Identification of Novel Wsf1 Mutations in a Sicilian Child with Wolfram Syndrome

Abstract

Wolfram Syndrome (WS) is a rare hereditary disease with autosomal recessive inheritance with incomplete penetrance. It is characterized by diabetes mellitus associated with progressive optic atrophy. The diagnosis is essentially clinical and mutation analysis is used to confirm the diagnosis. In the present study we describe the clinical and molecular features of a diabetic child carrying two novel WFS1 mutations. The Sicilian proband and his non-affected family were studied. Ophthalmologic examination included: visual acuity determination and funduscopy, optical coherent tomography, retinal fluorangiography, perimetry and electroretinogram. Molecular methods: automatic sequencing of PCR amplified WFS1 gene fragments and qRT-PCR analysis of WFS1 transcripts. 3 WSF1 mutations have been identified in the proband. One allele carries 2 paternally inherited mutations (c.1332 C>G and c.1631C>G) in exon-8, never annotated before, in heterozygosis with one "de novo" classic mutation (c.505 G>A) in exon-5. In addition, we report an unexpected molecular feature: higher WFS1 mRNA levels in the proband compared to the father. Milan, Italy) following manifacturer's instructions. A neuroblastoma cell line (LAN5) used as a positive control. WFS1 expression was analysed by real-time quantitative RT-PCR (qRT-PCR) in individual samples and WFS1 mRNA levels were quantified in comparison to β-actin mRNA expression. WFS1 PCR primers were purchased from Quiagen (Quantitect Primer Hs_WFS1_1_SG QT00082663) the amplicon size was 117 bp (Suppl. Figure 1); β-actin primers were obtained from RealTimePrimers.com (Elkins Park, PA, USA) with the following sequences: F 5'-GGACTTCGAGCAAGAGATGG-3'; R 5'-AGCACTGTGTTGGCGTACAG-3'; the amplicon size was 234 bp. All reactions were performed using the LightCycler 1.0 (Roche Diagnostics GMbH, Germany). Briefly, 2 g of total RNA was retrotranscribed using the ImProm-II™ Reverse Transcription System (Promega, Milan, Italy). Each RNA was combined with 1l of oligo16dT (Promega Milan, Italy) and thermally denatured at 70°C for 5 minutes and then chilled on ice. As a final step, the template-oligodT combination was added to the reaction mix (4 l ImProm-II™ 1X Reaction Buffer, 1l ImProm-II™ Reverse Transcriptase, 3.2 l Magnesium Chloride 3 mM, 1 l dNTPs mix 10 mM and 1 l Recombinant RNasin Ribonuclease Inhibitor 1u/l) and nuclease-free water was added to 20 l of final volume on ice. Following an initial annealing at 25°C for 5 minutes, the reaction was incubated at 42°C for one hour. In order to heat-inactivate the ImProm-II Reverse Transcriptase the reaction mix was finally incubated at 70°C for 15 minutes. Journal of Genetic Syndromes & Gene TherapyReal-time PCR was performed using 2 l of cDNA diluited 1:5, plus 10 l of Quantitech master mix (Qiagen), 2 l of Quantitect Primer Hs_WFS1_1_SG or β-actin primers, and nuclease-free water to 20 l of final volume. A standard reference curve was performed in parallel using RNAs extracted from LAN5 cell line serially 1:2 diluited starting from 100 ng reaction...

Identification of a novel WFS1 homozygous nonsense mutation in Jordanian children with Wolfram syndrome

“…Other clinical symptoms were found to be associated with WS including ataxia, peripheral neuropathy, psychiatric problems, and renal tract abnormalities ( Haghighi et al, 2013 ). WS is diagnosed early in life with a juvenile onset of DM and OA ( Hardy et al, 1999 ); patients die young at a median age of 30 years mostly due to respiratory failure caused by brainstem atrophy ( Pizzolanti et al, 2014 ). WS prevalence is estimated to be 1 in 770,000 with a carrier frequency of 1 in 354 ( Nakamura et al, 2006 ).…”

“…Wolframin is expressed in in the β-cells of pancreas, heart, brain, muscle tissues, liver, spleen and kidney ( Hofmann et al, 2003 ). Wolframin is an endoplasmic reticulum (ER)-localized protein and studies indicate a role in protein synthesis, protein folding and modification, membrane trafficking, and ER stress signaling ( Pizzolanti et al, 2014 ). Additionally, studies have shown that wolframin protein has a calmodulin (CaM) binding site at the N-terminal cytoplasmic domain between the residues Glu90 and Trp186 ( Yurimoto et al, 2009 ), suggesting a role for wolframin in maintaining the intracellular homeostasis of calcium ions by controlling their levels in the ER ( Qian et al, 2015 ).…”

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

“…Wolfram's syndrome (WFS; OMIM#222300) is a rare, hereditary, and neurodegenerative disease with an autosomal recessive pattern of inheritance 1 . The estimated prevalence of Wolfram's syndrome is 1 in 68,000 to 770,000 in overall population worldwide 2‐4 . WFS is described by the juvenile onset of diabetes mellitus and optic atrophy (at age < 16 years), and associated with sensorineural hearing loss, progressive neurologic abnormalities (peripheral neuropathy, cerebellar ataxia, psychiatric illness, dementia, and urinary tract atony) and other endocrine abnormalities 5 .…”

Endoplasmic reticulum stress inhibition ameliorated WFS1 expression alterations and reduced pancreatic islets’ insulin secretion induced by high-fat diet in rats

“…Wfs1 gene was first identified by Wolfram and Wagener (1983) in patients with Wolfram syndrome (i.e. diabetes mellitus and optic nerve atrophy) 12,15,16 . WFS1 is a glycoprotein expressed in the endoplasmic reticulum (ER) of pancreatic β cells, heart, placenta, lung, and brain [15][16][17] .…”

“…diabetes mellitus and optic nerve atrophy) 12,15,16 . WFS1 is a glycoprotein expressed in the endoplasmic reticulum (ER) of pancreatic β cells, heart, placenta, lung, and brain [15][16][17] . It is worth noting that the β cells of endocrine pancreas are the major site of WFS1 expression and no signs of its expression has been found in exocrine pancreas 18 .…”