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

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

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

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

The diverse molecular profiles of lynch syndrome-associated colorectal cancers are (highly) dependent on underlying germline mismatch repair mutations

Lynch syndrome (LS) is a hereditary cancer syndrome that accounts for 3% of all new colorectal cancer (CRC) cases. Patients carry a germline pathogenic variant in one of the mismatch repair (MMR) genes (MLH1, MSH2, MSH6 or PMS2), which encode proteins involved in a post-replicative proofreading and editing mechanism. The clinical presentation of LS is highly heterogeneous, showing high variability in age at onset and penetrance of cancer, which may be partly attributable to the molecular profiles of carcinomas. This review discusses the frequency of alterations in the WNT/B-CATENIN, RAF/MEK/ERK and PI3K/PTEN/AKT pathways identified in all four LS subgroups and how these changes may relate to the 'three pathway model' of carcinogenesis, in which LS CRCs develop from MMR-proficient adenomas, MMR-deficient adenomas or directly from MMR-deficient crypts. Understanding the specific differences in carcinogenesis for each LS subgroup will aid in the further optimization of guidelines for diagnosis, surveillance and treatment.Molecular tumour pathology - and tumour geneticsMTG2 - Moleculaire genetica van gastrointestinale tumore

Declining detection rates for APC and biallelic MUTYH variants in polyposis patients, implications for DNA testing policy

This study aimed to determine the prevalence of APC-associated familial adenomatous polyposis (FAP) and MUTYH-associated polyposis (MAP) in a large cohort, taking into account factors as adenoma count and year of diagnosis. All application forms used to send patients in for APC and MUTYH variant analysis between 1992 and 2017 were collected (n = 2082). Using the data provided on the application form, the APC and biallelic MUTYH prevalence was determined and possible predictive factors were examined using multivariate multinomial logistic regression analysis in SPSS. The prevalence of disease causing variants in the APC gene significantly increases with adenoma count while MAP shows a peak prevalence in individuals with 50–99 adenomas. Logistic regression analysis shows significant odds ratios for adenoma count, age at diagnosis, and, interestingly, a decline in the chance of finding a variant in either gene over time. Moreover, in 22% (43/200) of patients with FAP-related extracolonic manifestations a variant was identified. The overall detection rates are above 10% for patients with >10 adenomas aged 20 adenomas aged T variant in the tumor or a first-degree relative with >10 adenomas. Therefore, APC and MUTYH testing in patients with >10 adenomas aged 20 adenomas aged <70 is advised. Almost all FAP and MAP patients not meeting these criteria showed other characteristics that can be used as an indication to prompt genetic testing

Surgical Treatment for Unexplained Severe Pain of the Thyroid Gland: Report of Three Cases and Concise Review of the Literature

Painful thyroid has a limited differential diagnosis. In rare cases, no clear cause can be found after careful clinical, biochemical, and radiological analysis. This may lead to extensive patient morbidity and frustration when symptomatic treatment proves insufficient. Hemithyroidectomy or total thyroidectomy may then be the last resort for doctor and patient. Three cases of unexplained painful thyroid which were successfully treated with hemi or total thyroidectomy are presented. In two cases extensive histological evaluation did not yield a satisfactory explanation for the extreme thyroid pain. In one case histological evaluation of the thyroid revealed Hashimoto's thyroiditis. Review of the literature does not mention surgical treatment for unexplained painful thyroid, and only 15 cases of surgical treatment for painful Hashimoto's thyroiditis are presented. Surgical therapy is a successful final option in the treatment of unexplained painful thyroid and painful Hashimoto's thyroiditis

Dilemmas for the pathologist in the oncologic assessment of pancreatoduodenectomy specimens

A pancreatoduodenectomy specimen is complex, and there is much debate on how it is best approached by the pathologist. In this review, we provide an overview of topics relevant for current clinical practice in terms of gross dissection, and macro- and microscopic assessment of the pancreatoduodenectomy specimen with a suspicion of suspected pancreatic cancer. Tumor origin, tumor size, degree of differentiation, lymph node status, and resection margin status are universally accepted as prognostic for survival. However, different guidelines diverge on important issues, such as the diagnostic criteria for evaluating the completeness of resection. The macroscopic assessment of the site of origin in periampullary tumors and cystic lesions is influenced by the grossing method. Bi-sectioning of the head of the pancreas may offer an advantage in this respect, as this method allows for optimal visualization of the periampullary area. However, a head-to-head comparison of the assessment of clinically relevant parameters, using axial slicing versus bi-sectioning, is not available yet and the gold standard to compare both techniques prospectively might be subject of debate. Further studies are required to validate the various dissection protocols used for pancreatoduodenectomy specimens and their specific value in the assessment of pathological parameters relevant for prognosis

Disruption of mouse ERCC1 results in a novel repair syndrome with growth failure, nuclear abnormalities and senescence.

BACKGROUND: The structure-specific ERCC1/XPF endonuclease complex that contains the ERCC1 and XPF subunits is implicated in the repair of two distinct types of lesions in DNA: nucleotide excision repair (NER) for ultraviolet-induced lesions and bulky chemical adducts; and recombination repair of the very genotoxic interstrand cross-links. RESULTS: Here, we present a detailed analysis of two types of mice with mutations in ERCC1, one in which the gene is 'knocked out', and one in which the encoded protein contains a seven amino-acid carboxy-terminal truncation. In addition to the previously reported symptoms of severe runting, abnormalities of liver nuclei and greatly reduced lifespan (which appeared less severe in the truncation mutant), both types of ERCC1-mutant mouse exhibited an absence of subcutaneous fat, early onset of ferritin deposition in the spleen, kidney malfunction, gross abnormalities of ploidy and cytoplasmic invaginations in nuclei of liver and kidney, and compromised NER and cross-link repair. We also found that heterozygosity for ERCC1 mutations did not appear to provide a selective advantage for chemically induced tumorigenesis. An important clue to the cause of the very severe ERCC1-mutant phenotypes is our finding that ERCC1-mutant cells undergo premature replicative senescence, unlike cells from mice with a defect only in NER. CONCLUSIONS: Our results strongly suggest that the accumulation in ERCC1-mutant mice of endogenously generated DNA interstrand cross-links, which are normally repaired by ERCC1-dependent recombination repair, underlies both the early onset of cell cycle arrest and polyploidy in the liver and kidney. Thus, our work provides an insight into the molecular basis of ageing and highlights the role of ERCC1 and interstrand DNA cross-links