Assessment of the genetic causes of recessive childhood non-syndromic deafness in the UK - implications for genetic testing.

Approximately one in 2000 children is born with a genetic hearing impairment, mostly inherited as a non-syndromic, autosomal recessive trait, for which more than 30 different genes have been identified. Previous studies have shown that one of these genes, connexin 26 (GJB2), accounts for 30-60% of such deafness, but the relative contribution of the many other genes is not known, especially in the outbred UK population. This lack of knowledge hampers the development of diagnostic genetic services for deafness. In an effort to determine the molecular aetiology of deafness in the population, 142 sib pairs with early-onset, non-syndromic hearing impairment were recruited. Those in whom deafness could not be attributed to GJB2 mutations were investigated further for other mapped genes. The genetic basis of 55 cases (38.7%) was established, 33.1% being due to mutations in the GJB2 gene and 3.5% due to mutations in SLC26A4. None of the remaining 26 loci investigated made a significant contribution to deafness in a Caucasian population. We suggest that screening the GJB2 and SLC26A4 genes should form the basis of any genetic testing programme for childhood deafness and highlight a number of important issues for consideration and future work.

Erythroid Kruppel-like factor (EKLF) coordinates erythroid cell proliferation and hemoglobinization in cell lines derived from EKLF null mice.

Erythroid Kruppel-like factor (EKLF) is a transcription factor of the C2H2 zinc-finger class that is essential for definitive erythropoiesis. We generated immortal erythroid cell lines from EKLF(-/-) fetal liver progenitor cells that harbor a single copy of the entire human beta-globin locus and then reintroduced EKLF as a tamoxifen-inducible, EKLF-mutant estrogen receptor (EKLF-ER) fusion protein. Addition of tamoxifen resulted in enhanced differentiation and hemoglobinization, coupled with reduced proliferation. Human beta-globin gene expression increased significantly, whereas gamma-globin transcripts remained elevated at levels close to endogenous mouse alpha-globin transcript levels. We conclude that EKLF plays a role in regulation of the cell cycle and hemoglobinization in addition to its role in beta-globin gene expression. The cell lines we used will facilitate structural and functional analyses of EKLF in these processes and provide useful tools for the elucidation of nonglobin EKLF target genes.

A three-colour flow cytometry technique for measuring trophoblast intracellular antigens: the relative expression of TAP1 in human cytotrophoblast and decidual cells.

Flow cytometry is conventionally used to measure cell-surface antigen expression. However, many antigens are found within the cytoplasm, and it is necessary to fix and permeabilize cells to enable antibodies to gain access to them. In this study we have established the conditions for studying intracellular antigens in human trophoblast cells by flow cytometry using an antibody to TAP1 (a key molecule in the process of Class I MHC assembly). We have previously shown by immunocytochemistry that TAP1 expression is apparently greater on Class 1 positive extravillous cytotrophoblast than on any other fetal or maternal tissue. However, as immunohistochemistry is not quantitative we have used three-colour flow cytometry to measure the expression of TAP1 in different trophoblast populations. Villous and extravillous cytotrophoblast were identified in first trimester and term placental and decidual digests on the basis of their expression of cytokeratin and Class I MHC antigens. The level of expression of TAP1 for each population was investigated using a commercial kit that determines the number of antibody-binding sites per cell. TAP expression was found to be three- to fivefold higher in extravillous cytotrophoblast, confirming our previous findings. The techniques developed here are directly applicable to the measurement of other intracellular molecules in trophoblast, in particular cytokines.

Glycoforms modify the dynamic stability and functional activity of an enzyme.

Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide proton exchange rates for individual glycoforms of RNase B and unglycosylated RNase A showed that while the 3D structure was unaffected, glycosylation decreased dynamic fluctuations throughout the molecule. There was individual variation in the NH-ND exchange rates of the same protons in different glycoforms, demonstrating the effects of variable glycosylation on dynamic stability. Consistent with the overall decrease in flexibility, and with the possibility that all of the sugars may afford steric protection to susceptible sites, was the finding that each of the glycoforms tested showed increased resistance to Pronase compared with the unglycosylated protein. In a novel sensitive assay using double-stranded RNA substrate, the different glycoforms showed nearly a 4-fold variation in functional activity; molecular modeling suggested that steric factors may also play a role in modulating this interaction.

Separation and analysis of the glycoform populations of ribonuclease B using capillary electrophoresis.

The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomannose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B with A. saitoi alpha(1-2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.