two patients who were independently
ascertained by us. Although it needs to be proved, consanguineity is possible because their parents were from
the same small region.
Aside from silent substitutions and the reported I333V and H611R mutations, we identified two novel
changes (A684V and R708C) both associated with a deleted allele (see Table) and with unclear consequences.
The I333V substitution was also detected in 8% control chromosomes. In one case, the I333V and H611R
mutation were found on the same allele but also in controls.
None of our patients harbored commonly encountered pathogenic mtDNA point mutations nor deletions.
Table 1. Age at diagnosis, severity of clinical presentation, and spectrum of WFS1 mutations of Italian
Wolfram syndrome patients.
Patient IDAge/Sex Age at onset of main clinical
Case 1 20/FFam History; consanguineity; DM
3yrs; DEAF 18yrs; OA 12yrs; DI;
renal and psychiatric abnorm.
Case 219/MDM 10yrs; DEAF 8yrs; OA 6yrs; DI ;
1519del 16-bp (h)
Case 3 23/FDM 10yrs; DEAF 12yrs; OA 7yrs;
DI; structural renal-tract
1387del CTCT (ht) (father); I333V(ht)
1387delCTCT (ht) (father) ; I333V(ht)
Case 422/FDM 12yrs; DEAF 14yrs; OA 12yrs;
DI 14yrs; ataxic gait and psychiatric
1387del CTCT (ht);I333V (ht); H611R
(h) A684V (ht)
Case 518/MDM 12yrs; DEAF 13yrs; OA 13yrs;
S443I( (ht) (mother) ; IVS6+16g->a
(ht)); H611R (ht) (father)
Case 613/FFam History; DM 6yrs; OA 12yrs;
1519del 16-bp (h) ;R708C(ht)
*Novel variants are in boldface.
DM=diabetes mellitus; DEAF= hearing loss; OA= optic atrophy; DI= diabetes insipidus.
h= homozygous mutation; ht= heterozygous mutation.
Our study identified four new pathogenic mutations. All novel changes were confirmed by sequencing of a
second independent PCR product and additionally by either RFLP or SSCP methods (Figure 1).
Mutations in Italian WS Patients
SSCP analysis shows the paternal origin of the 1387delCTCT in family 2.
Each of the four novel pathogenic mutations has important consequences for WFS1 structure and function.
The 1519del16 mutation is an out-of-frame deletion resulting in premature stop codon at residue 454, removing
the last 437 amino. The 1387delCTCT produces a frameshift in the reading sequence leading to a premature stop
codon at residue Val 441, deleting the last 400 amino acids in wolframin. The homozygous 16-bp deletion and
the heterozygous deletion of CTCT were flanked by direct repeats and presumably resulted from DNA slippage
and mispairing during DNA replication. Both mutations would be predicted to lead to severe loss of wolframin,
removing six transmembrane domains and the entire intracytoplasmic C-terminal part.
The novel S443I mutation is likely pathogenic because of the following reasons: 1) it has never been
reported and it was heterozygous in the patient and her mother but absent in over 100 control chromosomes; 2)
the amino acid substitution replaces isoleucine for a polar residue; 3) it occurs within the highly preserved 4th
transmembrane segment, in a stretch of evolutionarily conserved residues (FTVTSYXSL) and it is close to the
G437R mutation, previously reported in a British WS patient. Moreover, prediction of secondary structure using
theNPS@ program, with prediction accepted only when there was a consensus between the PREDATOR, the
GOR IV, and the SOPM programs (http://pbil.ibcp.fr), revealed that the random coiled region flanking Ser443
would be replaced by an extended strand in the presence of Ile443. This is likely to perturbate the
transmembrane domains of wolframin.
The fourth novel mutation, a heterozygous point mutation at position +16 in intron 6, was paternally
inherited, absent in 100 control chromosomes, and it could well result in abnormal splicing of messanger RNA.
The functional role of additional WFS1 mutation is unclear. Patient 3 presented also a new A684V and
patient 5 a new R708C. Although never reported before, absent in control chromosomes, and affecting
conserved amino acids between human and murine WFS1, the pathogenic significance of these alterations is
provisonal. We detected a I333V in 3/12 alleles but also at low frequency (8%) in controls. In a British report
(Hardy, 1999), the conserved Ile at residue 333 is mutated in as much as 40% of individuals but this might relate
to different frequency among diverse populations. Although listed as a nonconservative substitution, the
I333V has currently unknown functional significance. We also detected the H611R mutation, an amino acid
changes previously reported in association with bipolar disorders. Historically, psychiatric illness has been
associated with as much as 26% of WS carriers and the H611R allele is considered a neutral polymorphism likely
in linkage dysequilibrium with affective disorders (Furlong, 1999), although data are contradictory (Swift, 2000).
In our series, it is notable that psychiatric features in the form of anorexia and mood disorders were found in
three patients and in six of their relatives. However, there was no excess of the abovementioned mutation in our
cases when compared to control chromosomes (FMS, unpublihed).
While representing the first molecular chracterization of Italian WS familes, our data expand the spectrum of
WFS1 allelic variants. To identify any possible mutational hot spots in the WFS1 protein sequence, we
combined ours with data from previous reports (Inoue, 1998; Strom, 1998; Hardy, 1999). Although conclusions
cannot be drawn because of the relatively small number of patients, it is interesting to note that the majority of
frameshifts and nonsense mutations encompass the predicted transmembrane domains of the protein. A 4-bp
deletion at position 2648 was found in four British families and we identified a deletion/frameshift mutation
shared by more than one patient but overall the WFS1 gene appears affected by multiple private mutations,
mostly in exon 8. Screening for mutations will thus require complete sequence of the WFS1 gene.
4 Tessa et al. Download full-text
We thank the expert technical assistance of Ms. Cristina Purificato. We are indebted to Dr. Charles Cattages
for revising the manuscript. This work was partially supported by grants from the Italian Ministry of Health
(OBG-Ricerca Corrente and G. Gaslini-Ricerca Corrente, 1998).
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