USH2A mutation analysis in 70 Dutch families with Usher syndrome type II

Usher syndrome type II (USH2) is characterised by moderate to severe high-frequency hearing impairment, progressive visual loss due to retinitis pigmentosa and intact vestibular responses. Three loci are known for USH2, however, only the gene for USH2a (USH2A) has been identified. Mutation analysis of USH2A was performed in 70 Dutch USH2 families. Ten mutations in USH2A were detected, of which three are novel, c.949C>A, c.2242C>T (p.Gln748X) and c.4405C>T (p.Gln1468X). Including 9 previously published Dutch USH2a families, estimates of the prevalence of USH2a in the Dutch USH2 population were made. Mutations were identified in 62% of the families. In 28% both mutated alleles were identified, whereas in 34% the mutation in only one allele was found. It is estimated that about 28% of the Dutch USH2 families have a different causative gene. Analysis of deduced haplotypes suggests that c.1256G>T (p.Cys419Phe) is a Dutch ancestral mutation, occurring in 16% of the alleles.     

Genetic heterogeneity of Usher syndrome type II.

Usher syndrome is an autosomal recessive disorder characterised by retinitis pigmentosa and congenital sensorineural hearing loss. A gene for Usher syndrome type II (USH2) has been localised to chromosome 1q32-q41. DNA from a family with four of seven sibs affected with clinical characteristics of Usher syndrome type II was genotyped using markers spanning the 1q32-1q41 region. These included D1S70 and D1S81, which are believed to flank USH2. Genotypic results and subsequent linkage analysis indicated non-linkage of this family to these markers. The A test analysis for heterogeneity with this family and 32 other Usher type II families was statistically significant at p < 0.05. Further clinical evaluation of this family was done in light of the linkage results to determine if any phenotypic characteristics would allow for clinical identification of the unlinked type. No clear phenotypic differences were observed; however, this unlinked family may represent a previously unreported subtype of Usher type II characterised by a milder form of retinitis pigmentosa and mild vestibular abnormalities. Heterogeneity of Usher syndrome type II complicates efforts to isolate and clone Usher syndrome genes using linkage analysis and limits the use of DNA markers in early detection of Usher type II.     

Permeation of human ovarian tissue with cryoprotective agents in preparation for cryopreservation

The recent improvements in the treatment of cancer by chemo- and radiotherapy have led to a significant increase in the survival rates of patients with malignant disease, but at the expense of distressing side effects. One major problem, especially for younger patients, is that aggressive therapy destroys a significant proportion of the follicular population, which can result in either temporary or permanent infertility. Freeze-banking pieces of ovarian cortex prior to treatment is one strategy for preserving fecundity. When the patient is in remission, fertility could, theoretically, be restored by autografting the thawed tissue at the orthotopic site or by growing isolated follicles to maturity in vitro. Recent studies have found good follicular survival in frozen-thawed human ovarian tissue but to optimize the process an effective cryopreservation method needs to be developed. An essential part of such a technique is to permeate the tissue with a cryoprotectant to minimize ice formation and the extent of this equilibration is an important determinant of post-thaw cellular survival. In the current study, we have investigated the diffusion of four cryoprotective agents into human tissue at both 4 degrees C and 37 degrees C. We have also studied the effect of adding different concentrations of the non penetrating cryoprotective agent, sucrose, to the freezing media using the release of lactate dehydrogenase as a measure of its protective effect. At 4 degrees C propylene glycol and glycerol penetrated the tissue significantly slower than either ethylene glycol or dimethyl sulphoxide. At the higher temperature of 37 degrees C all four cryoprotectants penetrated at a faster rate, however concern about enhanced toxicity prevents the use of these conditions in practice. Thus, the results suggest that the best method of preparing tissue for freezing is exposure for 30 min to 1.5 M solutions of ethylene glycol or dimethyl sulphoxide at 4 degrees C; this achieved a mean tissue concentration that was almost 80% that of the bathing solution. We also report that the addition of low concentrations of sucrose to the freezing medium does not have a significant protective effect against freezing injury.      

Sequencing of the alpha-synuclein gene in a large series of cases of familial Parkinson's disease fails to reveal any further mutations. The European Consortium on Genetic Susceptibility in Parkinson's Disease (GSPD)

A mutation in exon 4 of the human alpha-synuclein gene was reported recently in four families with autosomal dominant Parkinson's disease (PD). In order to examine whether mutations in this exon or elsewhere in the gene are common in familial PD, all seven exons of the alpha- synuclein gene were amplified by PCR from index cases of 30 European and American Caucasian kindreds affected with autosomal dominant PD. Each product was sequenced directly and examined for mutations in the open reading frame. No mutations were found in any of the samples examined. We conclude that the A53T change described in the alpha- synuclein gene is a rare cause of PD or may even be a rare variant. Mutations in the regulatory or intronic regions of the gene were not excluded by this study.