Human cytomegalovirus (HCMV) and hearing impairment: Infection of fibroblast cells with HCMV induces chromosome breaks at 1q23.3, between loci DFNA7 and DFNA49—Both involved in dominantly inherited, sensorineural, hearing impairment

Human cytomegalovirus (HCMV) infection is the most common congenital infection in developed countries and is responsible for a substantial fraction of sensorineural hearing impairment (SNHI) in children. The risk of hearing impairment is associated with viral load in urine and blood collected during the first postnatal month. However, although inner ear abnormalities are observed in some children with HCMV-induced SNHI, the exact mechanism whereby congenital HCMV infection causes hearing impairment is unknown. Earlier studies using standard cytogenetic mapping techniques showed that infection of S-phase human fibroblast cells with HCMV resulted in two specific, site-directed, chromosome breaks at band positions 1q21 and 1q42 which include loci involved in dominantly and recessively inherited hearing impairment, respectively. These findings suggested that cells infected with HCMV might provide a reservoir for genetic damage and, in a clinical perspective, a scenario could be envisioned whereby hearing impairment could result from early DNA damage of dividing fetal cells rather than viral replication and cell lysis. In this work we demonstrate, using fine mapping techniques, that HCMV infection in S-phase fibroblast cells induces genetic damage at 1q23.3, within a maximal region of 37 kb, containing five low copy repeat (LCR) elements. The breakpoint is situated between two hearing impairment (HI) loci, DFNA49 and DFNA7, and in close proximity to the MPZ gene previously shown to be involved in autosomal dominant Charcot-Marie-Tooth syndrome (CMT1B) with auditory neuropathy

Limb girdle muscular dystrophy type 2I: No correlation between clinical severity, histopathology and glycosylated α-dystroglycan levels in patients homozygous for common FKRP mutation

Limb girdle muscular dystrophy type 2I (LGMD2I) is a progressive disorder caused by mutations in the FuKutin-Related Protein gene (FKRP). LGMD2I displays clinical heterogeneity with onset of severe symptoms in early childhood to mild calf and thigh hypertrophy in the second or third decade. Patients homozygous for the common FKRP mutation c.826C>A (p.Leu276Ile) show phenotypes within the milder end of the clinical spectrum. However, this group also manifests substantial clinical variability. FKRP deficiency causes hypoglycosylation of α-dystroglycan; a component of the dystrophin associated glycoprotein complex. α-Dystroglycan hypoglycosylation is associated with loss of interaction with laminin α2, which in turn results in laminin α2 depletion. Here, we have attempted to clarify if the clinical variability seen in patients homozygous for c.826C>A is related to alterations in muscle fibre pathology, α-DG glycosylation levels, levels of laminin α2 as well as the capacity of α-DG to bind to laminin. We have assessed vastus lateralis muscle biopsies from 25 LGMD2I patients harbouring the c.826C>A/c.826C>A genotype by histological examination, immunohistochemistry and immunoblotting. No clear correlation was found between clinical severity, as determined by self-reported walking function, and the above features, suggesting that more complex molecular processes are contributing to the progression of disease

Fukutin-Related Protein Resides in the Golgi Cisternae of Skeletal Muscle Fibres and Forms Disulfide-Linked Homodimers via an N-Terminal Interaction

Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein a-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein

Usher syndrome in Denmark: mutation Spectrum and some clinical observations

Background: Usher syndrome (USH) is a genetically heterogeneous deafness-blindness syndrome, divided into three clinical subtypes: USH1, USH2 and USH3. Methods: Mutations in 21 out of 26 investigated Danish unrelated individuals with USH were identified, using a combination of molecular diagnostic methods. Results: Before Next Generation Sequencing (NGS) became available mutations in nine individuals (1 USH1, 7 USH2, 1 USH3) were identified by Sanger sequencing of USH1C, USH2A or CLRN1 or by Arrayed Primer EXtension (APEX) method. Mutations in 12 individuals (7 USH1, 5 USH2) were found by targeted NGS of ten known USH genes. Five novel pathogenic variants were identified. We combined our data with previously published, and obtained an overview of the USH mutation spectrum in Denmark, including 100 unrelated individuals; 32 with USH1, 67 with USH2, and 1 with USH3. Macular edema was observed in 44 of 117 individuals. Olfactory function was tested in 12 individuals and found to be within normal range in all. Conclusion: Mutations that lead to USH1 were predominantly identified in MYO7A (75%), whereas all mutations in USH2 cases were identified in USH2A. The MYO7A mutation c.93C>A, p.(Cys31*) accounted for 33% of all USH1 mutations and the USH2A c.2299delG, p.(Glu767Serfs*21) variant accounted for 45% of all USH2 mutations in the Danish cohort