We report clinical and molecular observations about nine unrelated patients with WS and their follow-up. Up to now a wide spectrum of WFS1
mutations were described 
. In our study 14 different molecular defects were found. Thirteen were identified in both alleles: five probands carried compound heterozygous for WFS1
mutations; three had WFS1
defects in homozygous state. In one proband (patient 7) only an heterozygous mutation (A684V) was detected. Two novel mutations the S888X, and the T461P were found in patients 2 and 9 respectively (). All cases exhibited the main clinical features of WS.
The median age of the patients at the time of DNA analysis was 15,1 years (range 12–23).
Diabetes Mellitus is usually the first symptom occurring before age of 10 years, with optic atrophy at a median age of 11 years 
. In our series, the average age at onset of diabetes was 8,7 years (range 4–14). WS-associated DM shows clear differences respect to autoimmune type 1 diabetes. WS-associated DM is due to loss of pancreatic β-cell function and number, as confirmed by autoptic studies, without markers for an autoimmune process 
Optic Atrophy occurs by definition in all patients with WS and is progressive. All our patients had OA and its identification was concomitant or after diabetes diagnosis except in case n° 1 and 5: where OA occurred before DM diagnosis. OA was diagnosed and confirmed by brain NMR in the first decade of life in 5 cases and between 11 and 16 years of age in the other 4. Although our patients are genetically heterogeneous, ophthalmological findings and age at detection are similar to other series 
. The pathogenesis of OA could result from the effects of WS mutation on the survival of retinal ganglion cells, and in that case the mutation would lead to anterograde atrophy of retinal axons and shrinkage of the optic nerve. Retinal ganglion cells and optic nerve glial cells were found to be strongly labelled, suggesting that dual dysfunction of Wolframin in these cells might explain the progressive optic nerve atrophy reported in WS 
The development of polyuria and/or enuresis can indicate diabetes insipidus, which usually appears during the second decade. In our series only three patients showed DI at younger age (n° 2, 5, 8 at age of 91/2, 16, 7 years respectively). ().
Sensorineural Deafness, confirmed by audiograms, was diagnosed in three patients at a mean age of 10.8 years (range 7–91/2
). Proband 7 had profound deafness from infancy requiring a cochlear implant. Such early onset of severe deafness has not been usually reported in Wolfram Syndrome. Hearing impairment reported in 66% of WS patients, ranging from congenital deafness to mild, progressive hearing impairment. Other case series reported deafness at a median age of 16 years in 60% of cases 
. Audiometric features include a severe auditory threshold shift, more evident for the medium/high frequencies. Auditory impairment could be a consequence not only of a dysfunction of cochlear neurons and VII nerve fibers, but also of the central nervous pathways in brainstem and inferior colliculus 
Involvement of urinary tract represents another serious complication in WS patients and is estimated to occur in up to 90% of patients, being diagnosed during adolescence 
or adulthood 
. In our cases has been found only one patient (n° 6) with renal tract abnormalities: the ultrasonography of the urinary tract showed a double left district.
Neuropsychiatric manifestations are reported in the third decade. However Chaussenot A et al, who showed that the onset of neurologic symptoms, with a median age of 15 years, is much earlier than previously reported (mean age 30 years) 
. In the majority of our patients behavioral problems or depression were evident at much younger ages. This could be due to the fact that previous studies focused on cerebellar ataxia and brainstem involvement as main neurological involvement in WS.
In many cases, it can be difficult to tease out the effect of multiple disabilities and reactive depression from a true depressive illness. It is important to involve a pediatric psychiatrist as part of the multidisciplinary team, particularly if there is a seminal event such as a first seizure. Such events can have devastating psychological consequences for an adolescent.
Wolframin in vivo
is organized as a tetramer which originates a membrane Ca2+
channel of the Endoplasmic Reticulum (ER) and lack of function of WFS1
determines apoptotic input signaling 
. The ER has many roles, which include post-translational modification, folding and assembly of newly synthesized proteins such as insulin. Perturbations in ER function cause an imbalance between these processes, leading to accumulation of misfolded and unfolded proteins in +the organelle, a state called ER stress 
Except the V142fsX251 found in exon 4, all the detected mutations are located in exon 8, corresponding to the transmembrane region and carboxytail of Wolframin protein. The majority of mutations described in our study were localized to the predicted transmembrane domains (64,3%, n
9/14). Mutations in the amino-terminal domain were identified on 1 (7,1%) and in the carboxy-tail on 4 (28,6%) respectively (). This is in agreement with other studies in Italian and world-wide populations 
. All causative mutations were identified on both alleles except in one case in which only one heterozygous mutation was found.
Schematic representation of Wolframin showing all our identified mutations.
The predicting information that genetic analysis can give regards the difference between inactivating and non-inactivating mutations. The age at onset of DM was chosen as an indicator of disease severity. Missense mutations and 3 bp deletions, resulting in a deletion of one amino acid, were considered non-inactivating mutations. Nonsense, frameshift mutations, deletions and insertions of more than 3 bp were considered inactivating mutations 
. Among the five patients with WFS1
compound heterozygous mutation, two carried 2 non-inactivating mutations, three carried 1 inactivating and 1 non-inactivating. Their ages at diabetes clinical onset were 11 years and 7,3 years, respectively.
Among the three patients with WFS1 homozygous mutation two carried inactivating and one non-activating; their mean ages at onset of diabetes were 5,5 and 10 years respectively.
Patient 7 carried only one non-inactivating mutation and the age at onset of diabetes was 14 years.
However, the only patient harbouring one mutations outside exon 8 showed a mild phenotype: the onset of diabetes mellitus and optic atrophy was 9 and 16 years respectively. A hearing deficit, diabetes insipidus, renal tract and neurologic abnormalities were absent. We could observe in our study that inactivating mutation give a more severe phenotype that non inactivating mutation, although clinical symptoms are different, according to a previous observation 
In our study, seven cases among 9 cases were identified mutations already published elsewhere. To clarify the actual genotype/phenotype correlation, we compared clinical manifestaions of the subjects sharing the same mutations. The clinical features caused by the missense mutation Y528D and the delection F646fs708X, both found in homozygous state in proband P5 and in P6, respectively; were similar to what reported by Zalluoa 2008 and Zalluoa 2008-Rigoli 2011. Unfortunately, the combination of compound heterozygous defects found in our series has never been reported in literature. So, it is difficult to compare the phenotype these our patients with what reported in other studies. However, validation of any genotype–phenotype correlation must await detailed functional analysis of mutations on a cellular and molecular level.
If the mutation causing the syndrome in a family is known, it is possible to offer genetic counselling and prenatal diagnosis. Prenatal diagnosis is possible analyzing DNA extracted from fetal cells by amniocentesis at approximately 15–18 weeks gestation or chorionic villus sampling at approximately 10–12 weeks gestation. 
WS is a progressive neurodegenerative disorder, that usually appears in adolescence and requires careful endocrinological evaluation aimed to start an early and adequate hormonal substitutive therapy. Even if WS is characterized by a wide differential diagnosis including other causes of neurodegeneration, it should be suspected in a young with diabetes mellitus and optic atrophy, hearing loss, polyuria and polydipsia but adequate glucose control 
. Genetic test represents the best opportunity to confirm the clinical diagnosis and to propose prenatal diagnosis.
In conclusion, we have analyzed the WFS1 in nine WS patients. Our study increases the spectrum of WFS1 mutations with two novel variants.