Targeted Audiological Surveillance Program in Campania, Italy

To identify children with postnatal hearing loss, a structured monitoring system is needed. The goal of this study was to describe a targeted surveillance program in Italy to identify children with postnatal hearing loss

A donor splice site mutation in CISD2 generates multiple truncated, non-functional isoforms in Wolfram syndrome type 2 patients

Abstract Background Mutations in the gene that encodes CDGSH iron sulfur domain 2 (CISD2) are causative of Wolfram syndrome type 2 (WFS2), a rare autosomal recessive neurodegenerative disorder mainly characterized by diabetes mellitus, optic atrophy, peptic ulcer bleeding and defective platelet aggregation. Four mutations in the CISD2 gene have been reported. Among these mutations, the homozygous c.103 + 1G > A substitution was identified in the donor splice site of intron 1 in two Italian sisters and was predicted to cause a exon 1 to be skipped. Methods Here, we employed molecular assays to characterize the c.103 + 1G > A mutation using the patient’s peripheral blood mononuclear cells (PBMCs). 5′-RACE coupled with RT-PCR were used to analyse the effect of the c.103 + 1G > A mutation on mRNA splicing. Western blot analysis was used to analyse the consequences of the CISD2 mutation on the encoded protein. Results We demonstrated that the c.103 + 1G > A mutation functionally impaired mRNA splicing, producing multiple splice variants characterized by the whole or partial absence of exon 1, which introduced amino acid changes and a premature stop. The affected mRNAs resulted in either predicted targets for nonsense mRNA decay (NMD) or non-functional isoforms. Conclusions We concluded that the c.103 + 1G > A mutation resulted in the loss of functional CISD2 protein in the two Italian WFS2 patients

Additional file 2: Figure S2. of A donor splice site mutation in CISD2 generates multiple truncated, non-functional isoforms in Wolfram syndrome type 2 patients

Chromatograms of the 400 nt-5′-RACE products not subjected to subcloning. (TIFF 155 kb

Immunofluorescence and Molecular analysis of <i>Calcium ion channel subunits</i> in wBM-hMSC.

<p>(A) Positive control normal Human Skeletal Muscle Myoblast (HSMM). Scale bar = 10 µm (alpha SMA) and 25 µm (SA, Myosin, Myogenin and Desmin). (B) Undifferentiated wBM hMSC analyzed at 28 days. Scale bar = 10 µm (a-SMA), 25 µm (SA, Myosin, and Desmin), 50 (Myogenin). (C) Original gels demonstrating amplification of calcium ion channel subunit transcripts in wBM hMSCs: -RT: control of reverse transcription without RT enzyme; C−: negative control (water); C+: positive control (HSMM); line 1: wBM hMSCs from first passage; line 2: wBM hMSCs from second passage; line 3: wBM hMSCs from fourth passage; β-actin, housekeeping gene.</p

Integration of BM-hMSCs in the striate muscle.

<p>(A) Bisbenzimide-labelled BM-hMSCs (in blue) appear integrated into desmin-positive striated muscle fibers (in green). (B) At 4 months, several BM-hMSCs are located in close vicinity (arrowheads) to acetylcholine receptors (α-BTX staining, in red) (C). Proliferative profile of transplanted BM-hMSCs at one and (D) four months after transplantation, as revealed by Ki67 immunohistochemistry (in purple). Scale bar = 50 µm.</p

Bisbenzimide-stained BM-hMSCs (in blue) transplanted into rat bulbocavernosus muscle.

<p>(A) 24 hours after transplantation BM-hMSCs appear undifferentiated with a typical round shape (inset in A, scale bar = 500 µm); (B) one month after engraftment many BM-hMSCs with elongated shape are recognizable among muscular fibers (inset in B, scale bar = 100 µm). (C) At 4 months, migration toward muscle fibers is confirmed by elongated appearance of cells occupying peripheral position (inset b), whereas undifferentiated cells are observed in the core of graft (inset a). Scale bar = 500 µm.</p

Myogenic differentiation on laminin matrix and <i>L-type Calcium ion channel subunits</i> analysis.

<p>(A) Phase contrast images of laminin cells: presence of some binucleated cells. Scale bar = 25 µm. (B) Immunofluorescence analysis confirmed the presence of a few binucleated structures positive for desmin, SA, myogenin in BM-hMSC cultured on laminin cells. Scale bar = 25 µm. (C) Original gels demonstrating amplification of <i>calcium ion channel subunit</i> transcripts in laminin cells: –RT: control of reverse transcription without RT enzyme; C−: negative control, water; C+: positive control, HSMM; line 1: control wBM-hMSCs; line 2: laminin cells cultured in DMEM-F12 supplemented with 15% FBS; line 3: laminin cells induced to myogenic differentiation with EGF for 7 days.</p

Mouse BM-mMSCs.

<p>(A) Desmin-positive multinucleated myotubes (nuclei labelled in blue with bisbenzimide) derived from the fusion of C2C12 myoblasts. (B) EGFP-MSCs, plated alone, display a fibroblast-like shape. (C–F) In co-culture with C2C12 cells (desmin-positive, labelled in red), MSCs (green) show long cytoplasmatic processes (arrow). (G–J) EGFP-MSCs adhere to desmin-positive myotubes (arrowhead). Scale bar = 50 µm.</p