Structural and functional analysis of lysosomal ss-galactosidase and its relation to the protective protein.

Lysosomal B-galactosidase is the glycosidase, that cleaves B-linked galactosyl mmenes from a variety of natural and synthetic substrates. In normal tissues of various species this enzyme appears to associate with two other hydrolases, N-acetyl-o:-neuraminidase and the protective protein. Mutations at the B-galactosidase locus on chromosome 3 are the basis of two lysosomal storage disorders, GMcgangliosidosis and Morquio B syndrome. The scope of our experimental work is to gain more insights into the structural and functional properties of these proteins, their mutual relation in lysosomes and their involvement in genetic disorders. Knowledge on the primary structure of lysosomal protective protein has recently brought to the identification of its multifunctional activities. This thesis deals with the isolation and characterization of the cDNAs encoding the classic lysosomal B-galactosidase and a £-galactosidase-related protein. The latter is derived from alternatively spliced pre-m.RNA, as deduced from analysis of the genomic organization of the £-galactosidase gene. These studies include the identification of the £-galactosidase promoter region. In order to introduce this part of the experimental work, the thesis starts with an overview of what is known to date about promoter elements of genes encoding lysosomal enzymes and their putative mode of regulation. The occurrence of alternative splicing among pre-mRNAs coding for other lysosomal enzymes is briefly reviewed. The thesis continues with the description of the genetic lesions found in two patients with the severe infantile type of GMcgangliosidosis. Curiously this mutation, affecting both alleles in the patients, impairs correct splicing of premRNA molecules. The final part of the thesis describes on one hand the two separable roles of the protective protein: a protective function towards £-galactosidase and neuraminidase and a catalytic activity resembling that of lysosomal cathepsin A On the other hand it gives new meaning to the association of £-galactosidase and protective protein, that seems to be important for the correct transport of £galactosidase out of the endoplasmic reticulum and to the lysosomes

Educational Interpreter Services for Hearing-Impaired Students: Provider and Consumer Disagreements

Thirteen supervisors of educational programs for hearing-impaired students completed an assessment designed to determine the need for educational interpreters in a midwestern state and how well it was being met. The results suggested that the trend toward the integration of hearing-impaired students in to regular programs continues and that, with the higher incidence of integration, there is an associated unmet need for educational interpreters. Nine supervisors, 24 teachers, 27 interpreters and 18 hearing-impaired college students rated the characteristics and skills of interpreters which they perceived to be most important. Significant differences existed between and within groups in the characteristics and skills perceived important to educational interpreting (p\u3c .05). Major differences existed between the skills and characteristics cited as most important by hearing-impaired persons and those cited by teachers and interpreters

The missing heritability of familial colorectal cancer

Pinpointing heritability factors is fundamental for the prevention and early detection of cancer. Up to one-quarter of colorectal cancers (CRCs) occur in the context of familial aggregation of this disease, suggesting a strong genetic component. Currently, only less than half of the heritability of CRC can be attributed to hereditary syndromes or common risk loci. Part of the missing heritability of this disease may be explained by the inheritance of elusive high-risk variants, polygenic inheritance, somatic mosaicism, as well as shared environmental factors, among others. A great deal of the missing heritability in CRC is expected to be addressed in the coming years with the increased application of cutting-edge next-generation sequencing technologies, routine multigene panel testing and tumour-focussed germline predisposition screening approaches. On the other hand, it will be important to define the contribution of environmental factors to familial aggregation of CRC incidence. This review provides an overview of the known genetic causes of familial CRC and aims at providing clues that explain the missing heritability of this disease.MTG2 - Moleculaire genetica van gastrointestinale tumore

Quantitative NMR analysis of intra-and extracellular metabolism of mammalian cells: A tutorial

MTG3 - Moleculaire genetica en pathologie van endocriene tumorenMolecular tumour pathology - and tumour genetic

Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH.

Comparative genomic hybridization by means of BAC microarrays (array CGH) allows high-resolution profiling of copy-number aberrations in tumor DNA. However, specific genetic lesions associated with small but clinically relevant tumor areas may pass undetected due to intra-tumor heterogeneity and/or the presence of contaminating normal cells. Here, we show that the combination of laser capture microdissection, phi29 DNA polymerase-mediated isothermal genomic DNA amplification, and array CGH allows genomic profiling of very limited numbers of cells. Moreover, by means of simple statistical models, we were able to bypass the exclusion of amplification distortions and variability prone areas, and to detect tumor-specific chromosomal gains and losses. We applied this new combined experimental and analytical approach to the genomic profiling of colorectal adenomatous polyps and demonstrated our ability to accurately detect single copy gains and losses affecting either whole chromosomes or small genomic regions from as little as 2 ng of DNA or 1000 microdissected cells

BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

Background: Cancer-specific hypermethylation of (promoter) CpG islands is common during the tumorigenesis of colon cancer. Although associations between certain genetic aberrations, such as BRAF mutation and microsatellite instability, and the CpG island methylator phenotype (CIMP), have been found, the mechanisms by which these associations are established are still unclear. We studied genome-wide DNA methylation differences between

MLPAinter for MLPA interpretation: An integrated approach for the analysis, visualisation and data management of Multiplex Ligation-dependent Probe Amplification

Background: Multiplex Ligation-Dependent Probe Amplification (MLPA) is an application that can be used for the detection of multiple chromosomal aberrations in a single experiment. In one reaction, up to 50 different genomic sequences can be analysed. For a reliable work-flow, tools are needed for administrative support, data management, normalisation, visualisation, reporting and interpretation.Results: Here, we developed a data management system, MLPAInter for MLPA interpretation, that is windows executable and has a stand-alone database for monitoring and interpreting the MLPA data stream that is generated from the experimental setup to analysis, quality control and visualisation. A statistical approach is applied for the normalisation and analysis of large series of MLPA traces, making use of multiple control samples and internal controls.Conclusions: MLPAinter visualises MLPA data in plots with information about sample replicates, normalisation settings, and sample characteristics. This integrated approach helps in the automated handling of large series of MLPA data and guarantees a quick and streamlined dataflow from the beginning of an experiment to an authorised report

Use of sanger and next-generation sequencing to screen for mosaic and intronic APC variants in unexplained colorectal polyposis patients

In addition to classic germline APC gene variants, APC mosaicism and deep intronic germline APC variants have also been reported to be causes of adenomatous polyposis. In this study, we investigated 80 unexplained colorectal polyposis patients without germline pathogenic variants in known polyposis predisposing genes to detect mosaic and deep intronic APC variants. All patients developed more than 50 colorectal polyps, with adenomas being predominantly observed. To detect APC mosaicism, we performed next-generation sequencing (NGS) in leukocyte DNA. Furthermore, using Sanger sequencing, the cohort was screened for the following previously reported deep intronic pathogenic germline APC variants: c.1408 + 731C > T, p.(Gly471Serfs*55), c.1408 + 735A > T, p.(Gly471Serfs*55), c.1408 + 729A > G, p.(Gly471Serfs*55) and c.532-941G > A, p.(Phe178Argfs*22). We did not detect mosaic or intronic APC variants in the screened unexplained colorectal polyposis patients. The results of this study indicate that the deep intronic APC variants investigated in this study are not a cause of colorectal polyposis in this Dutch population. In addition, NGS did not detect any further mosaic variants in our cohort.Molecular tumour pathology - and tumour geneticsMTG2 - Moleculaire genetica van gastrointestinale tumore