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.

BackgroundUsher syndrome (USH) is a genetically heterogeneous deafness-blindness syndrome, divided into three clinical subtypes: USH1, USH2 and USH3.MethodsMutations in 21 out of 26 investigated Danish unrelated individuals with USH were identified, using a combination of molecular diagnostic methods.ResultsBefore 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.ConclusionMutations 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.

To investigate if there is a correlation between clinical expression (duration of disease, age, walking function) and morphological alterations in affected muscle (structural changes, immunohistochemical and the level of alpha-DG glycosylation) among 25 patients with Limb Girdle Muscular Dystrophy type 2I (LGMD2I) all with the common C.826C>A mutation ). Muscle biopsies were obtained from 25 patients. Quantitative evaluation of morphological alterations in muscle sections, and immunohistochemistry (IHC) with antibodies directed against the alpha-dystroglycan glycan epitope, was performed by light microscopy. A semi-quantitative assessment of changes was recorded, point-graded and summarized as morphological sum-score for each biopsy.. Western blot (WB) analysis on muscle biopsy homogenates were carried with antibodies directed towards the core alpha-DG as well as the alpha-DG-glycan epitope. Laminin overlay was included. Muscle biopsies from 25 patients with LGMD2I presented large variation in morphological features. All biopsies presented reduced molecular weight of alpha-DG as detected with alpha-DG core antibody on WB analysis. Immunohistochemistry and WB analysis directed towards the alpha-DG-glycan epitope, as well as laminin overlay, demonstrated large variation in signal intensity among the LGMD2I patients. Results from IHC and WB/laminin overlay correlated poorly. There was no obvious correlation between ages at onset or duration of disease at biopsy versus the level of alpha-DG glycosylation. Likewise, there was no apparent association between severities of disease and the sum-scores of structural changes in the muscle biopsies. The clinical and morphological variability seen among LGMD2I patients with identical FKRP genotype must therefore be explained by other genetic or environmental mechanisms. To investigate if there is a correlation between clinical expression (duration of disease, age, walking function) and morphological alterations in affected muscle (structural changes, immunohistochemical and the level of alpha-DG glycosylation) among 25 patients with Limb Girdle Muscular Dystrophy type 2I (LGMD2I) all with the common C.826C>A mutation ). Muscle biopsies were obtained from 25 patients. Quantitative evaluation of morphological alterations in muscle sections, and immunohistochemistry (IHC) with antibodies directed against the alpha-dystroglycan glycan epitope, was performed by light microscopy. A semi-quantitative assessment of changes was recorded, point-graded and summarized as morphological sum-score for each biopsy.. Western blot (WB) analysis on muscle biopsy homogenates were carried with antibodies directed towards the core alpha-DG as well as the alpha-DG-glycan epitope. Laminin overlay was included. Muscle biopsies from 25 patients with LGMD2I presented large variation in morphological features. All biopsies presented reduced molecular weight of alpha-DG as detected with alpha-DG core antibody on WB analysis. Immunohistochemistry and WB analysis directed towards the alpha-DG-glycan epitope, as well as laminin overlay, demonstrated large variation in signal intensity among the LGMD2I patients. Results from IHC and WB/laminin overlay correlated poorly. There was no obvious correlation between ages at onset or duration of disease at biopsy versus the level of alpha-DG glycosylation. Likewise, there was no apparent association between severities of disease and the sum-scores of structural changes in the muscle biopsies. The clinical and morphological variability seen among LGMD2I patients with identical FKRP genotype must therefore be explained by other genetic or environmental mechanisms.

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 α-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 type II (USH2) is an autosomal recessive disorder, characterised by moderate to severe high-frequency hearing impairment, normal balance function and progressive visual impairment due to retinitis pigmentosa. Usher syndrome type IIa, the most common subtype, is defined by mutations in the USH2A gene encoding a short and a recently discovered long usherin isoform comprising 21 and 73 exons, respectively. More than 120 different disease-causing mutations have been reported, however, most of the previous reports concern mutations restricted to exons 1-21 of the USH2A gene. To explore the spectrum of USH2A disease-causing mutations among Scandinavian USH2 cases, patients from 118 unrelated families of which 27 previously had been found to carry mutations in exons 1-21 were subjected to extensive DNA sequence analysis of the full size USH2A gene. Altogether, 122 USH2A DNA sequence alterations were identified of which 57 were predicted to be disease-causing, 7 were considered to be of uncertain pathogenicity and 58 were predicted to be benign variants. Of 36 novel pathogenic USH2A mutations 31 were located in exons 22-73, specific to the long isoform. USH2A mutations were identified in 89/118 (75.4%) families. In 79/89 (88.8%) of these families two pathogenic mutations were identified whereas in 10/89 (11.2%) families the second mutation remained unidentified. In 5/118 (4.2%) families the USH phenotype could be explained by mutations in the USH3A gene. The results presented here provide a comprehensive picture of the genetic aetiology of Usher syndrome type IIA in Scandinavia as it is known to date. (C) 2008 Wiley-Liss, Inc.

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 37kb, 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.

To perform a quantitative analysis with gene-arrays, one must take into account inaccuracies (experimental variations, biological variations and other measurement errors) which are seldom known. In this paper we investigated amplification and noise propagation related errors by measuring intensity dependent variations. Based on a set of control samples, we create confidence intervals for up and down regulations. We validated our method through a qPCR experiment and compared it to standard analysis methods (including loess normalization and filtering methods based on genetic variability). The results reveal that amplification related errors are a major concern.

RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants. The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units. There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers. The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them. None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases. Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples. In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora. Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources. We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L. monocytogenes must be treated as potentially harmful.

Usher syndrome type IIa is an autosomal recessive disorder characterized by mild-to-severe hearing loss and progressive visual loss due to retinitis pigmentosa. The mutation that most commonly causes Usher syndrome type IIa is a 1-bp deletion, described as "2299delG," in the USH2A gene. The mutation has been identified in several patients from northern and southern Europe and from North America, and it has been found in single patients from South America, South Africa, and China. Various studies have reported a range of frequencies (.16-.44) among patients with Usher syndrome, depending on the geographic origin of the patients. The 2299delG mutation may be the one that most frequently causes retinitis pigmentosa in humans. Given the high frequencies and the wide geographic distribution of the mutation, it was of interest to determine whether the mutation resulted from an ancestral mutational event or represented a mutational hotspot in the USH2A gene. Haplotype analysis was performed on DNA samples from 116 unrelated patients with Usher syndrome type IIa; the patients were from 14 countries and represented 148 2299delG alleles. On the basis of six single-nucleotide polymorphisms within the USH2A gene, 12 core haplotypes were observed in a panel of normal chromosomes. However, in our analysis, only one core haplotype was found to be associated with the 2299delG mutation. The data indicate that the widespread geographic distribution of the 2299delG mutation is the result of an ancestral mutation that has spread throughout Europe and into the New World as a result of migration.

Usher syndrome is a group of autosomal recessive disorders characterised by progressive visual loss from retinitis pigmentosa and moderate to severe sensorineural hearing loss. Usher syndrome is estimated to account for 6-10% of all congenital sensorineural hearing loss. A gene locus in Usher type II (USH2) families has been assigned to a small region on chromosome 1q41 called the UHS2A locus. We have investigated two families with Usher syndrome from different isolated populations. One family is a Norwegian Saami family and the second family is from the Cayman Islands. They both come from relatively isolated populations and are inbred families suitable for linkage analysis. A lod score of 3.09 and 7.65 at zero recombination was reached respectively in the two families with two point linkage analysis to the USH2A locus on 1q41. Additional homozygosity mapping of the affected subjects concluded with a candidate region of 6.1 Mb. This region spans the previously published candidate region in USH2A. Our study emphasises that the mapped gene for USH2 is also involved in patients from other populations and will have implications for future mutation analysis once the USH2A gene is cloned.

Hereditary hearing impairment affects about 1 in 1000 newborns. In most cases hearing loss is non-syndromic with no other clinical features, while in other families deafness is associated with specific clinical abnormalities. Analysis of large families with non-syndromic and syndromic deafness have been used to identify genes or gene locations that cause hearing impairment. The present report describes a large Norwegian family with autosomal dominant non-syndromic, progressive high tone hearing loss with linkage to 1q21-q23. A maximum LOD score of 7.65 (theta = 0.00) was obtained with the microsatellite marker D1S196. Analysis of recombinant individuals maps the deafness gene (DFNA7) to a 22 cM region between D1S104 and D1S466. The region contains several attractive candidate genes. This report supports the idea of extensive genetic heterogeneity in hereditary hearing impairment and represents the first localization of a deafness gene in a Norwegian family.